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1
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Sobkowiak K, Kohzaki M, Böhm R, Mailler J, Huber F, Emamzadah S, Tropia L, Hiller S, Halazonetis TD. REV7 functions with REV3 as a checkpoint protein delaying mitotic entry until DNA replication is completed. Cell Rep 2025; 44:115431. [PMID: 40106439 DOI: 10.1016/j.celrep.2025.115431] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2024] [Revised: 12/18/2024] [Accepted: 02/24/2025] [Indexed: 03/22/2025] Open
Abstract
REV7, also named MAD2B or MAD2L2, is a subunit of the DNA translesion polymerase zeta and also part of the 53BP1-shieldin complex, which is present at sites of DNA double-strand breaks. REV7 has high sequence similarity to the MAD2 spindle assembly checkpoint protein, prompting us to examine whether REV7 has a checkpoint function. We observed that, in chicken and human cells exposed to agents that induce DNA replication stress, REV7 inhibits mitotic entry; this effect is most evident when the canonical DNA replication stress checkpoint, mediated by ATR, is inhibited. Similar to MAD2, REV7 undergoes conformational changes upon ligand binding, and its checkpoint function depends on its ability to homodimerize and bind its ligands. Notably, even in unchallenged cells, deletion of the REV7 gene leads to premature mitotic entry, raising the possibility that the REV7 checkpoint monitors ongoing DNA replication.
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Affiliation(s)
- Katarzyna Sobkowiak
- Department of Molecular and Cellular Biology, University of Geneva, 1205 Geneva, Switzerland
| | - Masaoki Kohzaki
- Department of Molecular and Cellular Biology, University of Geneva, 1205 Geneva, Switzerland.
| | - Raphael Böhm
- Biozentrum, University of Basel, 4056 Basel, Switzerland
| | - Jonathan Mailler
- Department of Molecular and Cellular Biology, University of Geneva, 1205 Geneva, Switzerland
| | - Florian Huber
- Department of Molecular and Cellular Biology, University of Geneva, 1205 Geneva, Switzerland
| | - Soheila Emamzadah
- Department of Molecular and Cellular Biology, University of Geneva, 1205 Geneva, Switzerland
| | - Laurence Tropia
- Department of Molecular and Cellular Biology, University of Geneva, 1205 Geneva, Switzerland
| | | | - Thanos D Halazonetis
- Department of Molecular and Cellular Biology, University of Geneva, 1205 Geneva, Switzerland.
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2
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Cordes J, Zhao S, Engel CM, Stingele J. Cellular responses to RNA damage. Cell 2025; 188:885-900. [PMID: 39983673 DOI: 10.1016/j.cell.2025.01.005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2024] [Revised: 11/26/2024] [Accepted: 01/02/2025] [Indexed: 02/23/2025]
Abstract
RNA plays a central role in protein biosynthesis and performs diverse regulatory and catalytic functions, making it essential for all processes of life. Like DNA, RNA is constantly subjected to damage from endogenous and environmental sources. However, while the DNA damage response has been extensively studied, it was long assumed that RNA lesions are relatively inconsequential due to the transient nature of most RNA molecules. Here, we review recent studies that challenge this view by revealing complex RNA damage responses that determine survival when cells are exposed to nucleic acid-damaging agents and promote the resolution of RNA lesions.
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Affiliation(s)
- Jacqueline Cordes
- Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, 81377 Munich, Germany
| | - Shubo Zhao
- Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, 81377 Munich, Germany; College of Basic Medical Sciences, Medical Basic Research Innovation Center of Airway Disease in North China, Key Laboratory of Pathobiology, Ministry of Education, Jilin University, Changchun 130021, China
| | - Carla M Engel
- Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, 81377 Munich, Germany
| | - Julian Stingele
- Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, 81377 Munich, Germany.
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3
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Lim HE, Lim HJ, Yoo HY. Interaction of DDB1 with NBS1 in a DNA Damage Checkpoint Pathway. Int J Mol Sci 2024; 25:13097. [PMID: 39684807 DOI: 10.3390/ijms252313097] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2024] [Revised: 11/19/2024] [Accepted: 12/04/2024] [Indexed: 12/18/2024] Open
Abstract
Various DNA damage checkpoint control mechanisms in eukaryotic cells help maintain genomic integrity. Among these, NBS1, a key component of the MRE11-RAD50-NBS1 (MRN) complex, is an essential protein involved in the DNA damage response (DDR). In this study, we discovered that DNA damage-binding protein 1 (DDB1) interacts with NBS1. DDB1 is a DDR sensor protein found in UV-induced DNA replication blocks. Through pull-down and immunoprecipitation assays conducted in Xenopus egg extracts and human cell lines, we demonstrated a specific interaction between NBS1 and DDB1. DDB1 was also found to associate with several proteins that interact with NBS1, including DNA topoisomerase 2-binding protein 1 (TopBP1) and Mediator of DNA damage checkpoint protein 1 (MDC1). Notably, the interaction between DDB1 and NBS1 is disrupted in MDC1-depleted egg extracts, indicating that MDC1 is necessary for this interaction. Furthermore, the depletion of DDB1 leads to increased Chk1 activation upon DNA damage. These novel findings regarding the interaction between NBS1 and DDB1 provide new insights into how DDB1 regulates DNA damage pathways.
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Affiliation(s)
- Hoe Eun Lim
- Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul 06351, Republic of Korea
- Research Institute for Future Medicine, Samsung Medical Center, Seoul 06351, Republic of Korea
| | - Hee Jung Lim
- Research Institute for Future Medicine, Samsung Medical Center, Seoul 06351, Republic of Korea
| | - Hae Yong Yoo
- Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul 06351, Republic of Korea
- Research Institute for Future Medicine, Samsung Medical Center, Seoul 06351, Republic of Korea
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4
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Shi JJ, Chen RY, Liu YJ, Li CY, Yu J, Tu FY, Sheng JX, Lu JF, Zhang LL, Yang GJ, Chen J. Unraveling the role of ubiquitin-conjugating enzyme 5 (UBC5) in disease pathogenesis: A comprehensive review. Cell Signal 2024; 124:111376. [PMID: 39236836 DOI: 10.1016/j.cellsig.2024.111376] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2024] [Revised: 08/22/2024] [Accepted: 08/30/2024] [Indexed: 09/07/2024]
Abstract
While certain members of ubiquitin-coupled enzymes (E2s) have garnered attention as potential therapeutic targets across diverse diseases, research progress on Ubiquitin-Conjugating Enzyme 5 (UBC5)-a pivotal member of the E2s family involved in crucial cellular processes such as apoptosis, DNA repair, and signal transduction-has been relatively sluggish. Previous findings suggest that UBC5 plays a vital role in the ubiquitination of various target proteins implicated in diseases and homeostasis, particularly in various cancer types. This review comprehensively introduces the structure and biological functions of UBC5, with a specific focus on its contributions to the onset and advancement of diverse diseases. It suggests that targeting UBC5 holds promise as a therapeutic approach for disease therapy. Recent discoveries highlighting the high homology between UBC5, UBC1, and UBC4 have provided insight into the mechanism of UBC5 in protein degradation and the regulation of cellular functions. As our comprehension of the structural distinctions among UBC5 and its homologues, namely UBC1 and UBC4, advances, our understanding of UBC5's functional significance also expands.
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Affiliation(s)
- Jin-Jin Shi
- State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, School of Marine Sciences, Ningbo University, Ningbo 315211, China
| | - Ru-Yi Chen
- State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, School of Marine Sciences, Ningbo University, Ningbo 315211, China
| | - Yan-Jun Liu
- State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, School of Marine Sciences, Ningbo University, Ningbo 315211, China
| | - Chang-Yun Li
- State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, School of Marine Sciences, Ningbo University, Ningbo 315211, China
| | - Jing Yu
- State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, School of Marine Sciences, Ningbo University, Ningbo 315211, China
| | - Fei-Yang Tu
- State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, School of Marine Sciences, Ningbo University, Ningbo 315211, China
| | - Jian-Xiang Sheng
- State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, School of Marine Sciences, Ningbo University, Ningbo 315211, China
| | - Jian-Fei Lu
- State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, School of Marine Sciences, Ningbo University, Ningbo 315211, China
| | - Le-Le Zhang
- School of Basic Medical Sciences, Chengdu University, Chengdu 610106, China.
| | - Guan-Jun Yang
- State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, School of Marine Sciences, Ningbo University, Ningbo 315211, China.
| | - Jiong Chen
- State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, School of Marine Sciences, Ningbo University, Ningbo 315211, China.
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5
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Li P, Yu X. The role of rRNA in maintaining genome stability. DNA Repair (Amst) 2024; 139:103692. [PMID: 38759435 DOI: 10.1016/j.dnarep.2024.103692] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2024] [Revised: 05/06/2024] [Accepted: 05/06/2024] [Indexed: 05/19/2024]
Abstract
Over the past few decades, unbiased approaches such as genetic screening and protein affinity purification have unveiled numerous proteins involved in DNA double-strand break (DSB) repair and maintaining genome stability. However, despite our knowledge of these protein factors, the underlying molecular mechanisms governing key cellular events during DSB repair remain elusive. Recent evidence has shed light on the role of non-protein factors, such as RNA, in several pivotal steps of DSB repair. In this review, we provide a comprehensive summary of these recent findings, highlighting the significance of ribosomal RNA (rRNA) as a critical mediator of DNA damage response, meiosis, and mitosis. Moreover, we discuss potential mechanisms through which rRNA may influence genome integrity.
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Affiliation(s)
- Peng Li
- Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang, China; School of Life Sciences, Westlake University, Hangzhou, Zhejiang, China; Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, Zhejiang, China
| | - Xiaochun Yu
- Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang, China; School of Life Sciences, Westlake University, Hangzhou, Zhejiang, China; Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, Zhejiang, China.
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6
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O'Brien S, Ubhi T, Wolf L, Gandhi K, Lin S, Chaudary N, Dhani NC, Milosevic M, Brown GW, Angers S. FBXW7-loss Sensitizes Cells to ATR Inhibition Through Induced Mitotic Catastrophe. CANCER RESEARCH COMMUNICATIONS 2023; 3:2596-2607. [PMID: 38032106 PMCID: PMC10734389 DOI: 10.1158/2767-9764.crc-23-0306] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/18/2023] [Revised: 09/27/2023] [Accepted: 11/28/2023] [Indexed: 12/01/2023]
Abstract
FBXW7 is a commonly mutated tumor suppressor gene that functions to regulate numerous oncogenes involved in cell-cycle regulation. Genome-wide CRISPR fitness screens identified a signature of DNA repair and DNA damage response genes as required for the growth of FBXW7-knockout cells. Guided by these findings, we show that FBXW7-mutant cells have high levels of replication stress, which results in a genotype-specific vulnerability to inhibition of the ATR signaling pathway, as these mutant cells become heavily reliant on a robust S-G2 checkpoint. ATR inhibition induces an accelerated S-phase, leading to mitotic catastrophe and cell death caused by the high replication stress present in FBXW7-/- cells. In addition, we provide evidence in cell and organoid studies, and mining of publicly available high-throughput drug screening efforts, that this genotype-specific vulnerability extends to multiple types of cancer, providing a rational means of identifying responsive patients for targeted therapy. SIGNIFICANCE We have elucidated the synthetic lethal interactions between FBXW7 mutation and DNA damage response genes, and highlighted the potential of ATR inhibitors as targeted therapies for cancers harboring FBXW7 alterations.
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Affiliation(s)
- Siobhan O'Brien
- Department of Biochemistry, University of Toronto, Ontario, Canada
- Terrence Donnelly Centre for Cellular and Biomolecular Research, Toronto, Ontario, Canada
| | - Tajinder Ubhi
- Department of Biochemistry, University of Toronto, Ontario, Canada
- Terrence Donnelly Centre for Cellular and Biomolecular Research, Toronto, Ontario, Canada
| | - Lucie Wolf
- Terrence Donnelly Centre for Cellular and Biomolecular Research, Toronto, Ontario, Canada
| | - Krishna Gandhi
- Terrence Donnelly Centre for Cellular and Biomolecular Research, Toronto, Ontario, Canada
| | - Sichun Lin
- Terrence Donnelly Centre for Cellular and Biomolecular Research, Toronto, Ontario, Canada
| | - Naz Chaudary
- Princess Margaret Cancer Centre, Toronto, Ontario, Canada
- Ontario Cancer Institute, Toronto, Ontario, Canada
| | | | - Michael Milosevic
- Princess Margaret Cancer Centre, Toronto, Ontario, Canada
- Department of Radiation Oncology, University of Toronto, Ontario, Canada
- Institute of Medical Science, University of Toronto, Ontario, Canada
| | - Grant W. Brown
- Department of Biochemistry, University of Toronto, Ontario, Canada
- Terrence Donnelly Centre for Cellular and Biomolecular Research, Toronto, Ontario, Canada
| | - Stephane Angers
- Department of Biochemistry, University of Toronto, Ontario, Canada
- Terrence Donnelly Centre for Cellular and Biomolecular Research, Toronto, Ontario, Canada
- Leslie Dan Faculty of Pharmacy, University of Toronto, Ontario, Canada
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7
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Liu S, Byrne BM, Byrne TN, Oakley GG. Role of RPA Phosphorylation in the ATR-Dependent G2 Cell Cycle Checkpoint. Genes (Basel) 2023; 14:2205. [PMID: 38137027 PMCID: PMC10742774 DOI: 10.3390/genes14122205] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2023] [Revised: 12/05/2023] [Accepted: 12/06/2023] [Indexed: 12/24/2023] Open
Abstract
Cells respond to DNA double-strand breaks by initiating DSB repair and ensuring a cell cycle checkpoint. The primary responder to DSB repair is non-homologous end joining, which is an error-prone repair pathway. However, when DSBs are generated after DNA replication in the G2 phase of the cell cycle, a second DSB repair pathway, homologous recombination, can come into action. Both ATM and ATR are important for DSB-induced DSB repair and checkpoint responses. One method of ATM and ATR working together is through the DNA end resection of DSBs. As a readout and marker of DNA end resection, RPA is phosphorylated at Ser4/Ser8 of the N-terminus of RPA32 in response to DSBs. Here, the significance of RPA32 Ser4/Ser8 phosphorylation in response to DNA damage, specifically in the S phase to G2 phase of the cell cycle, is examined. RPA32 Ser4/Ser8 phosphorylation in G2 synchronized cells is necessary for increases in TopBP1 and Rad9 accumulation on chromatin and full activation of the ATR-dependent G2 checkpoint. In addition, our data suggest that RPA Ser4/Ser8 phosphorylation modulates ATM-dependent KAP-1 phosphorylation and Rad51 chromatin loading in G2 cells. Through the phosphorylation of RPA Ser4/Ser8, ATM acts as a partner with ATR in the G2 phase checkpoint response, regulating key downstream events including Rad9, TopBP1 phosphorylation and KAP-1 phosphorylation/activation via the targeting of RPA32 Ser4/Ser8.
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Affiliation(s)
- Shengqin Liu
- Department of Oral Biology, University of Nebraska Medical Center College of Dentistry, Lincoln, NE 68583, USA
| | - Brendan M. Byrne
- Department of Oral Biology, University of Nebraska Medical Center College of Dentistry, Lincoln, NE 68583, USA
| | - Thomas N. Byrne
- Department of Oral Biology, University of Nebraska Medical Center College of Dentistry, Lincoln, NE 68583, USA
| | - Gregory G. Oakley
- Department of Oral Biology, University of Nebraska Medical Center College of Dentistry, Lincoln, NE 68583, USA
- Eppley Cancer Center, Omaha, NE 68198, USA
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8
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Wu X, Zhou X, Wang S, Mao G. DNA damage response(DDR): a link between cellular senescence and human cytomegalovirus. Virol J 2023; 20:250. [PMID: 37915066 PMCID: PMC10621139 DOI: 10.1186/s12985-023-02203-y] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2023] [Accepted: 10/04/2023] [Indexed: 11/03/2023] Open
Abstract
The DNA damage response (DDR) is a signaling cascade that is triggered by DNA damage, involving the halting of cell cycle progression and repair. It is a key event leading to senescence, which is characterized by irreversible cell cycle arrest and the senescence-associated secretory phenotype (SASP) that includes the expression of inflammatory cytokines. Human cytomegalovirus (HCMV) is a ubiquitous pathogen that plays an important role in the senescence process. It has been established that DDR is necessary for HCMV to replicate effectively. This paper reviews the relationship between DDR, cellular senescence, and HCMV, providing new sights for virus-induced senescence (VIS).
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Affiliation(s)
- Xinna Wu
- Affiliated Zhejiang Hospital, Zhejiang University School of Medicine, Hangzhou, 310030, China
| | - Xuqiang Zhou
- College of Life Science, Zhejiang Chinese Medical University, Hangzhou, 310053, China
| | - Sanying Wang
- Zhejiang Provincial Key Lab of Geriatrics & Geriatrics Institute of Zhejiang Province, Department of Geriatrics, Zhejiang Hospital, Hangzhou, 310030, China.
| | - Genxiang Mao
- Affiliated Zhejiang Hospital, Zhejiang University School of Medicine, Hangzhou, 310030, China.
- Zhejiang Provincial Key Lab of Geriatrics & Geriatrics Institute of Zhejiang Province, Department of Geriatrics, Zhejiang Hospital, Hangzhou, 310030, China.
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9
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Xin D, Gai X, Ma Y, Li Z, Li Q, Yu X. Pre-rRNA Facilitates TopBP1-Mediated DNA Double-Strand Break Response. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2023; 10:e2206931. [PMID: 37582658 PMCID: PMC10558638 DOI: 10.1002/advs.202206931] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/24/2022] [Revised: 06/28/2023] [Indexed: 08/17/2023]
Abstract
In response to genotoxic stress-induced DNA damage, TopBP1 mediates ATR activation for signaling transduction and DNA damage repair. However, the detailed molecular mechanism remains elusive. Here, using unbiased protein affinity purification and RNA sequencing, it is found that TopBP1 is associated with pre-ribosomal RNA (pre-rRNA). Pre-rRNA co-localized with TopBP1 at DNA double-strand breaks (DSBs). Similar to pre-rRNA, ribosomal proteins also colocalize with TopBP1 at DSBs. The recruitment of TopBP1 to DSBs is suppressed when cells are transiently treated with RNA polymerase I inhibitor (Pol I-i) to suppress pre-rRNA biogenesis but not protein translation. Moreover, the BRCT4-5 of TopBP1 recognizes pre-rRNA and forms liquid-liquid phase separation (LLPS) with pre-rRNA, which may be the molecular basis of DSB-induced foci of TopBP1. Finally, Pol I-i treatment impairs TopBP1-associated cell cycle checkpoint activation and homologous recombination repair. Collectively, this study reveals that pre-rRNA plays a key role in the TopBP1-dependent DNA damage response.
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Affiliation(s)
- Di Xin
- School of Life SciencesWestlake UniversityHangzhouZhejiang310024China
- Department of Hepatobiliary and Pancreatic Surgery and Zhejiang Provincial Key Laboratory of Pancreatic DiseaseThe First Affiliated HospitalZhejiang University School of MedicineHangzhouZhejiang310003China
- Westlake Laboratory of Life Sciences and BiomedicineHangzhouZhejiang310024China
- Institute of Basic Medical SciencesWestlake Institute for Advanced StudyHangzhouZhejiang310024China
| | - Xiaochen Gai
- School of Life SciencesWestlake UniversityHangzhouZhejiang310024China
- Westlake Laboratory of Life Sciences and BiomedicineHangzhouZhejiang310024China
- Institute of Basic Medical SciencesWestlake Institute for Advanced StudyHangzhouZhejiang310024China
| | - Yidi Ma
- School of Life SciencesWestlake UniversityHangzhouZhejiang310024China
- Westlake Laboratory of Life Sciences and BiomedicineHangzhouZhejiang310024China
- Institute of Basic Medical SciencesWestlake Institute for Advanced StudyHangzhouZhejiang310024China
| | - Zexing Li
- School of Life SciencesTianjin UniversityTianjin300072China
| | - Qilin Li
- School of Life SciencesWestlake UniversityHangzhouZhejiang310024China
- Westlake Laboratory of Life Sciences and BiomedicineHangzhouZhejiang310024China
- Institute of Basic Medical SciencesWestlake Institute for Advanced StudyHangzhouZhejiang310024China
| | - Xiaochun Yu
- School of Life SciencesWestlake UniversityHangzhouZhejiang310024China
- Westlake Laboratory of Life Sciences and BiomedicineHangzhouZhejiang310024China
- Institute of Basic Medical SciencesWestlake Institute for Advanced StudyHangzhouZhejiang310024China
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10
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Kang Y, Han YG, Khim KW, Choi WG, Ju MK, Park K, Shin KJ, Chae YC, Choi JH, Kim H, Lee JY. Alteration of replication protein A binding mode on single-stranded DNA by NSMF potentiates RPA phosphorylation by ATR kinase. Nucleic Acids Res 2023; 51:7936-7950. [PMID: 37378431 PMCID: PMC10450186 DOI: 10.1093/nar/gkad543] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2022] [Revised: 06/07/2023] [Accepted: 06/13/2023] [Indexed: 06/29/2023] Open
Abstract
Replication protein A (RPA), a eukaryotic single-stranded DNA (ssDNA) binding protein, dynamically interacts with ssDNA in different binding modes and plays essential roles in DNA metabolism such as replication, repair, and recombination. RPA accumulation on ssDNA due to replication stress triggers the DNA damage response (DDR) by activating the ataxia telangiectasia and RAD3-related (ATR) kinase, which phosphorylates itself and downstream DDR factors, including RPA. We recently reported that the N-methyl-D-aspartate receptor synaptonuclear signaling and neuronal migration factor (NSMF), a neuronal protein associated with Kallmann syndrome, promotes RPA32 phosphorylation via ATR upon replication stress. However, how NSMF enhances ATR-mediated RPA32 phosphorylation remains elusive. Here, we demonstrate that NSMF colocalizes and physically interacts with RPA at DNA damage sites in vivo and in vitro. Using purified RPA and NSMF in biochemical and single-molecule assays, we find that NSMF selectively displaces RPA in the more weakly bound 8- and 20-nucleotide binding modes from ssDNA, allowing the retention of more stable RPA molecules in the 30-nt binding mode. The 30-nt binding mode of RPA enhances RPA32 phosphorylation by ATR, and phosphorylated RPA becomes stabilized on ssDNA. Our findings provide new mechanistic insight into how NSMF facilitates the role of RPA in the ATR pathway.
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Affiliation(s)
- Yujin Kang
- Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan 44919, Republic of Korea
| | - Ye Gi Han
- Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan 44919, Republic of Korea
| | - Keon Woo Khim
- Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan 44919, Republic of Korea
| | - Woo Gyun Choi
- Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan 44919, Republic of Korea
| | - Min Kyung Ju
- Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan 44919, Republic of Korea
| | - Kibeom Park
- Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan 44919, Republic of Korea
| | - Kyeong Jin Shin
- Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan 44919, Republic of Korea
| | - Young Chan Chae
- Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan 44919, Republic of Korea
| | - Jang Hyun Choi
- Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan 44919, Republic of Korea
- Institute of Basic Science Center for Genomic Integrity, Ulsan 44919, Republic of Korea
| | - Hongtae Kim
- Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan 44919, Republic of Korea
- Institute of Basic Science Center for Genomic Integrity, Ulsan 44919, Republic of Korea
| | - Ja Yil Lee
- Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan 44919, Republic of Korea
- Institute of Basic Science Center for Genomic Integrity, Ulsan 44919, Republic of Korea
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11
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Lin Y, Li J, Zhao H, McMahon A, McGhee K, Yan S. APE1 recruits ATRIP to ssDNA in an RPA-dependent and -independent manner to promote the ATR DNA damage response. eLife 2023; 12:e82324. [PMID: 37216274 PMCID: PMC10202453 DOI: 10.7554/elife.82324] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2022] [Accepted: 05/08/2023] [Indexed: 05/24/2023] Open
Abstract
Cells have evolved the DNA damage response (DDR) pathways in response to DNA replication stress or DNA damage. In the ATR-Chk1 DDR pathway, it has been proposed that ATR is recruited to RPA-coated single-stranded DNA (ssDNA) by direct ATRIP-RPA interaction. However, it remains elusive how ATRIP is recruited to ssDNA in an RPA-independent manner. Here, we provide evidence that APE1 directly associates ssDNA to recruit ATRIP onto ssDNA in an RPA-independent fashion. The N-terminal motif within APE1 is required and sufficient for the APE1-ATRIP interaction in vitro and the distinct APE1-ATRIP interaction is required for ATRIP recruitment to ssDNA and the ATR-Chk1 DDR pathway activation in Xenopus egg extracts. In addition, APE1 directly associates with RPA70 and RPA32 via two distinct motifs. Taken together, our evidence suggests that APE1 recruits ATRIP onto ssDNA in an RPA-dependent and -independent manner in the ATR DDR pathway.
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Affiliation(s)
- Yunfeng Lin
- Department of Biological Sciences, University of North Carolina at CharlotteCharlotteUnited States
| | - Jia Li
- Department of Biological Sciences, University of North Carolina at CharlotteCharlotteUnited States
| | - Haichao Zhao
- Department of Biological Sciences, University of North Carolina at CharlotteCharlotteUnited States
| | - Anne McMahon
- Department of Biological Sciences, University of North Carolina at CharlotteCharlotteUnited States
| | - Kelly McGhee
- Department of Biological Sciences, University of North Carolina at CharlotteCharlotteUnited States
| | - Shan Yan
- Department of Biological Sciences, University of North Carolina at CharlotteCharlotteUnited States
- School of Data Science, University of North Carolina at CharlotteCharlotteUnited States
- Center for Biomedical Engineering and Science, University of North Carolina at CharlotteCharlotteUnited States
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12
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Sundararajan V, Tan TZ, Lim D, Peng Y, Wengner AM, Ngoi NYL, Jeyasekharan AD, Tan DSP. Nuclear pCHK1 as a potential biomarker of increased sensitivity to ATR inhibition. J Pathol 2023; 259:194-204. [PMID: 36373784 PMCID: PMC10107453 DOI: 10.1002/path.6032] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2022] [Revised: 10/28/2022] [Accepted: 11/10/2022] [Indexed: 11/16/2022]
Abstract
Excessive genomic instability coupled with abnormalities in DNA repair pathways induces high levels of 'replication stress' when cancer cells propagate. Rather than hampering cancer cell proliferation, novel treatment strategies are turning their attention towards targeting cell cycle checkpoint kinases (such as ATR, CHK1, WEE1, and others) along the DNA damage response and replicative stress response pathways, thereby allowing unrepaired DNA damage to be carried forward towards mitotic catastrophe and apoptosis. The selective ATR kinase inhibitor elimusertib (BAY 1895344) has demonstrated preclinical and clinical monotherapy activity; however, reliable predictive biomarkers of treatment benefit are still lacking. In this study, using gene expression profiling of 24 cell lines from different cancer types and in a panel of ovarian cancer cell lines, we found that nuclear-specific enrichment of checkpoint kinase 1 (CHK1) correlated with increased sensitivity to elimusertib. Using an advanced multispectral imaging system in subsequent cell line-derived xenograft specimens, we showed a trend between nuclear phosphorylated CHK1 (pCHK1) staining and increased sensitivity to the ATR inhibitor elimusertib, indicating the potential value of pCHK1 expression as a predictive biomarker of ATR inhibitor sensitivity. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
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Affiliation(s)
- Vignesh Sundararajan
- Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore
| | - Tuan Zea Tan
- Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore.,Genomics and Data Analytics Core (GeDaC), Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore
| | - Diana Lim
- Department of Pathology, National University Hospital, Singapore, Singapore
| | - Yanfen Peng
- Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore
| | | | - Natalie Yan Li Ngoi
- Department of Haematology-Oncology, National University Cancer Institute, Singapore, Singapore
| | - Anand D Jeyasekharan
- Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore
| | - David Shao Peng Tan
- Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore.,Department of Haematology-Oncology, National University Cancer Institute, Singapore, Singapore
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13
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Xu T, Ma Q, Li Y, Yu Q, Pan P, Zheng Y, Li Z, Xiong X, Hou T, Yu B, Liu H, Sun Y. A small molecule inhibitor of the UBE2F-CRL5 axis induces apoptosis and radiosensitization in lung cancer. Signal Transduct Target Ther 2022; 7:354. [PMID: 36253371 PMCID: PMC9576757 DOI: 10.1038/s41392-022-01182-w] [Citation(s) in RCA: 26] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2022] [Revised: 08/21/2022] [Accepted: 09/06/2022] [Indexed: 11/18/2022] Open
Abstract
Protein neddylation is catalyzed by a neddylation activating enzyme (NAE, E1), an E2 conjugating enzyme, and an E3 ligase. In various types of human cancers, the neddylation pathway is abnormally activated. Our previous study validated that the neddylation E2 UBE2F is a promising therapeutic target in lung cancer. Although the NAE inhibitor MLN4924/pevonedistat is currently under clinical investigation as an anti-cancer agent, there are no small molecules available that selectively target UBE2F. Here, we report, for the first time, the discovery, via structure-based virtual screen and chemical optimization, of such a small molecule, designated as HA-9104. HA-9104 binds to UBE2F, reduces its protein levels, and consequently inhibits cullin-5 neddylation. Blockage of cullin-5 neddylation inactivates cullin-RING ligase-5 (CRL5) activity, leading to accumulation of the CRL5 substrate, NOXA, to induce apoptosis. Moreover, HA-9104 appears to form the DNA adduct via its 7-azaindole group to induce DNA damage and G2/M arrest. Biologically, HA-9104 effectively suppresses the growth and survival of lung cancer cells and confers radiosensitization in both in vitro cell culture and in vivo xenograft tumor models. In summary, we discovered a small molecule, designated HA-9104, that targets the UBE2F-CRL5 axis with anti-cancer activity alone or in combination with radiation.
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Affiliation(s)
- Tiantian Xu
- Cancer Institute, the Second Affiliated Hospital, and Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, 310029, China.,Cancer Center, Zhejiang University, Hangzhou, 310058, China.,Research Center for Life Science and Human Health, Binjiang Institute of Zhejiang University, Hangzhou, 310053, China
| | - Qisheng Ma
- School of Pharmaceutical Sciences, Key Laboratory of Advanced Drug Preparation Technologies, Military of Education, Zhengzhou University, Zhengzhou, 450001, China
| | - Yanan Li
- Cancer Institute, the Second Affiliated Hospital, and Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, 310029, China.,Cancer Center, Zhejiang University, Hangzhou, 310058, China
| | - Qing Yu
- Cancer Institute, the Second Affiliated Hospital, and Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, 310029, China.,Cancer Center, Zhejiang University, Hangzhou, 310058, China
| | - Peichen Pan
- College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China
| | - Yawen Zheng
- Department of Oncology, Jinan Central Hospital, Cheeloo College of Medicine, Shandong University, Jinan, 250033, China
| | - Zhijian Li
- Cancer Institute, the Second Affiliated Hospital, and Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, 310029, China.,Cancer Center, Zhejiang University, Hangzhou, 310058, China.,Research Center for Life Science and Human Health, Binjiang Institute of Zhejiang University, Hangzhou, 310053, China
| | - Xiufang Xiong
- Cancer Institute, the Second Affiliated Hospital, and Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, 310029, China.,Cancer Center, Zhejiang University, Hangzhou, 310058, China.,Research Center for Life Science and Human Health, Binjiang Institute of Zhejiang University, Hangzhou, 310053, China
| | - Tingjun Hou
- College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China
| | - Bin Yu
- School of Pharmaceutical Sciences, Key Laboratory of Advanced Drug Preparation Technologies, Military of Education, Zhengzhou University, Zhengzhou, 450001, China
| | - Hongmin Liu
- School of Pharmaceutical Sciences, Key Laboratory of Advanced Drug Preparation Technologies, Military of Education, Zhengzhou University, Zhengzhou, 450001, China
| | - Yi Sun
- Cancer Institute, the Second Affiliated Hospital, and Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, 310029, China. .,Cancer Center, Zhejiang University, Hangzhou, 310058, China. .,Research Center for Life Science and Human Health, Binjiang Institute of Zhejiang University, Hangzhou, 310053, China.
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14
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ASPM promotes ATR-CHK1 activation and stabilizes stalled replication forks in response to replication stress. Proc Natl Acad Sci U S A 2022; 119:e2203783119. [PMID: 36161901 PMCID: PMC9546549 DOI: 10.1073/pnas.2203783119] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/02/2022] Open
Abstract
ASPM (encoded by MCPH5) is a frequently mutated protein, and such mutations occur in >40% of cases of primary microcephaly (MCPH). Here, we characterize a function of ASPM in DNA replication and the replication stress response. ASPM serves as a scaffold to load stimulators required for ATR-CHK1 checkpoint signaling upon replication stress, which protects stalled replication forks from degradation. ASPM deficiency leads to genomic instability and the sensitization of cancer cells to replication stressors. ASPM is a protein encoded by primary microcephaly 5 (MCPH5) and is responsible for ensuring spindle position during mitosis and the symmetrical division of neural stem cells. We recently reported that ASPM promotes homologous recombination (HR) repair of DNA double strand breaks. However, its potential role in DNA replication and replication stress response remains elusive. Interestingly, we found that ASPM is dispensable for DNA replication under unperturbed conditions. However, ASPM is enriched at stalled replication forks in a RAD17-dependent manner in response to replication stress and promotes RAD9 and TopBP1 loading onto chromatin, facilitating ATR-CHK1 activation. ASPM depletion results in failed fork restart and nuclease MRE11-mediated nascent DNA degradation at the stalled replication fork. The overall consequence is chromosome instability and the sensitization of cancer cells to replication stressors. These data support a role for ASPM in loading RAD17-RAD9/TopBP1 onto chromatin to activate the ATR-CHK1 checkpoint and ultimately ensure genome stability.
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15
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Day M, Oliver AW, Pearl LH. Structure of the human RAD17-RFC clamp loader and 9-1-1 checkpoint clamp bound to a dsDNA-ssDNA junction. Nucleic Acids Res 2022; 50:8279-8289. [PMID: 35819203 PMCID: PMC9371934 DOI: 10.1093/nar/gkac588] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2022] [Revised: 06/20/2022] [Accepted: 06/27/2022] [Indexed: 11/14/2022] Open
Abstract
The RAD9-RAD1-HUS1 (9-1-1) clamp forms one half of the DNA damage checkpoint system that signals the presence of substantial regions of single-stranded DNA arising from replication fork collapse or resection of DNA double strand breaks. Loaded at the 5'-recessed end of a dsDNA-ssDNA junction by the RAD17-RFC clamp loader complex, the phosphorylated C-terminal tail of the RAD9 subunit of 9-1-1 engages with the mediator scaffold TOPBP1 which in turn activates the ATR kinase, localised through the interaction of its constitutive partner ATRIP with RPA-coated ssDNA. Using cryogenic electron microscopy (cryoEM) we have determined the structure of a complex of the human RAD17-RFC clamp loader bound to human 9-1-1, engaged with a dsDNA-ssDNA junction. The structure answers the key questions of how RAD17 confers specificity for 9-1-1 over PCNA, and how the clamp loader specifically recognises the recessed 5' DNA end and fixes the orientation of 9-1-1 on the ssDNA.
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Affiliation(s)
- Matthew Day
- Cancer Research UK DNA Repair Enzymes Group, Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9RQ, UK
| | - Antony W Oliver
- Cancer Research UK DNA Repair Enzymes Group, Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9RQ, UK
| | - Laurence H Pearl
- Cancer Research UK DNA Repair Enzymes Group, Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9RQ, UK
- Division of Structural Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW1E 6BT, UK
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16
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Xiao Y, Xiang JW, Gao Q, Bai YY, Huang ZX, Hu XH, Wang L, Li DWC. MAB21L1 promotes survival of lens epithelial cells through control of αB-crystallin and ATR/CHK1/p53 pathway. Aging (Albany NY) 2022; 14:6128-6148. [PMID: 35951367 PMCID: PMC9417230 DOI: 10.18632/aging.204203] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2022] [Accepted: 07/25/2022] [Indexed: 11/25/2022]
Abstract
The male abnormal gene family 21 (mab21), was initially identified in C. elegans. Since its identification, studies from different groups have shown that it regulates development of ocular tissues, brain, heart and liver. However, its functional mechanism remains largely unknown. Here, we demonstrate that Mab21L1 promotes survival of lens epithelial cells. Mechanistically, Mab21L1 upregulates expression of αB-crystallin. Moreover, our results show that αB-crystallin prevents stress-induced phosphorylation of p53 at S-20 and S-37 through abrogating the activation of the upstream kinases, ATR and CHK1. As a result of suppressing p53 activity by αB-crystallin, Mab21L1 downregulates expression of Bak but upregulates Mcl-1 during stress insult. Taken together, our results demonstrate that Mab21L1 promotes survival of lens epithelial cells through upregulation of αB-crystallin to suppress ATR/CHK1/p53 pathway.
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Affiliation(s)
- Yuan Xiao
- College of Life Sciences, Hunan Normal University, Changsha 410080, Hunan, China.,The State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Tianhe, Guangzhou 510230, Guangdong, China
| | - Jia-Wen Xiang
- The State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Tianhe, Guangzhou 510230, Guangdong, China
| | - Qian Gao
- College of Life Sciences, Hunan Normal University, Changsha 410080, Hunan, China.,The State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Tianhe, Guangzhou 510230, Guangdong, China
| | - Yue-Yue Bai
- College of Life Sciences, Hunan Normal University, Changsha 410080, Hunan, China.,The State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Tianhe, Guangzhou 510230, Guangdong, China
| | - Zhao-Xia Huang
- Department of Basic Medicine, Guizhou University of Traditional Chinese Medicine, Guiyang 121212, Guizhou, China
| | - Xiao-Hui Hu
- College of Life Sciences, Hunan Normal University, Changsha 410080, Hunan, China
| | - Ling Wang
- The Academician Work Station, Changsha Medical University, Changsha 410219, Hunan, China
| | - David Wan-Cheng Li
- College of Life Sciences, Hunan Normal University, Changsha 410080, Hunan, China.,The State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Tianhe, Guangzhou 510230, Guangdong, China
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17
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Wang XL, Lin FL, Xu W, Wang C, Wang QQ, Jiang RW. Silybin B exerts protective effect on cisplatin-induced neurotoxicity by alleviating DNA damage and apoptosis. JOURNAL OF ETHNOPHARMACOLOGY 2022; 288:114938. [PMID: 34999144 DOI: 10.1016/j.jep.2021.114938] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/27/2021] [Revised: 12/10/2021] [Accepted: 12/21/2021] [Indexed: 06/14/2023]
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE Silybum marianum is a traditional Chinese medicine that has been used for treating liver disease. Silybin consisting of silybin A and silybin B, is a member of Silybum marianum, and exerts a therapeutic effect on many diseases. However, the protective effect of silybin on cisplatin-induced neurotoxicity and the stereoisomer contributing to the effect remain unknown. AIM OF THE STUDY The present study aimed to study the effect of silybin on cisplatin-induced neuronal injury, compare the difference of protective effect between silybin A and silybin B, and the potential mechanism. MATERIALS AND METHODS High performance liquid chromatography (HPLC) was used to separate silybin A and silybin B. X-ray crystallographic analysis in combination with experimental and calculated ECD were performed to identify the structure of silybin A and silybin B. The toxicity of the silybin or cisplatin against murine hippocampal neuronal HT22 cells was determined through MTT assay. The cell cycle and cell apoptosis were measured by PI staining and Annexin V-FITC/PI staining, respectively, and then subjected to flow cytometry. Western blot analysis was conducted to quantify the expression of proteins related to apoptosis and DNA damage. Immunofluorescence was used to evaluate the expression of DNA damage marker. In vivo experiment, the behavioral analysis was determined through pole test, swimming test and Morris water maze test. The index of superoxide dismutase (SOD), reduced glutathione (GSH), total antioxidant capacity (T-AOC) and lipid peroxidation (LPO) were examined to evaluate the antioxidant capacity in mice brain. Nissl staining and Tunel assay were used to detect the neuronal viability and apoptosis in hippocampus. RESULTS We successfully separated and identified silybin A and silybin B. We found both silybin A and silybin B alleviated cisplatin-induced apoptosis and cell cycle arrest in HT22 cells, and silybin B was more effective. We chose silybin B for further mechanism investigation, and found silybin B alleviated DNA damage by enhancing phosphorylation of ATR and decreasing expression of γ-H2AX. In the in vivo experiment, we observed that silybin B markedly improved the behavioral abnormalities in cisplatin-treated mice, reduced LPO level while increased SOD, GSH and T-AOC in mice brain tissue. Nissl staining and Tunel assay showed that silybin B alleviated cisplatin-induced hippocampal damage. CONCLUSIONS These results suggest that silybin B might serve as a promising drug candidate in mitigating cisplatin-induced neural injury in the brain and thereby improving the chemotherapeutic outcomes.
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Affiliation(s)
- Xiao-Lu Wang
- Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, College of Pharmacy, Jinan University, Guangzhou, 510632, PR China.
| | - Fo-Lan Lin
- Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, College of Pharmacy, Jinan University, Guangzhou, 510632, PR China.
| | - Wei Xu
- Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, College of Pharmacy, Jinan University, Guangzhou, 510632, PR China.
| | - Chen Wang
- Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, College of Pharmacy, Jinan University, Guangzhou, 510632, PR China.
| | - Qi-Qi Wang
- Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, College of Pharmacy, Jinan University, Guangzhou, 510632, PR China.
| | - Ren-Wang Jiang
- Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, College of Pharmacy, Jinan University, Guangzhou, 510632, PR China.
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18
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Choi SH, Cho K, Kim ES, Yoo HY. Proline-serine-threonine-repeat region of MDC1 mediates Chk1 phosphorylation and the DNA double-strand break repair. Int J Biochem Cell Biol 2021; 143:106152. [PMID: 34974185 DOI: 10.1016/j.biocel.2021.106152] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2021] [Revised: 12/27/2021] [Accepted: 12/28/2021] [Indexed: 11/29/2022]
Abstract
MDC1, a mediator of DNA damage response, recruits other repair proteins on double-strand break (DSB) sites. MDC1 is necessary for activating checkpoint kinases Chk1 and Chk2. It is unclear whether Chk1 interacts with MDC1. MDC1 also comprises many discrete domains. The role of the proline-serine-threonine (PST)-repeat domain of MDC1 in the DNA damage response is unclear. Here, we showed that MDC1 directly binds Chk1 through this PST-repeat region. Phosphorylation of Chk1 by ionizing radiation (IR) also required this PST-repeat domain. Degradation of intact MDC1 was accelerated depending on the PST-repeat domain after IR exposure. In the IR damage response, the PST-repeat-deleted MDC1 levels remained elevated with slow degradation. This abnormal regulation of MDC1 was F-box- and WD40 repeat-containing 7 (FBXW7)-dependent. The mutation of lysine 1413 within the PST-repeat of MDC1 deregulated MDC1 with or without damage. K1413R mutant and PST-deleted MDC1 displayed reduced ability to repair the damaged genome post-IR exposure. These results provide that the PST domain of MDC1 is involved in Chk1 and DNA repair activation. The findings suggest new insights into how MDC1 connects the checkpoint and DNA repair in the DNA damage response.
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Affiliation(s)
- Seung Ho Choi
- Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul 06355, Korea; Samsung Biomedical Research Institute, Research Institute for Future Medicine, Samsung Medical Center, Seoul 06355, Korea
| | - Kyoungjoo Cho
- Department of Life Science, College of Fusion Science, Kyonggi University, Suwon 16227, Korea
| | - Eun Seon Kim
- Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul 06355, Korea; Samsung Biomedical Research Institute, Research Institute for Future Medicine, Samsung Medical Center, Seoul 06355, Korea
| | - Hae Yong Yoo
- Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul 06355, Korea; Samsung Biomedical Research Institute, Research Institute for Future Medicine, Samsung Medical Center, Seoul 06355, Korea.
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19
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Broennimann K, Ricardo-Lax I, Adler J, Michailidis E, de Jong YP, Reuven N, Shaul Y. RNR-R2 Upregulation by a Short Non-Coding Viral Transcript. Biomolecules 2021; 11:biom11121822. [PMID: 34944466 PMCID: PMC8698843 DOI: 10.3390/biom11121822] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2021] [Revised: 11/29/2021] [Accepted: 12/01/2021] [Indexed: 01/12/2023] Open
Abstract
DNA viruses require dNTPs for replication and have developed different strategies to increase intracellular dNTP pools. Hepatitis B virus (HBV) infects non-dividing cells in which dNTPs are scarce and the question is how viral replication takes place. Previously we reported that the virus induces the DNA damage response (DDR) pathway culminating in RNR-R2 expression and the generation of an active RNR holoenzyme, the key regulator of dNTP levels, leading to an increase in dNTPs. How the virus induces DDR and RNR-R2 upregulation is not completely known. The viral HBx open reading frame (ORF) was believed to trigger this pathway. Unexpectedly, however, we report here that the production of HBx protein is dispensable. We found that a small conserved region of 125 bases within the HBx ORF is sufficient to upregulate RNR-R2 expression in growth-arrested HepG2 cells and primary human hepatocytes. The observed HBV mRNA embedded regulatory element is named ERE. ERE in isolation is sufficient to activate the ATR-Chk1-E2F1-RNR-R2 DDR pathway. These findings demonstrate a non-coding function of HBV transcripts to support its propagation in non-cycling cells.
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Affiliation(s)
- Karin Broennimann
- Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel; (K.B.); (I.R.-L.); (J.A.); (N.R.)
| | - Inna Ricardo-Lax
- Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel; (K.B.); (I.R.-L.); (J.A.); (N.R.)
- Laboratory of Virology and Infectious Disease, Rockefeller University, New York, NY 10065, USA; (E.M.); (Y.P.d.J.)
| | - Julia Adler
- Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel; (K.B.); (I.R.-L.); (J.A.); (N.R.)
| | - Eleftherios Michailidis
- Laboratory of Virology and Infectious Disease, Rockefeller University, New York, NY 10065, USA; (E.M.); (Y.P.d.J.)
| | - Ype P. de Jong
- Laboratory of Virology and Infectious Disease, Rockefeller University, New York, NY 10065, USA; (E.M.); (Y.P.d.J.)
- Division of Gastroenterology and Hepatology, Weill Cornell Medicine, New York, NY 10065, USA
| | - Nina Reuven
- Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel; (K.B.); (I.R.-L.); (J.A.); (N.R.)
| | - Yosef Shaul
- Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel; (K.B.); (I.R.-L.); (J.A.); (N.R.)
- Correspondence: ; Tel.: +972-8-934-2320
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20
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Cheng X, Zhao JX, Dong F, Cao XC. ARID1A Mutation in Metastatic Breast Cancer: A Potential Therapeutic Target. Front Oncol 2021; 11:759577. [PMID: 34804958 PMCID: PMC8599951 DOI: 10.3389/fonc.2021.759577] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2021] [Accepted: 10/15/2021] [Indexed: 12/05/2022] Open
Abstract
Distant metastasis is the principal cause of mortality for breast cancer patients. Targeting specific mutations that have been acquired during the evolution process of advanced breast cancer is a potential means of enhancing the clinical efficacy of treatment strategies. In metastatic breast cancer, ARID1A is the most prevalent mutation of the SWI/SNF complex, which regulates DNA repair, recombination, and gene transcription. The low expression of ARID1A is associated with poor disease-free survival and overall survival of patients with luminal A or HER2-rich breast cancer. In addition, ARID1A plays a prominent role in maintaining luminal characteristics and has an advantage for identifying responses to treatment, including endocrine therapies, HDAC inhibitors and CDK4/6 inhibitors. The therapeutic vulnerabilities initiated by ARID1A alterations encourage us to explore new approaches to cope with ARID1A mutant-related drug resistance or metastasis. In this review, we describe the mutation profiles of ARID1A in metastatic breast cancer and the structure and function of ARID1A and the SWI/SNF complex as well as discuss the potential mechanisms of ARID1A-mediated endocrine resistance and therapeutic potential.
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Affiliation(s)
- Xuan Cheng
- The First Department of Breast Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin, China
- Key Laboratory of Cancer Prevention and Therapy, Tianjin, China
- Tianjin’s Clinical Research Center for Cancer, Tianjin, China
- Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin, China
| | - Jian-Xiong Zhao
- The First Department of Breast Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin, China
- Key Laboratory of Cancer Prevention and Therapy, Tianjin, China
- Tianjin’s Clinical Research Center for Cancer, Tianjin, China
- Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin, China
| | - Feng Dong
- Department of Neurosurgery, Tianjin Medical University General Hospital and Laboratory of Neuro-Oncology, Tianjin Neurological Institute, Tianjin, China
- State Key Laboratory of Experimental Hematology, The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Tianjin Key Laboratory of Cellular Homeostasis and Human Diseases, Department of Cell Biology, Tianjin Medical University, Tianjin, China
| | - Xu-Chen Cao
- The First Department of Breast Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin, China
- Key Laboratory of Cancer Prevention and Therapy, Tianjin, China
- Tianjin’s Clinical Research Center for Cancer, Tianjin, China
- Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin, China
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21
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Role of Dietary Antioxidants in p53-Mediated Cancer Chemoprevention and Tumor Suppression. OXIDATIVE MEDICINE AND CELLULAR LONGEVITY 2021; 2021:9924328. [PMID: 34257824 PMCID: PMC8257365 DOI: 10.1155/2021/9924328] [Citation(s) in RCA: 36] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/19/2021] [Accepted: 05/31/2021] [Indexed: 02/07/2023]
Abstract
Cancer arises through a complex interplay between genetic, behavioral, metabolic, and environmental factors that combined trigger cellular changes that over time promote malignancy. In terms of cancer prevention, behavioral interventions such as diet can promote genetic programs that may facilitate tumor suppression; and one of the key tumor suppressors responsible for initiating such programs is p53. The p53 protein is activated by various cellular events such as DNA damage, hypoxia, heat shock, and overexpression of oncogenes. Due to its role in cell fate decisions after DNA damage, regulatory pathways controlled by p53 help to maintain genome stability and thus “guard the genome” against mutations that cause cancer. Dietary intake of flavonoids, a C15 group of polyphenols, is known to inhibit cancer progression and assist DNA repair through p53-mediated mechanisms in human cells via their antioxidant activities. For example, quercetin arrests human cervical cancer cell growth by blocking the G2/M phase cell cycle and inducing mitochondrial apoptosis through a p53-dependent mechanism. Other polyphenols such as resveratrol upregulate p53 expression in several cancer cell lines by promoting p53 stability, which in colon cancer cells results in the activation of p53-mediated apoptosis. Finally, among vitamins, folic acid seems to play an important role in the chemoprevention of gastric carcinogenesis by enhancing gastric epithelial apoptosis in patients with premalignant lesions by significantly increased expression of p53. In this review, we discuss the role of these and other dietary antioxidants in p53-mediated cell signaling in relation to cancer chemoprevention and tumor suppression in normal and cancer cells.
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22
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Blakemore D, Vilaplana‐Lopera N, Almaghrabi R, Gonzalez E, Moya M, Ward C, Murphy G, Gambus A, Petermann E, Stewart GS, García P. MYBL2 and ATM suppress replication stress in pluripotent stem cells. EMBO Rep 2021; 22:e51120. [PMID: 33779025 PMCID: PMC8097389 DOI: 10.15252/embr.202051120] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2020] [Revised: 02/10/2021] [Accepted: 02/19/2021] [Indexed: 12/30/2022] Open
Abstract
Replication stress, a major cause of genome instability in cycling cells, is mainly prevented by the ATR-dependent replication stress response pathway in somatic cells. However, the replication stress response pathway in embryonic stem cells (ESCs) may be different due to alterations in cell cycle phase length. The transcription factor MYBL2, which is implicated in cell cycle regulation, is expressed a hundred to a thousand-fold more in ESCs compared with somatic cells. Here we show that MYBL2 activates ATM and suppresses replication stress in ESCs. Consequently, loss of MYBL2 or inhibition of ATM or Mre11 in ESCs results in replication fork slowing, increased fork stalling and elevated origin firing. Additionally, we demonstrate that inhibition of CDC7 activity rescues replication stress induced by MYBL2 loss and ATM inhibition, suggesting that uncontrolled new origin firing may underlie the replication stress phenotype resulting from loss/inhibition of MYBL2 and ATM. Overall, our study proposes that in addition to ATR, a MYBL2-MRN-ATM replication stress response pathway functions in ESCs to control DNA replication initiation and prevent genome instability.
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Affiliation(s)
- Daniel Blakemore
- Institute of Cancer and Genomic ScienceCollege of Medical and Dental SciencesUniversity of BirminghamBirminghamUK
| | - Nuria Vilaplana‐Lopera
- Institute of Cancer and Genomic ScienceCollege of Medical and Dental SciencesUniversity of BirminghamBirminghamUK
| | - Ruba Almaghrabi
- Institute of Cancer and Genomic ScienceCollege of Medical and Dental SciencesUniversity of BirminghamBirminghamUK
| | - Elena Gonzalez
- Institute of Cancer and Genomic ScienceCollege of Medical and Dental SciencesUniversity of BirminghamBirminghamUK
| | - Miriam Moya
- Institute of Cancer and Genomic ScienceCollege of Medical and Dental SciencesUniversity of BirminghamBirminghamUK
| | - Carl Ward
- Laboratory of Integrative BiologyGuangzhou Institutes of Biomedicine and HealthChinese Academy of Sciences (CAS)GuangzhouChina
- Chinese Academy of Sciences (CAS)Key Laboratory of Regenerative Biology and Guangdong Provincial Key Laboratory of Stem Cell and regenerative MedicineGuangzhou Institutes of Biomedicine and HealthGuangzhouChina
| | - George Murphy
- Department of MedicineBoston University School of MedicineBostonMAUSA
| | - Agnieszka Gambus
- Institute of Cancer and Genomic ScienceCollege of Medical and Dental SciencesUniversity of BirminghamBirminghamUK
| | - Eva Petermann
- Institute of Cancer and Genomic ScienceCollege of Medical and Dental SciencesUniversity of BirminghamBirminghamUK
| | - Grant S Stewart
- Institute of Cancer and Genomic ScienceCollege of Medical and Dental SciencesUniversity of BirminghamBirminghamUK
| | - Paloma García
- Institute of Cancer and Genomic ScienceCollege of Medical and Dental SciencesUniversity of BirminghamBirminghamUK
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23
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Hauge S, Eek Mariampillai A, Rødland GE, Bay LTE, Landsverk HB, Syljuåsen RG. Expanding roles of cell cycle checkpoint inhibitors in radiation oncology. Int J Radiat Biol 2021; 99:941-950. [PMID: 33877959 DOI: 10.1080/09553002.2021.1913529] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
PURPOSE Radiation-induced activation of cell cycle checkpoints have been of long-standing interest. The WEE1, CHK1 and ATR kinases are key factors in cell cycle checkpoint regulation and are essential for the S and G2 checkpoints. Here, we review the rationale for why inhibitors of WEE1, CHK1 and ATR could be beneficial in combination with radiation. CONCLUSIONS Combined treatment with radiation and inhibitors of these kinases results in checkpoint abrogation and subsequent mitotic catastrophe. This might selectively radiosensitize tumor cells, as they often lack the p53-dependent G1 checkpoint and therefore rely more on the G2 checkpoint to repair DNA damage. Further affecting the repair of radiation damage, inhibition of WEE1, CHK1 or ATR also specifically suppresses the homologous recombination repair pathway. Moreover, inhibition of these kinases can induce massive replication stress during S phase of the cell cycle, likely contributing to eliminate radioresistant S phase cells. Intriguingly, recent findings suggest that cell cycle checkpoint inhibitors in combination with radiation can also enhance anti-tumor immune effects. Altogether, the expanding knowledge about the functional roles of WEE1, CHK1 and ATR inhibitors support that they are promising candidates for use in combination with radiation treatment.
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Affiliation(s)
- Sissel Hauge
- Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
| | - Adrian Eek Mariampillai
- Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
| | - Gro Elise Rødland
- Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
| | - Lilli T E Bay
- Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
| | - Helga B Landsverk
- Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
| | - Randi G Syljuåsen
- Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
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24
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Structure-function analysis of TOPBP1's role in ATR signaling using the DSB-mediated ATR activation in Xenopus egg extracts (DMAX) system. Sci Rep 2021; 11:467. [PMID: 33432091 PMCID: PMC7801695 DOI: 10.1038/s41598-020-80626-1] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2020] [Accepted: 12/23/2020] [Indexed: 11/20/2022] Open
Abstract
The protein kinase ATR is activated at sites of DNA double-strand breaks where it plays important roles in promoting DNA end resection and regulating cell cycle progression. TOPBP1 is a multi BRCT repeat containing protein that activates ATR at DSBs. Here we have developed an experimental tool, the DMAX system, to study the biochemical mechanism for TOPBP1-mediated ATR signalling. DMAX combines simple, linear dsDNA molecules with Xenopus egg extracts and results in a physiologically relevant, DSB-induced activation of ATR. We find that DNAs of 5000 nucleotides, at femtomolar concentration, potently activate ATR in this system. By combining immunodepletion and add-back of TOPBP1 point mutants we use DMAX to determine which of TOPBP1’s nine BRCT domains are required for recruitment of TOPBP1 to DSBs and which domains are needed for ATR-mediated phosphorylation of CHK1. We find that BRCT1 and BRCT7 are important for recruitment and that BRCT5 functions downstream of recruitment to promote ATR-mediated phosphorylation of CHK1. We also show that BRCT7 plays a second role, independent of recruitment, in promoting ATR signalling. These findings supply a new research tool for, and new insights into, ATR biology.
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25
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Biochemical analysis of TOPBP1 oligomerization. DNA Repair (Amst) 2020; 96:102973. [PMID: 32987353 DOI: 10.1016/j.dnarep.2020.102973] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2020] [Revised: 08/20/2020] [Accepted: 09/09/2020] [Indexed: 11/24/2022]
Abstract
TOPBP1 is an important scaffold protein that helps orchestrate the cellular response to DNA damage. Although it has been previously appreciated that TOPBP1 can form oligomers, how this occurs and the functional consequences for oligomerization were not yet known. Here, we use protein binding assays and other biochemical techniques to study how TOPBP1 self associates. TOPBP1 contains 9 copies of the BRCT domain, and we report that a subset of these BRCT domains interact with one another to drive oligomerization. An intact BRCT 2 domain is required for TOPBP1 oligomerization and we find that the BRCT1&2 region of TOPBP1 interacts with itself and with the BRCT4&5 pair. RAD9 and RHINO are two heterologous binding partners for TOPBP1's BRCT 1&2 domains, and we show that binding of these partners does not come at the expense of TOPBP1 oligomerization. Furthermore, we show that a TOPBP1 oligomer can simultaneously interact with both RAD9 and RHINO. Lastly, we find that the oligomeric state necessary for TOPBP1 to activate the ATR protein kinase is likely to be a tetramer.
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26
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Ren X, Jiang K, Zhang F. The Multifaceted Roles of RCC1 in Tumorigenesis. Front Mol Biosci 2020; 7:225. [PMID: 33102517 PMCID: PMC7522611 DOI: 10.3389/fmolb.2020.00225] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2020] [Accepted: 08/11/2020] [Indexed: 01/31/2023] Open
Abstract
RCC1 (regulator of chromosome condensation 1) is the only known guanine nucleotide exchange factor of Ran, a nuclear Ras-like G protein. RCC1 combines with chromatin and Ran to establish a concentration gradient of RanGTP, thereby participating in a series of cell physiological activities. In this review, we discuss the structure of RCC1 and describe how RCC1 affects the formation and function of the nuclear envelope, spindle formation, and nuclear transport. We mainly focus on the effect of RCC1 on the cell cycle during tumorigenesis and the recent research progress that has been made in relation to different tumor types.
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Affiliation(s)
- Xuanqi Ren
- College of Life Sciences, Shanghai Normal University, Shanghai, China
| | - Kai Jiang
- College of Life Sciences, Shanghai Normal University, Shanghai, China
| | - Feng Zhang
- College of Life Sciences, Shanghai Normal University, Shanghai, China
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27
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Ciardo D, Haccard O, Narassimprakash H, Chiodelli V, Goldar A, Marheineke K. Polo-like kinase 1 (Plk1) is a positive regulator of DNA replication in the Xenopus in vitro system. Cell Cycle 2020; 19:1817-1832. [PMID: 32573322 PMCID: PMC7469467 DOI: 10.1080/15384101.2020.1782589] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2019] [Revised: 03/24/2020] [Accepted: 06/07/2020] [Indexed: 12/18/2022] Open
Abstract
Polo-like kinase 1 (Plk1) is a cell cycle kinase essential for mitosis progression, but also important for checkpoint recovery and adaptation in response to DNA damage and replication stress. However, although Plk1 is expressed in S phase, little is known about its function during unperturbed DNA replication. Using Xenopus laevis egg extracts, mimicking early embryonic replication, we demonstrate that Plk1 is simultaneously recruited to chromatin with pre-replication proteins where it accumulates throughout S phase. Further, we found that chromatin-bound Plk1 is phosphorylated on its activating site T201, which appears to be sensitive to dephosphorylation by protein phosphatase 2A. Extracts immunodepleted of Plk1 showed a decrease in DNA replication, rescued by wild type recombinant Plk1. Inversely, modest Plk1 overexpression accelerated DNA replication. Plk1 depletion led to an increase in Chk1 phosphorylation and to a decrease in Cdk2 activity, which strongly suggests that Plk1 could inhibit the ATR/Chk1-dependent intra-S phase checkpoint during normal S phase. In addition, we observed that phosphorylated Plk1 levels are high during the rapid, early cell cycles of Xenopus development but decrease after the mid-blastula transition when the cell cycle and the replication program slow down along with more active checkpoints. These data shed new light on the role of Plk1 as a positive regulating factor for DNA replication in early, rapidly dividing embryos.
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Affiliation(s)
- Diletta Ciardo
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France
| | - Olivier Haccard
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France
| | - Hemalatha Narassimprakash
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France
| | - Virginie Chiodelli
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France
| | - Arach Goldar
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France
| | - Kathrin Marheineke
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France
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28
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Vigneswara V, Ahmed Z. The Role of Caspase-2 in Regulating Cell Fate. Cells 2020; 9:cells9051259. [PMID: 32438737 PMCID: PMC7290664 DOI: 10.3390/cells9051259] [Citation(s) in RCA: 48] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2020] [Revised: 05/11/2020] [Accepted: 05/12/2020] [Indexed: 12/13/2022] Open
Abstract
Caspase-2 is the most evolutionarily conserved member of the mammalian caspase family and has been implicated in both apoptotic and non-apoptotic signaling pathways, including tumor suppression, cell cycle regulation, and DNA repair. A myriad of signaling molecules is associated with the tight regulation of caspase-2 to mediate multiple cellular processes far beyond apoptotic cell death. This review provides a comprehensive overview of the literature pertaining to possible sophisticated molecular mechanisms underlying the multifaceted process of caspase-2 activation and to highlight its interplay between factors that promote or suppress apoptosis in a complicated regulatory network that determines the fate of a cell from its birth and throughout its life.
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29
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Dibitetto D, Sims JR, Ascenção CFR, Feng K, Kim D, Oberly S, Freire R, Smolka MB. Intrinsic ATR signaling shapes DNA end resection and suppresses toxic DNA-PKcs signaling. NAR Cancer 2020; 2:zcaa006. [PMID: 32743550 PMCID: PMC7380482 DOI: 10.1093/narcan/zcaa006] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2020] [Revised: 04/08/2020] [Accepted: 04/16/2020] [Indexed: 12/13/2022] Open
Abstract
Most cancer cells experience oncogene-induced replication stress and, as a result, exhibit high intrinsic activation of the ATR kinase. Although cancer cells often become more dependent on ATR for survival, the precise mechanism by which ATR signaling ensures cancer cell fitness and viability remains incompletely understood. Here, we find that intrinsic ATR signaling is crucial for the ability of cancer cells to promote DNA end resection, the first step in homology-directed DNA repair. Inhibition of ATR over multiple cell division cycles depletes the pool of pro-resection factors and prevents the engagement of RAD51 as well as RAD52 at nuclear foci, leading to toxic DNA-PKcs signaling and hypersensitivity to PARP inhibitors. The effect is markedly distinct from acute ATR inhibition, which blocks RAD51-mediated repair but not resection and engagement of RAD52. Our findings reveal a key pro-resection function for ATR and define how ATR inhibitors can be used for effective manipulation of DNA end resection capacity and DNA repair outcomes in cancer cells.
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Affiliation(s)
- Diego Dibitetto
- Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853, USA
| | - Jennie R Sims
- Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853, USA
| | - Carolline F R Ascenção
- Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853, USA
| | - Kevin Feng
- Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853, USA
| | - Dongsung Kim
- Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853, USA
| | - Susannah Oberly
- Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853, USA
| | - Raimundo Freire
- Unidad de Investigación, Hospital Universitario de Canarias, Ofra s/n, La Cuesta, 38320 La Laguna, Tenerife, Spain.,Instituto de Tecnologías Biomédicas, Universidad de La Laguna, 38200 La Laguna, Tenerife, Spain.,Universidad Fernando Pessoa Canarias, 35450 Las Palmas de Gran Canaria, Spain
| | - Marcus B Smolka
- Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853, USA
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30
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Basbous J, Aze A, Chaloin L, Lebdy R, Hodroj D, Ribeyre C, Larroque M, Shepard C, Kim B, Pruvost A, Moreaux J, Maiorano D, Mechali M, Constantinou A. Dihydropyrimidinase protects from DNA replication stress caused by cytotoxic metabolites. Nucleic Acids Res 2020; 48:1886-1904. [PMID: 31853544 PMCID: PMC7038975 DOI: 10.1093/nar/gkz1162] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2019] [Revised: 11/27/2019] [Accepted: 11/29/2019] [Indexed: 01/28/2023] Open
Abstract
Imbalance in the level of the pyrimidine degradation products dihydrouracil and dihydrothymine is associated with cellular transformation and cancer progression. Dihydropyrimidines are degraded by dihydropyrimidinase (DHP), a zinc metalloenzyme that is upregulated in solid tumors but not in the corresponding normal tissues. How dihydropyrimidine metabolites affect cellular phenotypes remains elusive. Here we show that the accumulation of dihydropyrimidines induces the formation of DNA-protein crosslinks (DPCs) and causes DNA replication and transcriptional stress. We used Xenopus egg extracts to recapitulate DNA replication invitro. We found that dihydropyrimidines interfere directly with the replication of both plasmid and chromosomal DNA. Furthermore, we show that the plant flavonoid dihydromyricetin inhibits human DHP activity. Cellular exposure to dihydromyricetin triggered DPCs-dependent DNA replication stress in cancer cells. This study defines dihydropyrimidines as potentially cytotoxic metabolites that may offer an opportunity for therapeutic-targeting of DHP activity in solid tumors.
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Affiliation(s)
- Jihane Basbous
- Institute of Human Genetics (IGH), CNRS, Université de Montpellier, 34396 Montpellier Cedex 5, France
| | - Antoine Aze
- Institute of Human Genetics (IGH), CNRS, Université de Montpellier, 34396 Montpellier Cedex 5, France
| | - Laurent Chaloin
- Institut de Recherche en Infectiologie de Montpellier, CNRS, Université de Montpellier, 34293 Montpellier Cedex 5, France
| | - Rana Lebdy
- Institute of Human Genetics (IGH), CNRS, Université de Montpellier, 34396 Montpellier Cedex 5, France
| | - Dana Hodroj
- Institute of Human Genetics (IGH), CNRS, Université de Montpellier, 34396 Montpellier Cedex 5, France.,Cancer Research Center of Toulouse (CRCT), 31037 Toulouse Cedex 1, France
| | - Cyril Ribeyre
- Institute of Human Genetics (IGH), CNRS, Université de Montpellier, 34396 Montpellier Cedex 5, France
| | - Marion Larroque
- Institute of Human Genetics (IGH), CNRS, Université de Montpellier, 34396 Montpellier Cedex 5, France.,Institut du Cancer de Montpellier (ICM),34298 Montpellier Cedex 5, France
| | - Caitlin Shepard
- School of Medicine, Emory University, Atlanta, GA 30322, USA
| | - Baek Kim
- School of Medicine, Emory University, Atlanta, GA 30322, USA
| | - Alain Pruvost
- Service de Pharmacologie et Immunoanalyse (SPI), Plateforme SMArt-MS, CEA, INRA, Université Paris-Saclay, 91191 Gif-sur-Yvette Cedex, France
| | - Jérôme Moreaux
- Institute of Human Genetics (IGH), CNRS, Université de Montpellier, 34396 Montpellier Cedex 5, France
| | - Domenico Maiorano
- Institute of Human Genetics (IGH), CNRS, Université de Montpellier, 34396 Montpellier Cedex 5, France
| | - Marcel Mechali
- Institute of Human Genetics (IGH), CNRS, Université de Montpellier, 34396 Montpellier Cedex 5, France
| | - Angelos Constantinou
- Institute of Human Genetics (IGH), CNRS, Université de Montpellier, 34396 Montpellier Cedex 5, France
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31
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Menolfi D, Zha S. ATM, ATR and DNA-PKcs kinases-the lessons from the mouse models: inhibition ≠ deletion. Cell Biosci 2020; 10:8. [PMID: 32015826 PMCID: PMC6990542 DOI: 10.1186/s13578-020-0376-x] [Citation(s) in RCA: 137] [Impact Index Per Article: 27.4] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2019] [Accepted: 01/14/2020] [Indexed: 01/11/2023] Open
Abstract
DNA damage, especially DNA double strand breaks (DSBs) and replication stress, activates a complex post-translational network termed DNA damage response (DDR). Our review focuses on three PI3-kinase related protein kinases-ATM, ATR and DNA-PKcs, which situate at the apex of the mammalian DDR. They are recruited to and activated at the DNA damage sites by their respective sensor protein complexes-MRE11/RAD50/NBS1 for ATM, RPA/ATRIP for ATR and KU70-KU80/86 (XRCC6/XRCC5) for DNA-PKcs. Upon activation, ATM, ATR and DNA-PKcs phosphorylate a large number of partially overlapping substrates to promote efficient and accurate DNA repair and to coordinate DNA repair with other DNA metabolic events (e.g., transcription, replication and mitosis). At the organism level, robust DDR is critical for normal development, aging, stem cell maintenance and regeneration, and physiological genomic rearrangements in lymphocytes and germ cells. In addition to endogenous damage, oncogene-induced replication stresses and genotoxic chemotherapies also activate DDR. On one hand, DDR factors suppress genomic instability to prevent malignant transformation. On the other hand, targeting DDR enhances the therapeutic effects of anti-cancer chemotherapy, which led to the development of specific kinase inhibitors for ATM, ATR and DNA-PKcs. Using mouse models expressing kinase dead ATM, ATR and DNA-PKcs, an unexpected structural function of these kinases was revealed, where the expression of catalytically inactive kinases causes more genomic instability than the loss of the proteins themselves. The spectrum of genomic instabilities and physiological consequences are unique for each kinase and depends on their activating complexes, suggesting a model in which the catalysis is coupled with DNA/chromatin release and catalytic inhibition leads to the persistence of the kinases at the DNA lesion, which in turn affects repair pathway choice and outcomes. Here we discuss the experimental evidences supporting this mode of action and their implications in the design and use of specific kinase inhibitors for ATM, ATR and DNA-PKcs for cancer therapy.
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Affiliation(s)
- Demis Menolfi
- Institute for Cancer Genetics, College of Physicians & Surgeons, Columbia University, New York, NY 10032 USA
| | - Shan Zha
- Institute for Cancer Genetics, College of Physicians & Surgeons, Columbia University, New York, NY 10032 USA
- Department of Pathology and Cell Biology, College of Physicians & Surgeons, Columbia University, New York, NY 10032 USA
- Division of Pediatric Oncology, Hematology and Stem Cell Transplantation, Department of Pediatrics, College of Physicians & Surgeons, Columbia University, New York, NY 10032 USA
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32
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Cartel M, Didier C. Regulation of CHK1 by the Ubiquitin-Proteasome System. FEBS J 2020; 287:1982-1984. [PMID: 31904911 DOI: 10.1111/febs.15173] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2019] [Accepted: 12/09/2019] [Indexed: 11/28/2022]
Abstract
The checkpoint kinase 1 (CHK1) is a master regulator of genome integrity in vertebrate cells. Despite its important cell cycle functions, its regulation is still incompletely understood. Cassidy et al. provide novel insights on the regulation of the CHK1 abundance by the HECT E3 ligase HUWE1 during unperturbed cell cycle as well as in response to replicative stress. These results may help us to apprehend the underlying mechanism of tumorigenesis.
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Affiliation(s)
- Maëlle Cartel
- Cancer Research Center of Toulouse, INSERM U1037, CNRS ERL 5294, Université de Toulouse, France.,Équipe Labellisée 2016, Ligue Nationale Contre le Cancer, Toulouse, France
| | - Christine Didier
- Cancer Research Center of Toulouse, INSERM U1037, CNRS ERL 5294, Université de Toulouse, France.,Équipe Labellisée 2016, Ligue Nationale Contre le Cancer, Toulouse, France
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33
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Mladenov E, Fan X, Paul-Konietzko K, Soni A, Iliakis G. DNA-PKcs and ATM epistatically suppress DNA end resection and hyperactivation of ATR-dependent G 2-checkpoint in S-phase irradiated cells. Sci Rep 2019; 9:14597. [PMID: 31601897 PMCID: PMC6787047 DOI: 10.1038/s41598-019-51071-6] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2019] [Accepted: 09/20/2019] [Indexed: 11/29/2022] Open
Abstract
We previously reported that cells exposed to low doses of ionizing radiation (IR) in the G2-phase of the cell cycle activate a checkpoint that is epistatically regulated by ATM and ATR operating as an integrated module. In this module, ATR interphases exclusively with the cell cycle to implement the checkpoint, mainly using CHK1. The ATM/ATR module similarly regulates DNA end-resection at low IR-doses. Strikingly, at high IR-doses, the ATM/ATR coupling relaxes and each kinase exerts independent contributions to resection and the G2-checkpoint. DNA-PKcs links to the ATM/ATR module and defects cause hyper-resection and hyperactivation of G2-checkpoint at all doses examined. Surprisingly, our present report reveals that cells irradiated in S-phase utilize a different form of wiring between DNA-PKcs/ATM/ATR: The checkpoint activated in G2-phase is regulated exclusively by ATR/CHK1; similarly at high and low IR-doses. DNA end-resection supports ATR-activation, but inhibition of ATR leaves resection unchanged. DNA-PKcs and ATM link now epistatically to resection and their inhibition causes hyper-resection and ATR-dependent G2-checkpoint hyperactivation at all IR-doses. We propose that DNA-PKcs, ATM and ATR form a modular unit to regulate DSB processing with their crosstalk distinctly organized in S- and G2- phase, with strong dependence on DSB load only in G2-phase.
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Affiliation(s)
- Emil Mladenov
- Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, 45122, Essen, Germany.
| | - Xiaoxiang Fan
- Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, 45122, Essen, Germany
| | - Katja Paul-Konietzko
- Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, 45122, Essen, Germany
| | - Aashish Soni
- Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, 45122, Essen, Germany
| | - George Iliakis
- Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, 45122, Essen, Germany.
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34
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Hustedt N, Álvarez-Quilón A, McEwan A, Yuan JY, Cho T, Koob L, Hart T, Durocher D. A consensus set of genetic vulnerabilities to ATR inhibition. Open Biol 2019; 9:190156. [PMID: 31506018 PMCID: PMC6769295 DOI: 10.1098/rsob.190156] [Citation(s) in RCA: 75] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2019] [Accepted: 08/22/2019] [Indexed: 12/12/2022] Open
Abstract
The response to DNA replication stress in eukaryotes is under the control of the ataxia-telangiectasia and Rad3-related (ATR) kinase. ATR responds to single-stranded (ss) DNA to stabilize distressed DNA replication forks, modulate DNA replication firing and prevent cells with damaged DNA or incomplete DNA replication from entering into mitosis. Furthermore, inhibitors of ATR are currently in clinical development either as monotherapies or in combination with agents that perturb DNA replication. To gain a genetic view of the cellular pathways requiring ATR kinase function, we mapped genes whose mutation causes hypersensitivity to ATR inhibitors with genome-scale CRISPR/Cas9 screens. We delineate a consensus set of 117 genes enriched in DNA replication, DNA repair and cell cycle regulators that promote survival when ATR kinase activity is suppressed. We validate 14 genes from this set and report genes not previously described to modulate response to ATR inhibitors. In particular we found that the loss of the POLE3/POLE4 proteins, which are DNA polymerase ε accessory subunits, results in marked hypersensitivity to ATR inhibition. We anticipate that this 117-gene set will be useful for the identification of genes involved in the regulation of genome integrity and the characterization of new biological processes involving ATR, and may reveal biomarkers of ATR inhibitor response in the clinic.
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Affiliation(s)
- Nicole Hustedt
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, CanadaM5G 1X5
| | - Alejandro Álvarez-Quilón
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, CanadaM5G 1X5
| | - Andrea McEwan
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, CanadaM5G 1X5
| | - Jing Yi Yuan
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, CanadaM5G 1X5
| | - Tiffany Cho
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, CanadaM5G 1X5
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario, CanadaM5S 1A8
| | - Lisa Koob
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, CanadaM5G 1X5
| | - Traver Hart
- Department of Bioinformatics and Computational Biology, University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Daniel Durocher
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, CanadaM5G 1X5
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario, CanadaM5S 1A8
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Lin Y, Bai L, Cupello S, Hossain MA, Deem B, McLeod M, Raj J, Yan S. APE2 promotes DNA damage response pathway from a single-strand break. Nucleic Acids Res 2019; 46:2479-2494. [PMID: 29361157 PMCID: PMC5861430 DOI: 10.1093/nar/gky020] [Citation(s) in RCA: 37] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2017] [Accepted: 01/09/2018] [Indexed: 02/06/2023] Open
Abstract
As the most common type of DNA damage, DNA single-strand breaks (SSBs) are primarily repaired by the SSB repair mechanism. If not repaired properly or promptly, unrepaired SSBs lead to genome stability and have been implicated in cancer and neurodegenerative diseases. However, it remains unknown how unrepaired SSBs are recognized by DNA damage response (DDR) pathway, largely because of the lack of a feasible experimental system. Here, we demonstrate evidence showing that an ATR-dependent checkpoint signaling is activated by a defined plasmid-based site-specific SSB structure in Xenopus HSS (high-speed supernatant) system. Notably, the distinct SSB signaling requires APE2 and canonical checkpoint proteins, including ATR, ATRIP, TopBP1, Rad9 and Claspin. Importantly, the SSB-induced ATR DDR is essential for SSB repair. We and others show that APE2 interacts with PCNA via its PIP box and preferentially interacts with ssDNA via its C-terminus Zf–GRF domain, a conserved motif found in >100 proteins involved in DNA/RNA metabolism. Here, we identify a novel mode of APE2–PCNA interaction via APE2 Zf–GRF and PCNA C-terminus. Mechanistically, the APE2 Zf–GRF–PCNA interaction facilitates 3′-5′ SSB end resection, checkpoint protein complex assembly, and SSB-induced DDR pathway. Together, we propose that APE2 promotes ATR–Chk1 DDR pathway from a single-strand break.
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Affiliation(s)
- Yunfeng Lin
- Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC 28223, USA
| | - Liping Bai
- Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC 28223, USA
| | - Steven Cupello
- Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC 28223, USA
| | - Md Akram Hossain
- Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC 28223, USA
| | - Bradley Deem
- Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC 28223, USA
| | - Melissa McLeod
- Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC 28223, USA
| | - Jude Raj
- Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC 28223, USA
| | - Shan Yan
- Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC 28223, USA
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Mladenov E, Fan X, Dueva R, Soni A, Iliakis G. Radiation-dose-dependent functional synergisms between ATM, ATR and DNA-PKcs in checkpoint control and resection in G 2-phase. Sci Rep 2019; 9:8255. [PMID: 31164689 PMCID: PMC6547644 DOI: 10.1038/s41598-019-44771-6] [Citation(s) in RCA: 42] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2019] [Accepted: 04/23/2019] [Indexed: 12/31/2022] Open
Abstract
Using data generated with cells exposed to ionizing-radiation (IR) in G2-phase of the cell cycle, we describe dose-dependent interactions between ATM, ATR and DNA-PKcs revealing unknown mechanistic underpinnings for two key facets of the DNA damage response: DSB end-resection and G2-checkpoint activation. At low IR-doses that induce low DSB-numbers in the genome, ATM and ATR regulate epistatically the G2-checkpoint, with ATR at the output-node, interfacing with the cell-cycle predominantly through Chk1. Strikingly, at low IR-doses, ATM and ATR epistatically regulate also resection, and inhibition of either activity fully suppresses resection. At high IR-doses that induce high DSB-numbers in the genome, the tight ATM/ATR coupling relaxes and independent outputs to G2-checkpoint and resection occur. Consequently, both kinases must be inhibited to fully suppress checkpoint activation and resection. DNA-PKcs integrates to the ATM/ATR module by regulating resection at all IR-doses, with defects in DNA-PKcs causing hyper-resection and G2-checkpoint hyper-activation. Notably, hyper-resection is absent from other c-NHEJ mutants. Thus, DNA-PKcs specifically regulates resection and adjusts the activation of the ATM/ATR module. We propose that selected DSBs are shepherd by DNA-PKcs from c-NHEJ to resection-dependent pathways for processing under the regulatory supervision of the ATM/ATR module.
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Affiliation(s)
- Emil Mladenov
- Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, 45122, Essen, Germany
| | - Xiaoxiang Fan
- Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, 45122, Essen, Germany
| | - Rositsa Dueva
- Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, 45122, Essen, Germany
| | - Aashish Soni
- Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, 45122, Essen, Germany
| | - George Iliakis
- Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, 45122, Essen, Germany.
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37
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ATM, DNA-PKcs and ATR: shaping development through the regulation of the DNA damage responses. ACTA ACUST UNITED AC 2019. [DOI: 10.1007/s42764-019-00003-9] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
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Abstract
Besides TopBP1, ETAA1 has been identified more recently as an activator of the ATR-ATRIP complex in human cells. We have examined the role of ETAA1 in the Xenopus egg-extract system, which has been instrumental in the study of ATR-ATRIP. Depletion of ETAA1 from egg extracts did not noticeably reduce the activation of ATR-ATRIP in response to replication stress, as monitored by the ATR-dependent phosphorylation of Chk1 and RPA. Moreover, lack of ETAA1 did not appear to affect DNA replication during an unperturbed S-phase. Significantly, we find that TopBP1 is considerably more abundant than ETAA1 in egg extracts. We proceeded to show that ETAA1 could support the activation of ATR-ATRIP in response to replication stress if we increased its concentration in egg extracts by adding extra full-length recombinant ETAA1. Thus, TopBP1 appears to be the predominant activator of ATR-ATRIP in response to replication stress in this system. We have also explored the biochemical mechanism by which ETAA1 activates ATR-ATRIP. We have developed an in vitro system in which full-length recombinant ETAA1 supports activation of ATR-ATRIP in the presence of defined components. We find that binding of ETAA1 to RPA associated with single-stranded DNA (ssDNA) greatly stimulates its ability to activate ATR-ATRIP. Thus, RPA-coated ssDNA serves as a direct positive effector in the ETAA1-mediated activation of ATR-ATRIP.
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Affiliation(s)
- Ke Lyu
- a Division of Biology and Biological Engineering , California Institute of Technology , Pasadena , CA , USA
| | - Akiko Kumagai
- a Division of Biology and Biological Engineering , California Institute of Technology , Pasadena , CA , USA
| | - William G Dunphy
- a Division of Biology and Biological Engineering , California Institute of Technology , Pasadena , CA , USA
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39
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Moussa RS, Park KC, Kovacevic Z, Richardson DR. Ironing out the role of the cyclin-dependent kinase inhibitor, p21 in cancer: Novel iron chelating agents to target p21 expression and activity. Free Radic Biol Med 2019; 133:276-294. [PMID: 29572098 DOI: 10.1016/j.freeradbiomed.2018.03.027] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/21/2017] [Revised: 03/02/2018] [Accepted: 03/14/2018] [Indexed: 12/12/2022]
Abstract
Iron (Fe) has become an important target for the development of anti-cancer therapeutics with a number of Fe chelators entering human clinical trials for advanced and resistant cancer. An important aspect of the activity of these compounds is their multiple molecular targets, including those that play roles in arresting the cell cycle, such as the cyclin-dependent kinase inhibitor, p21. At present, the exact mechanism by which Fe chelators regulate p21 expression remains unclear. However, recent studies indicate the ability of chelators to up-regulate p21 at the mRNA level was dependent on the chelator and cell-type investigated. Analysis of the p21 promoter identified that the Sp1-3-binding site played a significant role in the activation of p21 transcription by Fe chelators. Furthermore, there was increased Sp1/ER-α and Sp1/c-Jun complex formation in melanoma cells, suggesting these complexes were involved in p21 promoter activation. Elucidating the mechanisms involved in the regulation of p21 expression in response to Fe chelator treatment in neoplastic cells will further clarify how these agents achieve their anti-tumor activity. It will also enhance our understanding of the complex roles p21 may play in neoplastic cells and lead to the development of more effective and specific anti-cancer therapies.
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Affiliation(s)
- Rayan S Moussa
- Molecular Pharmacology and Pathology Program, Discipline of Pathology and Bosch Institute, Medical Foundation Building (K25), The University of Sydney, Sydney, New South Wales 2006, Australia
| | - Kyung Chan Park
- Molecular Pharmacology and Pathology Program, Discipline of Pathology and Bosch Institute, Medical Foundation Building (K25), The University of Sydney, Sydney, New South Wales 2006, Australia
| | - Zaklina Kovacevic
- Molecular Pharmacology and Pathology Program, Discipline of Pathology and Bosch Institute, Medical Foundation Building (K25), The University of Sydney, Sydney, New South Wales 2006, Australia
| | - Des R Richardson
- Molecular Pharmacology and Pathology Program, Discipline of Pathology and Bosch Institute, Medical Foundation Building (K25), The University of Sydney, Sydney, New South Wales 2006, Australia; Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-8550, Japan.
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40
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Tang C, Tang S, Zhang S, Luo M, Jia G, Zhi H, Diao X. SiSTL1, encoding a large subunit of ribonucleotide reductase, is crucial for plant growth, chloroplast biogenesis, and cell cycle progression in Setaria italica. JOURNAL OF EXPERIMENTAL BOTANY 2019; 70:1167-1182. [PMID: 30534992 PMCID: PMC6382339 DOI: 10.1093/jxb/ery429] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/27/2018] [Accepted: 11/30/2018] [Indexed: 05/15/2023]
Abstract
The activity of ribonucleotide reductase (RNR), which catalyses the transformation of four ribonucleoside diphosphates (NDPs) to their corresponding deoxyribonucleoside diphosphates (dNDPs), is the main determiner of the cellular concentration of dNTP pools and should be tightly coordinated with DNA synthesis and cell-cycle progression. Constitutively increased or decreased RNR activity interferes with DNA replication and leads to arrested cell cycle progression; however, the mechanisms underlying these disruptive effects in higher plants remain to be uncovered. In this study, we identified a RNR large subunit mutant, sistl1, in Setaria italica (foxtail millet), which exhibited growth retardation as well as striped leaf phenotype, i.e. irregularly reduced leaf vein distances and decreased chloroplast biogenesis. We determined that a Gly737 to Glu substitution occurring in the C-terminus of the SiSTL1 protein slightly affected its optimal function, leading in turn to the reduced expression of genes variously involved in the assembly and activation of the DNA pre-replicative complex, elongation of replication forks and S phase entry. Our study provides new insights into how SiSTL1 regulates plant growth, chloroplast biogenesis, and cell cycle progression in Poaceae crops.
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Affiliation(s)
- Chanjuan Tang
- Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Sha Tang
- Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Shuo Zhang
- Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Mingzhao Luo
- Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Guanqing Jia
- Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Hui Zhi
- Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
- Correspondence: or
| | - Xianmin Diao
- Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
- Correspondence: or
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41
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Tang C, Tang S, Zhang S, Luo M, Jia G, Zhi H, Diao X. SiSTL1, encoding a large subunit of ribonucleotide reductase, is crucial for plant growth, chloroplast biogenesis, and cell cycle progression in Setaria italica. JOURNAL OF EXPERIMENTAL BOTANY 2019. [PMID: 30534992 DOI: 10.5061/dryad.t6v7t8s] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Subscribe] [Scholar Register] [Indexed: 05/15/2023]
Abstract
The activity of ribonucleotide reductase (RNR), which catalyses the transformation of four ribonucleoside diphosphates (NDPs) to their corresponding deoxyribonucleoside diphosphates (dNDPs), is the main determiner of the cellular concentration of dNTP pools and should be tightly coordinated with DNA synthesis and cell-cycle progression. Constitutively increased or decreased RNR activity interferes with DNA replication and leads to arrested cell cycle progression; however, the mechanisms underlying these disruptive effects in higher plants remain to be uncovered. In this study, we identified a RNR large subunit mutant, sistl1, in Setaria italica (foxtail millet), which exhibited growth retardation as well as striped leaf phenotype, i.e. irregularly reduced leaf vein distances and decreased chloroplast biogenesis. We determined that a Gly737 to Glu substitution occurring in the C-terminus of the SiSTL1 protein slightly affected its optimal function, leading in turn to the reduced expression of genes variously involved in the assembly and activation of the DNA pre-replicative complex, elongation of replication forks and S phase entry. Our study provides new insights into how SiSTL1 regulates plant growth, chloroplast biogenesis, and cell cycle progression in Poaceae crops.
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Affiliation(s)
- Chanjuan Tang
- Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Sha Tang
- Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Shuo Zhang
- Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Mingzhao Luo
- Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Guanqing Jia
- Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Hui Zhi
- Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Xianmin Diao
- Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
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42
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Croft LV, Bolderson E, Adams MN, El-Kamand S, Kariawasam R, Cubeddu L, Gamsjaeger R, Richard DJ. Human single-stranded DNA binding protein 1 (hSSB1, OBFC2B), a critical component of the DNA damage response. Semin Cell Dev Biol 2019; 86:121-128. [DOI: 10.1016/j.semcdb.2018.03.014] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2017] [Revised: 03/21/2018] [Accepted: 03/22/2018] [Indexed: 12/18/2022]
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43
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Abstract
The chemical treatment of cancer started with the realization that DNA damaging agents such as mustard gas present notable antitumoural properties. Consequently, early drug development focused on genotoxic chemicals, some of which are still widely used in the clinic. However, the efficacy of such therapies is often limited by the side effects of these drugs on healthy cells. A refinement to this approach is to use compounds that can exploit the presence of DNA damage in cancer cells. Given that replication stress (RS) is a major source of genomic instability in cancer, targeting the RS-response kinase ataxia telangiectasia and Rad3-related protein (ATR) has emerged as a promising alternative. With ATR inhibitors now entering clinical trials, we here revisit the biology behind this strategy and discuss potential biomarkers that could be used for a better selection of patients who respond to therapy.
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Affiliation(s)
- Emilio Lecona
- Genomic Instability Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain
| | - Oscar Fernandez-Capetillo
- Genomic Instability Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.
- Science for Life Laboratory, Division of Genome Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
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44
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Dhuppar S, Mazumder A. Measuring cell cycle-dependent DNA damage responses and p53 regulation on a cell-by-cell basis from image analysis. Cell Cycle 2018; 17:1358-1371. [PMID: 29963960 DOI: 10.1080/15384101.2018.1482136] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Abstract
DNA damage in cells occurs from both endogenous and exogenous sources, and failure to repair such damage is associated with the emergence of different cancers, neurological disorders and aging. DNA damage responses (DDR) in cells are closely associated with the cell cycle. While most of our knowledge of DDR comes from bulk biochemistry, such methods require cells to be arrested at specific stages for cell cycle studies, potentially altering measured responses; nor is cell to cell variability in DDR or direct cell-level correlation of two response metrics measured in such methods. To overcome these limitations we developed a microscopy-based assay for determining cell cycle stages over large cell numbers. This method can be used to study cell-cycle-dependent DDR in cultured cells without the need for cell synchronization. Upon DNA damage γH2A.X induction was correlated to nuclear enrichment of p53 on a cell-by-cell basis and in a cell cycle dependent manner. Imaging-based cell cycle staging was combined with single molecule P53 mRNA detection and immunofluorescence for p53 protein in the very same cells to reveal an intriguing repression of P53 transcript numbers due to reduced transcription across different stages of the cell cycle during DNA damage. Our study hints at an unexplored mechanism for p53 regulation and underscores the importance of measuring single cell level responses to DNA damage.
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Affiliation(s)
- Shivnarayan Dhuppar
- a TIFR Centre for Interdisciplinary Sciences , TIFR Hyderabad , Hyderabad , India
| | - Aprotim Mazumder
- a TIFR Centre for Interdisciplinary Sciences , TIFR Hyderabad , Hyderabad , India
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45
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Kumbhar R, Vidal-Eychenié S, Kontopoulos DG, Larroque M, Larroque C, Basbous J, Kossida S, Ribeyre C, Constantinou A. Recruitment of ubiquitin-activating enzyme UBA1 to DNA by poly(ADP-ribose) promotes ATR signalling. Life Sci Alliance 2018; 1:e201800096. [PMID: 30456359 PMCID: PMC6238597 DOI: 10.26508/lsa.201800096] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2018] [Revised: 06/14/2018] [Accepted: 06/14/2018] [Indexed: 01/23/2023] Open
Abstract
The DNA damage response (DDR) ensures cellular adaptation to genotoxic insults. In the crowded environment of the nucleus, the assembly of productive DDR complexes requires multiple protein modifications. How the apical E1 ubiquitin activation enzyme UBA1 integrates spatially and temporally in the DDR remains elusive. Using a human cell-free system, we show that poly(ADP-ribose) polymerase 1 promotes the recruitment of UBA1 to DNA. We find that the association of UBA1 with poly(ADP-ribosyl)ated protein-DNA complexes is necessary for the phosphorylation replication protein A and checkpoint kinase 1 by the serine/threonine protein kinase ataxia-telangiectasia and RAD3-related, a prototypal response to DNA damage. UBA1 interacts directly with poly(ADP-ribose) via a solvent-accessible and positively charged patch conserved in the Animalia kingdom but not in Fungi. Thus, ubiquitin activation can anchor to poly(ADP-ribose)-seeded protein assemblies, ensuring the formation of functional ataxia-telangiectasia mutated and RAD3-related-signalling complexes.
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Affiliation(s)
- Ramhari Kumbhar
- Institut de Génétique Humaine, Centre National de la Recherche Scientifique, Université de Montpellier, Montpellier, France
| | - Sophie Vidal-Eychenié
- Institut de Génétique Humaine, Centre National de la Recherche Scientifique, Université de Montpellier, Montpellier, France
| | | | | | - Christian Larroque
- Institut de Recherche en Cancérologie de Montpellier, Université de Montpellier, Institut National de la Santé et de la Recherche Médicale, Montpellier, France
| | - Jihane Basbous
- Institut de Génétique Humaine, Centre National de la Recherche Scientifique, Université de Montpellier, Montpellier, France
| | - Sofia Kossida
- Institut de Génétique Humaine, Centre National de la Recherche Scientifique, Université de Montpellier, Montpellier, France.,IMGT, The International ImMunoGeneTics Information System, Montpellier, France
| | - Cyril Ribeyre
- Institut de Génétique Humaine, Centre National de la Recherche Scientifique, Université de Montpellier, Montpellier, France
| | - Angelos Constantinou
- Institut de Génétique Humaine, Centre National de la Recherche Scientifique, Université de Montpellier, Montpellier, France
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46
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Prakash A, Garcia-Moreno JF, Brown JAL, Bourke E. Clinically Applicable Inhibitors Impacting Genome Stability. Molecules 2018; 23:E1166. [PMID: 29757235 PMCID: PMC6100577 DOI: 10.3390/molecules23051166] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2018] [Revised: 04/27/2018] [Accepted: 05/01/2018] [Indexed: 12/14/2022] Open
Abstract
Advances in technology have facilitated the molecular profiling (genomic and transcriptomic) of tumours, and has led to improved stratification of patients and the individualisation of treatment regimes. To fully realize the potential of truly personalised treatment options, we need targeted therapies that precisely disrupt the compensatory pathways identified by profiling which allow tumours to survive or gain resistance to treatments. Here, we discuss recent advances in novel therapies that impact the genome (chromosomes and chromatin), pathways targeted and the stage of the pathways targeted. The current state of research will be discussed, with a focus on compounds that have advanced into trials (clinical and pre-clinical). We will discuss inhibitors of specific DNA damage responses and other genome stability pathways, including those in development, which are likely to synergistically combine with current therapeutic options. Tumour profiling data, combined with the knowledge of new treatments that affect the regulation of essential tumour signalling pathways, is revealing fundamental insights into cancer progression and resistance mechanisms. This is the forefront of the next evolution of advanced oncology medicine that will ultimately lead to improved survival and may, one day, result in many cancers becoming chronic conditions, rather than fatal diseases.
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Affiliation(s)
- Anu Prakash
- Discipline of Pathology, Lambe Institute for Translational Research, School of Medicine, National University of Ireland Galway, H91 YR71 Galway, Ireland.
| | - Juan F Garcia-Moreno
- Discipline of Surgery, Lambe Institute for Translational Research, School of Medicine, National University of Ireland Galway, H91 YR71 Galway, Ireland.
| | - James A L Brown
- Discipline of Surgery, Lambe Institute for Translational Research, School of Medicine, National University of Ireland Galway, H91 YR71 Galway, Ireland.
| | - Emer Bourke
- Discipline of Pathology, Lambe Institute for Translational Research, School of Medicine, National University of Ireland Galway, H91 YR71 Galway, Ireland.
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47
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Lahusen TJ, Kim SJ, Miao K, Huang Z, Xu X, Deng CX. BRCA1 function in the intra-S checkpoint is activated by acetylation via a pCAF/SIRT1 axis. Oncogene 2018; 37:2343-2350. [PMID: 29440709 DOI: 10.1038/s41388-018-0127-1] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2017] [Revised: 11/14/2017] [Accepted: 12/09/2017] [Indexed: 12/15/2022]
Abstract
Breast cancer associated gene 1 (BRCA1) function has been shown to be regulated by phosphorylation but the role of acetylation has not been determined. Therefore, we tested whether BRCA1 can be acetylated by the acetyltransferases P300/CBP-associated factor (pCAF), GCN5, and p300. p300 exhibited the highest level of BRCA1 acetylation; however, there was also a decrease in the total level of BRCA1. Therefore, we focused on pCAF and GCN5 because they both acetylated BRCA1 without affecting BRCA1 expression. Further analysis indicated that the acetylated form of BRCA1 is deacetylated by wild-type (WT) SIRT1, but not deacetylase mutant SIRT1, suggesting that SIRT1 is a specific deacetylase of BRCA1. We demonstrated that lysine 830 of BRCA1 is a preferential acetylation site by pCAF and tested its function in embryonic stem (ES) cells by changing lysine 830 to arginine using a transcription activator-like effector nuclease (TALEN) system. After exposure to DNA damage-inducing UV radiation, the viability of BRCA1 K830R mutant cells is greater than the WT ES cells. Further analysis using additional cell lines indicated that the BRCA1 K830R mutation impairs the intra-S checkpoint. Also, checkpoint kinase 1 (CHK1) phosphorylation was less in K830R cells as compared with WT cells after UV exposure. These data suggest that acetylation of BRCA1 on lysine 830 activates BRCA1 function at the intra-S checkpoint after DNA damage.
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Affiliation(s)
- Tyler J Lahusen
- Genetics of Development and Disease Branch, 10/9N105, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Seung-Jin Kim
- Genetics of Development and Disease Branch, 10/9N105, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Kai Miao
- Faculty of Health Sciences, University of Macau, Macau, SAR, China
| | - Zebin Huang
- Faculty of Health Sciences, University of Macau, Macau, SAR, China
| | - Xiaoling Xu
- Faculty of Health Sciences, University of Macau, Macau, SAR, China
| | - Chu-Xia Deng
- Genetics of Development and Disease Branch, 10/9N105, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892, USA. .,Faculty of Health Sciences, University of Macau, Macau, SAR, China.
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Cupello S, Richardson C, Yan S. Cell-free Xenopus egg extracts for studying DNA damage response pathways. THE INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY 2018; 60:229-236. [PMID: 27160070 DOI: 10.1387/ijdb.160113sy] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
In response to a variety of DNA replication stress or DNA damaging agents, the DNA damage response (DDR) pathways are triggered for cells to coordinate DNA repair, cell cycle checkpoints, apoptosis, and senescence. Cell-free Xenopus egg extracts, derived from the eggs of African clawed frogs (Xenopus laevis), have been widely used for studies concerning DDR pathways. In this review, we focus on how different experimental systems have been established using Xenopus egg extracts to investigate the DDR pathways that are activated in response to DNA replication stress, double-strand breaks (DSBs), inter-strand crosslinks (ICLs), and oxidative stress. We summarize how molecular details of DDR pathways are dissected by the mechanistic studies with Xenopus egg extracts. We also provide an update on the regulation of translesion DNA synthesis (TLS) polymerases (Pol ĸ and REV1) in the DDR pathways. A better understanding of DDR pathways using Xenopus egg extracts has opened new avenues for future cancer therapeutics. Finally, we offer our perspectives of future directions for studies of DDR pathways with Xenopus egg extracts.
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Affiliation(s)
- Steven Cupello
- Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC, USA
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Shen T, Zhou H, Shang C, Luo Y, Wu Y, Huang S. Ciclopirox activates ATR-Chk1 signaling pathway leading to Cdc25A protein degradation. Genes Cancer 2018; 9:39-52. [PMID: 29725502 PMCID: PMC5931253 DOI: 10.18632/genesandcancer.166] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2018] [Accepted: 02/11/2018] [Indexed: 02/05/2023] Open
Abstract
Ciclopirox olamine (CPX), an off-patent anti-fungal drug, has been found to inhibit the G1-cyclin dependent kinases partly by increasing the phosphorylation and degradation of Cdc25A. However, little is known about the molecular target(s) of CPX responsible for Cdc25A degradation. Here, we show that CPX induced the degradation of Cdc25A neither by increasing CK1α or decreasing DUB3 expression, nor via activating GSK3β, but through activating Chk1 in rhabdomyosarcoma (Rh30) and breast carcinoma (MDA-MB-231) cells. This is strongly supported by the findings that inhibition of Chk1 with TCS2312 or knockdown of Chk1 profoundly attenuated CPX-induced Cdc25A degradation in the cells. Furthermore, we observed that CPX caused DNA damage, which was independent of reactive oxygen species (ROS) induction, but related to iron chelation. CPX treatment resulted in the activation of ataxia telangiectasia mutated (ATM) and ATM-and RAD3-related (ATR) kinases. Treatment with Ku55933 (a selective ATM inhibitor) failed to prevent CPX-induced Chk1 phosphorylation and Cdc25A degradation. In contrast, knockdown of ATR conferred high resistance to CPX-induced Chk1 phosphorylation and Cdc25A degradation. Therefore, the results suggest that CPX-induced degradation of Cdc25A is attributed to the activation of ATR-Chk1 signaling pathway, a consequence of iron chelation-induced DNA damage.
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Affiliation(s)
- Tao Shen
- Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA, USA
- Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, LA, USA
| | - Hongyu Zhou
- Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA, USA
| | - Chaowei Shang
- Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA, USA
- Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, LA, USA
| | - Yan Luo
- Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA, USA
- State Key Laboratory of Biotherapy / Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan, P.R. China
| | - Yang Wu
- Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA, USA
- State Key Laboratory of Biotherapy / Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan, P.R. China
| | - Shile Huang
- Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA, USA
- Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, LA, USA
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50
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Echinacoside Alleviates UVB Irradiation-Mediated Skin Damage via Inhibition of Oxidative Stress, DNA Damage, and Apoptosis. OXIDATIVE MEDICINE AND CELLULAR LONGEVITY 2017; 2017:6851464. [PMID: 29213352 PMCID: PMC5682084 DOI: 10.1155/2017/6851464] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/12/2017] [Accepted: 08/30/2017] [Indexed: 12/20/2022]
Abstract
Ultraviolet B (UVB) irradiation has been known to cause skin damage, which is associated with oxidative stress, DNA damage, and apoptosis. Echinacoside is a phenylethanoid glycoside isolated from Herba Cistanches, which exhibits strong antioxidant activity. In this study, we evaluate the photoprotective effect of echinacoside on UVB-induced skin damage and explore the potential molecular mechanism. BALB/c mice and HaCaT cells were treated with echinacoside before UVB exposure. Histopathological examination was used to evaluate the skin damage. Cell viability, lactate dehydrogenase (LDH) levels, antioxidant enzyme activities, reactive oxygen species (ROS) generation, DNA damage, and apoptosis were measured as well. Western blot was used to measure the expression of related proteins. The results revealed that pretreatment of echinacoside ameliorated the skin injury; attenuated oxidative stress, DNA damage, and apoptosis caused by UVB exposure; and normalized the protein levels of ATR, p53, PIAS3, hnRNP K, PARP, and XPA. To summarize, echinacoside is beneficial in the prevention of UVB-induced DNA damage and apoptosis of the skin in vivo and in vitro.
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