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Davis BEM, Snedeker J, Ranjan R, Wooten M, Barton SS, Blundon J, Chen X. Increased levels of lagging strand polymerase α in an adult stem cell lineage affect replication-coupled histone incorporation. SCIENCE ADVANCES 2025; 11:eadu6799. [PMID: 40020063 PMCID: PMC11870066 DOI: 10.1126/sciadv.adu6799] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 11/17/2024] [Accepted: 01/29/2025] [Indexed: 03/03/2025]
Abstract
Stem cells display asymmetric histone inheritance, while nonstem progenitor cells exhibit symmetric patterns in the Drosophila male germ line. Here, we report that components involved in lagging strand synthesis, DNA polymerases α and δ, have substantially reduced levels in stem cells compared to progenitor cells, and this promotes local asymmetry of parental histone incorporation at the replication fork. Compromising Polα genetically induces the local replication-coupled histone incorporation pattern in progenitor cells to resemble that in stem cells, seen by both nuclear localization patterns and chromatin fibers. This is recapitulated using a Polα inhibitor in a concentration-dependent manner. The local old versus new histone asymmetry is comparable between stem cells and progenitor cells at both S phase and M phase. Together, these results indicate that developmentally programmed expression of key DNA replication components is important to shape stem cell chromatin. Furthermore, manipulating one crucial DNA replication component can induce replication-coupled histone dynamics in nonstem cells to resemble those in stem cells.
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Affiliation(s)
- Brendon E. M. Davis
- Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA
| | - Jonathan Snedeker
- Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA
| | - Rajesh Ranjan
- Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA
- Howard Hughes Medical Institute, Department of Biology, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA
| | - Matthew Wooten
- Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA
| | - Savannah Sáde Barton
- Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA
- Howard Hughes Medical Institute, Department of Biology, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA
| | - Joshua Blundon
- Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA
| | - Xin Chen
- Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA
- Howard Hughes Medical Institute, Department of Biology, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA
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2
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Guo S, An HM, Tian Z, Liu W, Wang XP. Downregulation of the DNA replication pathway genes stimulate reproductive diapause preparation in the cabbage beetle, Colaphellus bowringi. Int J Biol Macromol 2025; 286:138464. [PMID: 39645116 DOI: 10.1016/j.ijbiomac.2024.138464] [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: 09/03/2024] [Revised: 12/02/2024] [Accepted: 12/04/2024] [Indexed: 12/09/2024]
Abstract
Diapause is a prevalent strategy employed by insects to survive adverse environmental conditions, by halting development and reducing metabolic activity. Although the suppression of DNA replication aligns logically with these reduced developmental and physiological activities, the role of DNA replication in regulating insect diapause remains largely unknown. In this study, we used the cabbage beetle, Colaphellus bowringi, to investigate the role of DNA replication pathway in regulating reproductive diapause. Transcriptome analysis identified DNA replication as a key pathway during diapause preparation in female adults. Fourteen DNA replication genes were isolated, encompassing essential stages of DNA replication, including DNA unwinding, primer synthesis, and DNA synthesis. These genes exhibited consistently reduced expression in diapause females compared to those in reproductive females. RNA interference knockdown of these genes in reproductive female adults resulted in 11 out of 14 gene depletions, manifesting typical diapause traits such as suppressed vitellogenesis, arrested ovary growth, and increased lipid accumulation. Furthermore, we demonstrated 20-hydroxyecdysone (20E), through canonical signaling pathway, regulates the differential expression of DNA replication genes between reproductive and diapause states. Our findings suggest 20E deficiency suppresses DNA replication to induce reproductive diapause, and highlighting the DNA replication process as a potential target for pest management.
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Affiliation(s)
- Shuang Guo
- Hubei Key Laboratory of Insect Resources Utilization and Sustainable Pest Management, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
| | - Hao-Min An
- Hubei Key Laboratory of Insect Resources Utilization and Sustainable Pest Management, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China; Institute for Advanced Study, Shenzhen University, Shenzhen 518060, China
| | - Zhong Tian
- Hubei Key Laboratory of Insect Resources Utilization and Sustainable Pest Management, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China; Innovative Institute of Chinese Medicine and Pharmacy, Academy for Interdiscipline, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China
| | - Wen Liu
- Department of Biological Sciences, University of Alberta, G-504, Biological Sciences Bldg., Edmonton, Alberta T6G 2E9, Canada
| | - Xiao-Ping Wang
- Hubei Key Laboratory of Insect Resources Utilization and Sustainable Pest Management, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
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3
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Bhowmik D, Bhuyan A, Gunalan S, Kothandan G, Kumar D. In silico and immunoinformatics based multiepitope subunit vaccine design for protection against visceral leishmaniasis. J Biomol Struct Dyn 2024; 42:9731-9752. [PMID: 37655736 DOI: 10.1080/07391102.2023.2252901] [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: 02/12/2023] [Accepted: 08/22/2023] [Indexed: 09/02/2023]
Abstract
Visceral leishmaniasis (VL) is a vector-borne neglected tropical protozoan disease with high fatality and no certified vaccine. Conventional vaccine preparation is challenging and tedious. Here in this work, we created a global multiepitope subunit vaccination against VL utilizing innovative immunoinformatics technique based on the extensively conserved epitopic regions of the PrimPol protein of Leishmania donovani consisting of four subunits which were analyzed and studied, out of which DNA primase large subunit and DNA polymerase α subunit B were evaluated as antigens by Vaxijen 2.0. The multiepitope vaccine design includes a single adjuvant β-defensins, eight CTL epitopes, eight HTL epitopes, seven linear BCL epitopes and one discontinuous BCL epitope to induce innate, cellular and humoral immune responses against VL. The Expasy ProtParam tool characterized the physiochemical parameters of the vaccine. At the same time, SOLpro evaluated our vaccine constructs to be soluble upon expression. We also modeled the stable tertiary structure of our vaccine construct through Robetta modeling for molecular docking studies with toll-like receptor proteins through HADDOCK 2.4. Simulations based on molecular dynamics revealed an intact vaccine and TLR8 complex, supporting our vaccine design's immunogenicity. Also, the immune simulation of our vaccine by the C-ImmSim server demonstrated the potency of the multiepitope vaccine construct to induce proper immune response for host defense. Codon optimization and in silico cloning of our vaccine further assured high expression. The outcomes of our study on multiepitope vaccine design significantly produced a potential candidate against VL and can potentially eradicate the disease in the future after clinical investigations.Communicated by Ramaswamy H. Sarma.
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Affiliation(s)
- Deep Bhowmik
- Deparment of Microbiology, Assam University, Silchar, Assam, India
| | - Achyut Bhuyan
- Deparment of Microbiology, Assam University, Silchar, Assam, India
| | - Seshan Gunalan
- Biopolymer Modelling Laboratory, Centre of Advanced Study in Crystallography and Biophysics, Guindy Campus, University of Madras, Chennai, India
| | - Gugan Kothandan
- Biopolymer Modelling Laboratory, Centre of Advanced Study in Crystallography and Biophysics, Guindy Campus, University of Madras, Chennai, India
| | - Diwakar Kumar
- Deparment of Microbiology, Assam University, Silchar, Assam, India
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Snedeker J, Davis BEM, Ranjan R, Wooten M, Blundon J, Chen X. Reduced Levels of Lagging Strand Polymerases Shape Stem Cell Chromatin. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.04.26.591383. [PMID: 38746451 PMCID: PMC11092439 DOI: 10.1101/2024.04.26.591383] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/16/2024]
Abstract
Stem cells display asymmetric histone inheritance while non-stem progenitor cells exhibit symmetric patterns in the Drosophila male germline lineage. Here, we report that components involved in lagging strand synthesis, such as DNA polymerase α and δ (Polα and Polδ), have significantly reduced levels in stem cells compared to progenitor cells. Compromising Polα genetically induces the replication-coupled histone incorporation pattern in progenitor cells to be indistinguishable from that in stem cells, which can be recapitulated using a Polα inhibitor in a concentration-dependent manner. Furthermore, stem cell-derived chromatin fibers display a higher degree of old histone recycling by the leading strand compared to progenitor cell-derived chromatin fibers. However, upon reducing Polα levels in progenitor cells, the chromatin fibers now display asymmetric old histone recycling just like GSC-derived fibers. The old versus new histone asymmetry is comparable between stem cells and progenitor cells at both S-phase and M-phase. Together, these results indicate that developmentally programmed expression of key DNA replication components is important to shape stem cell chromatin. Furthermore, manipulating one crucial DNA replication component can induce replication-coupled histone dynamics in non-stem cells in a manner similar to that in stem cells.
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Affiliation(s)
- Jonathan Snedeker
- Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA
| | - Brendon E. M. Davis
- Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA
| | - Rajesh Ranjan
- Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA
- Howard Hughes Medical Institute, Department of Biology, The Johns Hopkins University, 3400 North Charles Street, Baltimore, Baltimore, MD 21218, USA
| | - Matthew Wooten
- Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA
- Current address: Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA
| | - Joshua Blundon
- Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA
| | - Xin Chen
- Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA
- Howard Hughes Medical Institute, Department of Biology, The Johns Hopkins University, 3400 North Charles Street, Baltimore, Baltimore, MD 21218, USA
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5
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Yin Z, Kilkenny ML, Ker DS, Pellegrini L. CryoEM insights into RNA primer synthesis by the human primosome. FEBS J 2024; 291:1813-1829. [PMID: 38335062 DOI: 10.1111/febs.17082] [Citation(s) in RCA: 4] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2023] [Revised: 11/24/2023] [Accepted: 01/26/2024] [Indexed: 02/12/2024]
Abstract
Eukaryotic DNA replication depends on the primosome - a complex of DNA polymerase alpha (Pol α) and primase - to initiate DNA synthesis by polymerisation of an RNA-DNA primer. Primer synthesis requires the tight coordination of primase and polymerase activities. Recent cryo-electron microscopy (cryoEM) analyses have elucidated the extensive conformational transitions required for RNA primer handover between primase and Pol α and primer elongation by Pol α. Because of the intrinsic flexibility of the primosome, however, structural information about the initiation of RNA primer synthesis is still lacking. Here, we capture cryoEM snapshots of the priming reaction to reveal the conformational trajectory of the human primosome that brings DNA primase subunits 1 and 2 (PRIM1 and PRIM2, respectively) together, poised for RNA synthesis. Furthermore, we provide experimental evidence for the continuous association of primase subunit PRIM2 with the RNA primer during primer synthesis, and for how both initiation and termination of RNA primer polymerisation are licenced by specific rearrangements of DNA polymerase alpha catalytic subunit (POLA1), the polymerase subunit of Pol α. Our findings fill a critical gap in our understanding of the conformational changes that underpin the synthesis of the RNA primer by the primosome. Together with existing evidence, they provide a complete description of the structural dynamics of the human primosome during DNA replication initiation.
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Affiliation(s)
- Zhan Yin
- Department of Biochemistry, University of Cambridge, UK
| | | | - De-Sheng Ker
- Department of Biochemistry, University of Cambridge, UK
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6
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Nasheuer HP, Meaney AM. Starting DNA Synthesis: Initiation Processes during the Replication of Chromosomal DNA in Humans. Genes (Basel) 2024; 15:360. [PMID: 38540419 PMCID: PMC10969946 DOI: 10.3390/genes15030360] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2024] [Revised: 03/09/2024] [Accepted: 03/11/2024] [Indexed: 06/14/2024] Open
Abstract
The initiation reactions of DNA synthesis are central processes during human chromosomal DNA replication. They are separated into two main processes: the initiation events at replication origins, the start of the leading strand synthesis for each replicon, and the numerous initiation events taking place during lagging strand DNA synthesis. In addition, a third mechanism is the re-initiation of DNA synthesis after replication fork stalling, which takes place when DNA lesions hinder the progression of DNA synthesis. The initiation of leading strand synthesis at replication origins is regulated at multiple levels, from the origin recognition to the assembly and activation of replicative helicase, the Cdc45-MCM2-7-GINS (CMG) complex. In addition, the multiple interactions of the CMG complex with the eukaryotic replicative DNA polymerases, DNA polymerase α-primase, DNA polymerase δ and ε, at replication forks play pivotal roles in the mechanism of the initiation reactions of leading and lagging strand DNA synthesis. These interactions are also important for the initiation of signalling at unperturbed and stalled replication forks, "replication stress" events, via ATR (ATM-Rad 3-related protein kinase). These processes are essential for the accurate transfer of the cells' genetic information to their daughters. Thus, failures and dysfunctions in these processes give rise to genome instability causing genetic diseases, including cancer. In their influential review "Hallmarks of Cancer: New Dimensions", Hanahan and Weinberg (2022) therefore call genome instability a fundamental function in the development process of cancer cells. In recent years, the understanding of the initiation processes and mechanisms of human DNA replication has made substantial progress at all levels, which will be discussed in the review.
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Affiliation(s)
- Heinz Peter Nasheuer
- Centre for Chromosome Biology, School of Biological and Chemical Sciences, Biochemistry, University of Galway, H91 TK33 Galway, Ireland;
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Lim CJ. Telomere C-Strand Fill-In Machinery: New Insights into the Human CST-DNA Polymerase Alpha-Primase Structures and Functions. Subcell Biochem 2024; 104:73-100. [PMID: 38963484 DOI: 10.1007/978-3-031-58843-3_5] [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] [Indexed: 07/05/2024]
Abstract
Telomeres at the end of eukaryotic chromosomes are extended by a specialized set of enzymes and telomere-associated proteins, collectively termed here the telomere "replisome." The telomere replisome acts on a unique replicon at each chromosomal end of the telomeres, the 3' DNA overhang. This telomere replication process is distinct from the replisome mechanism deployed to duplicate the human genome. The G-rich overhang is first extended before the complementary C-strand is filled in. This overhang is extended by telomerase, a specialized ribonucleoprotein and reverse transcriptase. The overhang extension process is terminated when telomerase is displaced by CTC1-STN1-TEN1 (CST), a single-stranded DNA-binding protein complex. CST then recruits DNA polymerase α-primase to complete the telomere replication process by filling in the complementary C-strand. In this chapter, the recent structure-function insights into the human telomere C-strand fill-in machinery (DNA polymerase α-primase and CST) will be discussed.
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Affiliation(s)
- Ci Ji Lim
- Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, WI, USA.
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8
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Spegg V, Altmeyer M. Genome maintenance meets mechanobiology. Chromosoma 2024; 133:15-36. [PMID: 37581649 PMCID: PMC10904543 DOI: 10.1007/s00412-023-00807-5] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2023] [Revised: 06/20/2023] [Accepted: 07/26/2023] [Indexed: 08/16/2023]
Abstract
Genome stability is key for healthy cells in healthy organisms, and deregulated maintenance of genome integrity is a hallmark of aging and of age-associated diseases including cancer and neurodegeneration. To maintain a stable genome, genome surveillance and repair pathways are closely intertwined with cell cycle regulation and with DNA transactions that occur during transcription and DNA replication. Coordination of these processes across different time and length scales involves dynamic changes of chromatin topology, clustering of fragile genomic regions and repair factors into nuclear repair centers, mobilization of the nuclear cytoskeleton, and activation of cell cycle checkpoints. Here, we provide a general overview of cell cycle regulation and of the processes involved in genome duplication in human cells, followed by an introduction to replication stress and to the cellular responses elicited by perturbed DNA synthesis. We discuss fragile genomic regions that experience high levels of replication stress, with a particular focus on telomere fragility caused by replication stress at the ends of linear chromosomes. Using alternative lengthening of telomeres (ALT) in cancer cells and ALT-associated PML bodies (APBs) as examples of replication stress-associated clustered DNA damage, we discuss compartmentalization of DNA repair reactions and the role of protein properties implicated in phase separation. Finally, we highlight emerging connections between DNA repair and mechanobiology and discuss how biomolecular condensates, components of the nuclear cytoskeleton, and interfaces between membrane-bound organelles and membraneless macromolecular condensates may cooperate to coordinate genome maintenance in space and time.
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Affiliation(s)
- Vincent Spegg
- Department of Molecular Mechanisms of Disease, University of Zurich, Zurich, Switzerland
| | - Matthias Altmeyer
- Department of Molecular Mechanisms of Disease, University of Zurich, Zurich, Switzerland.
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9
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Cordoba JJ, Mullins EA, Salay LE, Eichman BF, Chazin WJ. Flexibility and Distributive Synthesis Regulate RNA Priming and Handoff in Human DNA Polymerase α-Primase. J Mol Biol 2023; 435:168330. [PMID: 37884206 PMCID: PMC10872500 DOI: 10.1016/j.jmb.2023.168330] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2023] [Revised: 09/22/2023] [Accepted: 10/18/2023] [Indexed: 10/28/2023]
Abstract
DNA replication in eukaryotes relies on the synthesis of a ∼30-nucleotide RNA/DNA primer strand through the dual action of the heterotetrameric polymerase α-primase (pol-prim) enzyme. Synthesis of the 7-10-nucleotide RNA primer is regulated by the C-terminal domain of the primase regulatory subunit (PRIM2C) and is followed by intramolecular handoff of the primer to pol α for extension by ∼20 nucleotides of DNA. Here, we provide evidence that RNA primer synthesis is governed by a combination of the high affinity and flexible linkage of the PRIM2C domain and the surprisingly low affinity of the primase catalytic domain (PRIM1) for substrate. Using a combination of small angle X-ray scattering and electron microscopy, we found significant variability in the organization of PRIM2C and PRIM1 in the absence and presence of substrate, and that the population of structures with both PRIM2C and PRIM1 in a configuration aligned for synthesis is low. Crosslinking was used to visualize the orientation of PRIM2C and PRIM1 when engaged by substrate as observed by electron microscopy. Microscale thermophoresis was used to measure substrate affinities for a series of pol-prim constructs, which showed that the PRIM1 catalytic domain does not bind the template or emergent RNA-primed templates with appreciable affinity. Together, these findings support a model of RNA primer synthesis in which generation of the nascent RNA strand and handoff of the RNA-primed template from primase to polymerase α is mediated by the high degree of inter-domain flexibility of pol-prim, the ready dissociation of PRIM1 from its substrate, and the much higher affinity of the POLA1cat domain of polymerase α for full-length RNA-primed templates.
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Affiliation(s)
- John J Cordoba
- Chemical and Physical Biology Program, Vanderbilt University, Nashville, TN, USA; Center for Structural Biology, Vanderbilt University, Nashville, TN, USA
| | - Elwood A Mullins
- Center for Structural Biology, Vanderbilt University, Nashville, TN, USA; Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA
| | - Lauren E Salay
- Chemical and Physical Biology Program, Vanderbilt University, Nashville, TN, USA; Center for Structural Biology, Vanderbilt University, Nashville, TN, USA; Department of Biochemistry, Vanderbilt University, Nashville, TN, USA
| | - Brandt F Eichman
- Chemical and Physical Biology Program, Vanderbilt University, Nashville, TN, USA; Center for Structural Biology, Vanderbilt University, Nashville, TN, USA; Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA; Department of Biochemistry, Vanderbilt University, Nashville, TN, USA
| | - Walter J Chazin
- Chemical and Physical Biology Program, Vanderbilt University, Nashville, TN, USA; Center for Structural Biology, Vanderbilt University, Nashville, TN, USA; Department of Biochemistry, Vanderbilt University, Nashville, TN, USA; Department of Chemistry, Vanderbilt University, Nashville, TN, USA.
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10
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Cordoba JJ, Mullins EA, Salay LE, Eichman BF, Chazin WJ. Flexibility and distributive synthesis regulate RNA priming and handoff in human DNA polymerase α-primase. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.08.01.551538. [PMID: 37577606 PMCID: PMC10418221 DOI: 10.1101/2023.08.01.551538] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 08/15/2023]
Abstract
DNA replication in eukaryotes relies on the synthesis of a ~30-nucleotide RNA/DNA primer strand through the dual action of the heterotetrameric polymerase α-primase (pol-prim) enzyme. Synthesis of the 7-10-nucleotide RNA primer is regulated by the C-terminal domain of the primase regulatory subunit (PRIM2C) and is followed by intramolecular handoff of the primer to pol α for extension by ~20 nucleotides of DNA. Here we provide evidence that RNA primer synthesis is governed by a combination of the high affinity and flexible linkage of the PRIM2C domain and the low affinity of the primase catalytic domain (PRIM1) for substrate. Using a combination of small angle X-ray scattering and electron microscopy, we found significant variability in the organization of PRIM2C and PRIM1 in the absence and presence of substrate, and that the population of structures with both PRIM2C and PRIM1 in a configuration aligned for synthesis is low. Crosslinking was used to visualize the orientation of PRIM2C and PRIM1 when engaged by substrate as observed by electron microscopy. Microscale thermophoresis was used to measure substrate affinities for a series of pol-prim constructs, which showed that the PRIM1 catalytic domain does not bind the template or emergent RNA-primed templates with appreciable affinity. Together, these findings support a model of RNA primer synthesis in which generation of the nascent RNA strand and handoff of the RNA-primed template from primase to polymerase α is mediated by the high degree of inter-domain flexibility of pol-prim, the ready dissociation of PRIM1 from its substrate, and the much higher affinity of the POLA1cat domain of polymerase α for full-length RNA-primed templates.
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Affiliation(s)
- John J. Cordoba
- Chemical and Physical Biology Program, Vanderbilt University, Nashville, Tennessee, USA
- Center for Structural Biology, Vanderbilt University, Nashville, Tennessee, USA
| | - Elwood A. Mullins
- Center for Structural Biology, Vanderbilt University, Nashville, Tennessee, USA
- Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee, USA
| | - Lauren E. Salay
- Chemical and Physical Biology Program, Vanderbilt University, Nashville, Tennessee, USA
- Center for Structural Biology, Vanderbilt University, Nashville, Tennessee, USA
- Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, USA
| | - Brandt F. Eichman
- Chemical and Physical Biology Program, Vanderbilt University, Nashville, Tennessee, USA
- Center for Structural Biology, Vanderbilt University, Nashville, Tennessee, USA
- Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee, USA
- Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, USA
| | - Walter J. Chazin
- Chemical and Physical Biology Program, Vanderbilt University, Nashville, Tennessee, USA
- Center for Structural Biology, Vanderbilt University, Nashville, Tennessee, USA
- Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, USA
- Department of Chemistry, Vanderbilt University, Nashville, Tennessee, USA
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11
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Yuan Z, Georgescu R, Li H, O'Donnell ME. Molecular choreography of primer synthesis by the eukaryotic Pol α-primase. Nat Commun 2023; 14:3697. [PMID: 37344454 PMCID: PMC10284912 DOI: 10.1038/s41467-023-39441-1] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2023] [Accepted: 06/14/2023] [Indexed: 06/23/2023] Open
Abstract
The eukaryotic polymerase α (Pol α) synthesizes an RNA-DNA hybrid primer of 20-30 nucleotides. Pol α is composed of Pol1, Pol12, Primase 1 (Pri1), and Pri2. Pol1 and Pri1 contain the DNA polymerase and RNA primase activities, respectively. It has been unclear how Pol α hands over an RNA primer from Pri1 to Pol1 for DNA primer extension, and how the primer length is defined. Here we report the cryo-EM analysis of yeast Pol α in the apo, primer initiation, primer elongation, RNA primer hand-off from Pri1 to Pol1, and DNA extension states, revealing a series of very large movements. We reveal a critical point at which Pol1-core moves to take over the 3'-end of the RNA from Pri1. DNA extension is limited by a spiral motion of Pol1-core. Since both Pri1 and Pol1-core are flexibly attached to a stable platform, primer growth produces stress that limits the primer length.
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Affiliation(s)
- Zuanning Yuan
- Department of Structural Biology, Van Andel Institute, Grand Rapids, MI, USA
| | - Roxana Georgescu
- DNA Replication Laboratory and Howard Hughes Medical Institute, Rockefeller University, New York, NY, USA
| | - Huilin Li
- Department of Structural Biology, Van Andel Institute, Grand Rapids, MI, USA.
| | - Michael E O'Donnell
- DNA Replication Laboratory and Howard Hughes Medical Institute, Rockefeller University, New York, NY, USA.
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12
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Schneider A, Bergsch J, Lipps G. The monomeric archaeal primase from Nanoarchaeum equitans harbours the features of heterodimeric archaeoeukaryotic primases and primes sequence-specifically. Nucleic Acids Res 2023; 51:5087-5105. [PMID: 37099378 PMCID: PMC10250227 DOI: 10.1093/nar/gkad261] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2022] [Revised: 03/17/2023] [Accepted: 03/29/2023] [Indexed: 04/27/2023] Open
Abstract
The marine thermophilic archaeon Nanoarchaeum equitans possesses a monomeric primase encompassing the conserved domains of the small catalytic and the large regulatory subunits of archaeoeukaryotic heterodimeric primases in one protein chain. The recombinant protein primes on templates containing a triplet with a central thymidine, thus displaying a pronounced sequence specificity typically observed with bacterial type primases only. The N. equitans primase (NEQ395) is a highly active primase enzyme synthesizing short RNA primers. Termination occurs preferentially at about nine nucleotides, as determined by HPLC analysis and confirmed with mass spectrometry. Possibly, the compact monomeric primase NEQ395 represents the minimal archaeoeukaryotic primase and could serve as a functional and structural model of the heterodimeric archaeoeukaryotic primases, whose study is hindered by engagement in protein assemblies and rather low activity.
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Affiliation(s)
- Andy Schneider
- Institute of Chemistry and Bioanalytics, University of Applied Sciences Northwestern Switzerland, 4132 Muttenz, Switzerland
| | - Jan Bergsch
- Institute of Chemistry and Bioanalytics, University of Applied Sciences Northwestern Switzerland, 4132 Muttenz, Switzerland
| | - Georg Lipps
- Institute of Chemistry and Bioanalytics, University of Applied Sciences Northwestern Switzerland, 4132 Muttenz, Switzerland
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13
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Yuan Z, Georgescu R, Li H, O'Donnell ME. Molecular choreography of primer synthesis by the eukaryotic Pol α-primase. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.05.03.539257. [PMID: 37205351 PMCID: PMC10187153 DOI: 10.1101/2023.05.03.539257] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/21/2023]
Abstract
The eukaryotic polymerase α (Pol α) is a dual-function DNA polymerase/primase complex that synthesizes an RNA-DNA hybrid primer of 20-30 nucleotides for DNA replication. Pol α is composed of Pol1, Pol12, Primase 1 (Pri1), and Pri2, with Pol1 and Pri1 containing the DNA polymerase activity and RNA primase activity, respectively, whereas Pol12 and Pri2 serve a structural role. It has been unclear how Pol α hands over an RNA primer made by Pri1 to Pol1 for DNA primer extension, and how the primer length is defined, perhaps due to the difficulty in studying the highly mobile structure. Here we report a comprehensive cryo-EM analysis of the intact 4-subunit yeast Pol α in the apo, primer initiation, primer elongation, RNA primer hand-off from Pri1 to Pol1, and DNA extension states in a 3.5 Å - 5.6 Å resolution range. We found that Pol α is a three-lobed flexible structure. Pri2 functions as a flexible hinge that holds together the catalytic Pol1-core, and the noncatalytic Pol1 CTD that binds to Pol 12 to form a stable platform upon which the other components are organized. In the apo state, Pol1-core is sequestered on the Pol12-Pol1-CTD platform, and Pri1 is mobile perhaps in search of a template. Upon binding a ssDNA template, a large conformation change is induced that enables Pri1 to perform RNA synthesis, and positions Pol1-core to accept the future RNA primed site 50 Å upstream of where Pri1 binds. We reveal in detail the critical point at which Pol1-core takes over the 3'-end of the RNA from Pri1. DNA primer extension appears limited by the spiral motion of Pol1-core while Pri2-CTD stably holds onto the 5' end of the RNA primer. Since both Pri1 and Pol1-core are attached via two linkers to the platform, primer growth will produce stress within this "two-point" attachment that may limit the length of the RNA-DNA hybrid primer. Hence, this study reveals the large and dynamic series of movements that Pol α undergoes to synthesize a primer for DNA replication.
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Human primase hangs on the primer-template and Polα to facilitate primer termination. Nat Struct Mol Biol 2023; 30:575-576. [PMID: 37081321 DOI: 10.1038/s41594-023-00972-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/22/2023]
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15
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Díaz-Talavera A, Montero-Conde C, Leandro-García LJ, Robledo M. PrimPol: A Breakthrough among DNA Replication Enzymes and a Potential New Target for Cancer Therapy. Biomolecules 2022; 12:248. [PMID: 35204749 PMCID: PMC8961649 DOI: 10.3390/biom12020248] [Citation(s) in RCA: 8] [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: 12/28/2021] [Revised: 01/29/2022] [Accepted: 02/02/2022] [Indexed: 02/01/2023] Open
Abstract
DNA replication can encounter blocking obstacles, leading to replication stress and genome instability. There are several mechanisms for evading this blockade. One mechanism consists of repriming ahead of the obstacles, creating a new starting point; in humans, PrimPol is responsible for carrying out this task. PrimPol is a primase that operates in both the nucleus and mitochondria. In contrast with conventional primases, PrimPol is a DNA primase able to initiate DNA synthesis de novo using deoxynucleotides, discriminating against ribonucleotides. In vitro, PrimPol can act as a DNA primase, elongating primers that PrimPol itself sythesizes, or as translesion synthesis (TLS) DNA polymerase, elongating pre-existing primers across lesions. However, the lack of evidence for PrimPol polymerase activity in vivo suggests that PrimPol only acts as a DNA primase. Here, we provide a comprehensive review of human PrimPol covering its biochemical properties and structure, in vivo function and regulation, and the processes that take place to fill the gap-containing lesion that PrimPol leaves behind. Finally, we explore the available data on human PrimPol expression in different tissues in physiological conditions and its role in cancer.
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Affiliation(s)
- Alberto Díaz-Talavera
- Hereditary Endocrine Cancer Group, Human Cancer Genetics Program, Spanish National Cancer Research Centre (CNIO), 28029 Madrid, Spain; (C.M.-C.); (L.J.L.-G.); (M.R.)
- Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III (ISCIII), 28029 Madrid, Spain
| | - Cristina Montero-Conde
- Hereditary Endocrine Cancer Group, Human Cancer Genetics Program, Spanish National Cancer Research Centre (CNIO), 28029 Madrid, Spain; (C.M.-C.); (L.J.L.-G.); (M.R.)
- Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III (ISCIII), 28029 Madrid, Spain
| | - Luis Javier Leandro-García
- Hereditary Endocrine Cancer Group, Human Cancer Genetics Program, Spanish National Cancer Research Centre (CNIO), 28029 Madrid, Spain; (C.M.-C.); (L.J.L.-G.); (M.R.)
| | - Mercedes Robledo
- Hereditary Endocrine Cancer Group, Human Cancer Genetics Program, Spanish National Cancer Research Centre (CNIO), 28029 Madrid, Spain; (C.M.-C.); (L.J.L.-G.); (M.R.)
- Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III (ISCIII), 28029 Madrid, Spain
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16
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Eekhout T, Pedroza-Garcia JA, Kalhorzadeh P, De Jaeger G, De Veylder L. A Mutation in DNA Polymerase α Rescues WEE1KO Sensitivity to HU. Int J Mol Sci 2021; 22:9409. [PMID: 34502313 PMCID: PMC8430855 DOI: 10.3390/ijms22179409] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2021] [Revised: 08/25/2021] [Accepted: 08/26/2021] [Indexed: 11/16/2022] Open
Abstract
During DNA replication, the WEE1 kinase is responsible for safeguarding genomic integrity by phosphorylating and thus inhibiting cyclin-dependent kinases (CDKs), which are the driving force of the cell cycle. Consequentially, wee1 mutant plants fail to respond properly to problems arising during DNA replication and are hypersensitive to replication stress. Here, we report the identification of the polα-2 mutant, mutated in the catalytic subunit of DNA polymerase α, as a suppressor mutant of wee1. The mutated protein appears to be less stable, causing a loss of interaction with its subunits and resulting in a prolonged S-phase.
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Affiliation(s)
- Thomas Eekhout
- Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Gent, Belgium; (T.E.); (J.A.P.-G.); (P.K.); (G.D.J.)
- Center for Plant Systems Biology, VIB, 9052 Gent, Belgium
| | - José Antonio Pedroza-Garcia
- Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Gent, Belgium; (T.E.); (J.A.P.-G.); (P.K.); (G.D.J.)
- Center for Plant Systems Biology, VIB, 9052 Gent, Belgium
| | - Pooneh Kalhorzadeh
- Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Gent, Belgium; (T.E.); (J.A.P.-G.); (P.K.); (G.D.J.)
- Center for Plant Systems Biology, VIB, 9052 Gent, Belgium
| | - Geert De Jaeger
- Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Gent, Belgium; (T.E.); (J.A.P.-G.); (P.K.); (G.D.J.)
- Center for Plant Systems Biology, VIB, 9052 Gent, Belgium
| | - Lieven De Veylder
- Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Gent, Belgium; (T.E.); (J.A.P.-G.); (P.K.); (G.D.J.)
- Center for Plant Systems Biology, VIB, 9052 Gent, Belgium
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17
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Calvo P, Martínez-Jiménez MI, Díaz M, Stojkovic G, Kasho K, Guerra S, Wanrooij S, Méndez J, Blanco L. Motif WFYY of human PrimPol is crucial to stabilize the incoming 3'-nucleotide during replication fork restart. Nucleic Acids Res 2021; 49:8199-8213. [PMID: 34302490 PMCID: PMC8373064 DOI: 10.1093/nar/gkab634] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2021] [Revised: 07/06/2021] [Accepted: 07/12/2021] [Indexed: 11/12/2022] Open
Abstract
PrimPol is the second primase in human cells, the first with the ability to start DNA chains with dNTPs. PrimPol contributes to DNA damage tolerance by restarting DNA synthesis beyond stalling lesions, acting as a TLS primase. Multiple alignment of eukaryotic PrimPols allowed us to identify a highly conserved motif, WxxY near the invariant motif A, which contains two active site metal ligands in all members of the archeo-eukaryotic primase (AEP) superfamily. In vivo and in vitro analysis of single variants of the WFYY motif of human PrimPol demonstrated that the invariant Trp87 and Tyr90 residues are essential for both primase and polymerase activities, mainly due to their crucial role in binding incoming nucleotides. Accordingly, the human variant F88L, altering the WFYY motif, displayed reduced binding of incoming nucleotides, affecting its primase/polymerase activities especially during TLS reactions on UV-damaged DNA. Conversely, the Y89D mutation initially associated with High Myopia did not affect the ability to rescue stalled replication forks in human cells. Collectively, our data suggest that the WFYY motif has a fundamental role in stabilizing the incoming 3′-nucleotide, an essential requisite for both its primase and TLS abilities during replication fork restart.
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Affiliation(s)
- Patricia A Calvo
- Centro de Biología Molecular Severo Ochoa, CSIC-UAM, 28049, Madrid, Spain
| | | | - Marcos Díaz
- Spanish National Cancer Research Centre (CNIO), 28029, Madrid, Spain
| | - Gorazd Stojkovic
- Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden
| | - Kazutoshi Kasho
- Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden
- Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan
| | - Susana Guerra
- Centro de Biología Molecular Severo Ochoa, CSIC-UAM, 28049, Madrid, Spain
| | - Sjoerd Wanrooij
- Correspondence may also be addressed to Sjoerd Wanrooij. Tel: +46 722460309;
| | - Juan Méndez
- Correspondence may also be addressed to Juan Méndez. Tel: +34 917328000; Fax: +34 917328033;
| | - Luis Blanco
- To whom correspondence should be addressed. Tel: +34 911964685; Fax: +34 911964401;
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18
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Bainbridge LJ, Teague R, Doherty AJ. Repriming DNA synthesis: an intrinsic restart pathway that maintains efficient genome replication. Nucleic Acids Res 2021; 49:4831-4847. [PMID: 33744934 PMCID: PMC8136793 DOI: 10.1093/nar/gkab176] [Citation(s) in RCA: 30] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2021] [Revised: 03/01/2021] [Accepted: 03/05/2021] [Indexed: 12/25/2022] Open
Abstract
To bypass a diverse range of fork stalling impediments encountered during genome replication, cells possess a variety of DNA damage tolerance (DDT) mechanisms including translesion synthesis, template switching, and fork reversal. These pathways function to bypass obstacles and allow efficient DNA synthesis to be maintained. In addition, lagging strand obstacles can also be circumvented by downstream priming during Okazaki fragment generation, leaving gaps to be filled post-replication. Whether repriming occurs on the leading strand has been intensely debated over the past half-century. Early studies indicated that both DNA strands were synthesised discontinuously. Although later studies suggested that leading strand synthesis was continuous, leading to the preferred semi-discontinuous replication model. However, more recently it has been established that replicative primases can perform leading strand repriming in prokaryotes. An analogous fork restart mechanism has also been identified in most eukaryotes, which possess a specialist primase called PrimPol that conducts repriming downstream of stalling lesions and structures. PrimPol also plays a more general role in maintaining efficient fork progression. Here, we review and discuss the historical evidence and recent discoveries that substantiate repriming as an intrinsic replication restart pathway for maintaining efficient genome duplication across all domains of life.
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Affiliation(s)
- Lewis J Bainbridge
- Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, BN1 9RQ, UK
| | - Rebecca Teague
- Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, BN1 9RQ, UK
| | - Aidan J Doherty
- Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, BN1 9RQ, UK
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Pérez-Arnaiz P, Dattani A, Smith V, Allers T. Haloferax volcanii-a model archaeon for studying DNA replication and repair. Open Biol 2020; 10:200293. [PMID: 33259746 PMCID: PMC7776575 DOI: 10.1098/rsob.200293] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2020] [Accepted: 11/09/2020] [Indexed: 12/16/2022] Open
Abstract
The tree of life shows the relationship between all organisms based on their common ancestry. Until 1977, it comprised two major branches: prokaryotes and eukaryotes. Work by Carl Woese and other microbiologists led to the recategorization of prokaryotes and the proposal of three primary domains: Eukarya, Bacteria and Archaea. Microbiological, genetic and biochemical techniques were then needed to study the third domain of life. Haloferax volcanii, a halophilic species belonging to the phylum Euryarchaeota, has provided many useful tools to study Archaea, including easy culturing methods, genetic manipulation and phenotypic screening. This review will focus on DNA replication and DNA repair pathways in H. volcanii, how this work has advanced our knowledge of archaeal cellular biology, and how it may deepen our understanding of bacterial and eukaryotic processes.
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Affiliation(s)
| | | | | | - Thorsten Allers
- School of Life Sciences, University of Nottingham, Queen's Medical Centre, Nottingham, UK
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20
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Zhang M, Li W, Feng J, Gong Z, Yao Y, Zheng C. Integrative transcriptomics and proteomics analysis constructs a new molecular model for ovule abortion in the female-sterile line of Pinus tabuliformis Carr. PLANT SCIENCE : AN INTERNATIONAL JOURNAL OF EXPERIMENTAL PLANT BIOLOGY 2020; 294:110462. [PMID: 32234230 DOI: 10.1016/j.plantsci.2020.110462] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/05/2019] [Revised: 02/26/2020] [Accepted: 02/28/2020] [Indexed: 06/11/2023]
Abstract
Ovule development is critical to plant reproduction and free nuclear mitosis of megagametophyte (FNMM) is vital for ovule development. However, most results of ovule development were based on the studies in angiosperms, and its molecular regulation remained largely unknown in gymnosperms, particularly, during FNMM. In this context, we studied the genome-wide difference between sterile line (SL) and fertile line (FL) ovules using transcriptomics and proteomics approaches in Pinus tabuliformis Carr. Comparative analyses revealed that genes involved in DNA replication, DNA damage repair, Cell cycle, Apoptosis and Energy metabolism were highlighted. Further results showed the low expressions of MCM 2-7, RRM1, etc. perhaps led to abnormal DNA replication and damage repair, and the significantly different expressions of PARP2, CCs1, CCs3, etc. implied that the accumulated DNA double-stranded breaks were failed to be repaired and the cell cycle was arrested at G2/M in SL ovules, potentially resulting in the occurrence of apoptosis. Moreover, the deficiency of ETF-QO might hinder FNMM. Consequently, FNMM stopped and ovule aborted in SL ovules. Our results suggested a selective regulatory mechanism led to FNMM half-stop and ovule abortion in P. tabuliformis and these insights could be exploited to investigate the molecular regulations of ovule development in woody gymnosperms.
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Affiliation(s)
- Min Zhang
- College of Biological Sciences and Technology, Beijing Forestry University, No. 35, Tsinghua East Road, Beijing, 100083, China
| | - Wenhai Li
- College of Biological Sciences and Technology, Beijing Forestry University, No. 35, Tsinghua East Road, Beijing, 100083, China
| | - Jun Feng
- College of Biological Sciences and Technology, Beijing Forestry University, No. 35, Tsinghua East Road, Beijing, 100083, China
| | - Zaixin Gong
- College of Biological Sciences and Technology, Beijing Forestry University, No. 35, Tsinghua East Road, Beijing, 100083, China
| | - Yang Yao
- College of Biological Sciences and Technology, Beijing Forestry University, No. 35, Tsinghua East Road, Beijing, 100083, China
| | - Caixia Zheng
- College of Biological Sciences and Technology, Beijing Forestry University, No. 35, Tsinghua East Road, Beijing, 100083, China.
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21
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Acharya N, Khandagale P, Thakur S, Sahu JK, Utkalaja BG. Quaternary structural diversity in eukaryotic DNA polymerases: monomeric to multimeric form. Curr Genet 2020; 66:635-655. [PMID: 32236653 DOI: 10.1007/s00294-020-01071-1] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2020] [Revised: 03/13/2020] [Accepted: 03/24/2020] [Indexed: 12/14/2022]
Abstract
Sixteen eukaryotic DNA polymerases have been identified and studied so far. Based on the sequence similarity of the catalytic subunits of DNA polymerases, these have been classified into four A, B, X and Y families except PrimPol, which belongs to the AEP family. The quaternary structure of these polymerases also varies depending upon whether they are composed of one or more subunits. Therefore, in this review, we used a quaternary structure-based classification approach to group DNA polymerases as either monomeric or multimeric and highlighted functional significance of their accessory subunits. Additionally, we have briefly summarized various DNA polymerase discoveries from a historical perspective, emphasized unique catalytic mechanism of each DNA polymerase and highlighted recent advances in understanding their cellular functions.
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Affiliation(s)
- Narottam Acharya
- Laboratory of Genomic Instability and Diseases, Department of Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, 751023, India.
| | - Prashant Khandagale
- Laboratory of Genomic Instability and Diseases, Department of Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, 751023, India
| | - Shweta Thakur
- Laboratory of Genomic Instability and Diseases, Department of Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, 751023, India
| | - Jugal Kishor Sahu
- Laboratory of Genomic Instability and Diseases, Department of Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, 751023, India
| | - Bhabasha Gyanadeep Utkalaja
- Laboratory of Genomic Instability and Diseases, Department of Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, 751023, India
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22
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Bhowmik D, Jagadeesan R, Rai P, Nandi R, Gugan K, Kumar D. Evaluation of potential drugs against leishmaniasis targeting catalytic subunit of Leishmania donovani nuclear DNA primase using ligand based virtual screening, docking and molecular dynamics approaches. J Biomol Struct Dyn 2020; 39:1838-1852. [PMID: 32141397 DOI: 10.1080/07391102.2020.1739557] [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] [Indexed: 12/11/2022]
Abstract
Leishmania donovani, causes leishmaniasis, a global health trouble with around 89 different countries and its population under its risk. Replication initiation events have been instrumental in regulating the DNA duplication and as the small subunit of L. donovani nuclear DNA primase (Ld-PriS) inherits the catalytic site, it plays a vital role in DNA replication. In this study we have aimed Ld-PriS for the first time as a prospective target for the application of drug against Leishmania parasite. 3-D structures of Ld-PriS were built and ligand-based virtual screening was performed using hybrid similarity recognition techniques. Ligands from the ZINC database were used for the screening purposes based on known DNA primase inhibitor Sphingosine as a query. Top 150 ligands were taken into consideration for molecular docking against the query protein (Ld-PriS) using PyRx and iGEMDOCK softwares. Top five compounds with the best docking score were selected for pharmacokinetic investigation and molecular dynamic simulation. These top five screened inhibitors showed very poor binding affinity toward the catalytic subunit of human primase indicating their safety toward the host normal replication mechanism. The top five compounds showed good pharmacokinetic profiles and ADMET predictions revealed good absorption, solubility, permeability, uniform distribution, proper metabolism, minimal toxicity and good bioavailability. Simulation studies upto 50 ns revealed the three leads ZINC000009219046, ZINC000025998119 and ZINC000004677901 bind with Ld-PriS throughout the simulation and there were no huge variations in their backbone suggesting that these three may play as potential lead compounds for developing new drug against leishmaniasis.Communicated by Ramaswamy H. Sarma.
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Affiliation(s)
- Deep Bhowmik
- Department of Microbiology, Assam University, Silchar, Assam, India
| | - Rahul Jagadeesan
- CAS in Crystallography and Biophysics, Guindy Campus, University of Madras, Chennai, India
| | - Praveen Rai
- Department of Biotechnology, Central University of Rajasthan, Bandarsindri, India
| | - Rajat Nandi
- Department of Microbiology, Assam University, Silchar, Assam, India
| | - Kothandan Gugan
- CAS in Crystallography and Biophysics, Guindy Campus, University of Madras, Chennai, India
| | - Diwakar Kumar
- Department of Microbiology, Assam University, Silchar, Assam, India
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23
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Barton JK, Silva RMB, O'Brien E. Redox Chemistry in the Genome: Emergence of the [4Fe4S] Cofactor in Repair and Replication. Annu Rev Biochem 2019; 88:163-190. [PMID: 31220976 PMCID: PMC6590699 DOI: 10.1146/annurev-biochem-013118-110644] [Citation(s) in RCA: 36] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Many DNA-processing enzymes have been shown to contain a [4Fe4S] cluster, a common redox cofactor in biology. Using DNA electrochemistry, we find that binding of the DNA polyanion promotes a negative shift in [4Fe4S] cluster potential, which corresponds thermodynamically to a ∼500-fold increase in DNA-binding affinity for the oxidized [4Fe4S]3+ cluster versus the reduced [4Fe4S]2+ cluster. This redox switch can be activated from a distance using DNA charge transport (DNA CT) chemistry. DNA-processing proteins containing the [4Fe4S] cluster are enumerated, with possible roles for the redox switch highlighted. A model is described where repair proteins may signal one another using DNA-mediated charge transport as a first step in their search for lesions. The redox switch in eukaryotic DNA primases appears to regulate polymerase handoff, and in DNA polymerase δ, the redox switch provides a means to modulate replication in response to oxidative stress. We thus describe redox signaling interactions of DNA-processing [4Fe4S] enzymes, as well as the most interesting potential players to consider in delineating new DNA-mediated redox signaling networks.
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Affiliation(s)
- Jacqueline K Barton
- Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA;
| | - Rebekah M B Silva
- Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA;
| | - Elizabeth O'Brien
- Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA;
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Boudet J, Devillier JC, Wiegand T, Salmon L, Meier BH, Lipps G, Allain FHT. A Small Helical Bundle Prepares Primer Synthesis by Binding Two Nucleotides that Enhance Sequence-Specific Recognition of the DNA Template. Cell 2018; 176:154-166.e13. [PMID: 30595448 DOI: 10.1016/j.cell.2018.11.031] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2018] [Revised: 09/20/2018] [Accepted: 11/17/2018] [Indexed: 02/08/2023]
Abstract
Primases have a fundamental role in DNA replication. They synthesize a primer that is then extended by DNA polymerases. Archaeoeukaryotic primases require for synthesis a catalytic and an accessory domain, the exact contribution of the latter being unresolved. For the pRN1 archaeal primase, this domain is a 115-amino acid helix bundle domain (HBD). Our structural investigations of this small HBD by liquid- and solid-state nuclear magnetic resonance (NMR) revealed that only the HBD binds the DNA template. DNA binding becomes sequence-specific after a major allosteric change in the HBD, triggered by the binding of two nucleotide triphosphates. The spatial proximity of the two nucleotides and the DNA template in the quaternary structure of the HBD strongly suggests that this small domain brings together the substrates to prepare the first catalytic step of primer synthesis. This efficient mechanism is likely general for all archaeoeukaryotic primases.
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Affiliation(s)
- Julien Boudet
- Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zürich, 8093 Zürich, Switzerland.
| | - Jean-Christophe Devillier
- Institute of Chemistry and Bioanalytics, University of Applied Sciences Northwestern Switzerland, Hofackerstrasses 30, 4132 Muttenz, Switzerland
| | - Thomas Wiegand
- Physical Chemistry, ETH Zürich, 8093 Zürich, Switzerland
| | - Loic Salmon
- Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zürich, 8093 Zürich, Switzerland
| | - Beat H Meier
- Physical Chemistry, ETH Zürich, 8093 Zürich, Switzerland
| | - Georg Lipps
- Institute of Chemistry and Bioanalytics, University of Applied Sciences Northwestern Switzerland, Hofackerstrasses 30, 4132 Muttenz, Switzerland.
| | - Frédéric H-T Allain
- Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zürich, 8093 Zürich, Switzerland.
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25
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O'Brien E, Holt ME, Salay LE, Chazin WJ, Barton JK. Substrate Binding Regulates Redox Signaling in Human DNA Primase. J Am Chem Soc 2018; 140:17153-17162. [PMID: 30433774 DOI: 10.1021/jacs.8b09914] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Generation of daughter strands during DNA replication requires the action of DNA primase to synthesize an initial short RNA primer on the single-stranded DNA template. Primase is a heterodimeric enzyme containing two domains whose activity must be coordinated during primer synthesis: an RNA polymerase domain in the small subunit (p48) and a [4Fe4S] cluster-containing C-terminal domain of the large subunit (p58C). Here we examine the redox switching properties of the [4Fe4S] cluster in the full p48/p58 heterodimer using DNA electrochemistry. Unlike with isolated p58C, robust redox signaling in the primase heterodimer requires binding of both DNA and NTPs; NTP binding shifts the p48/p58 cluster redox potential into the physiological range, generating a signal near 160 mV vs NHE. Preloading of primase with NTPs enhances catalytic activity on primed DNA, suggesting that primase configurations promoting activity are more highly populated in the NTP-bound protein. We propose that p48/p58 binding of anionic DNA and NTPs affects the redox properties of the [4Fe4S] cluster; this electrostatic change is likely influenced by the alignment of primase subunits during activity because the configuration affects the [4Fe4S] cluster environment and coupling to DNA bases for redox signaling. Thus, both binding of polyanionic substrates and configurational dynamics appear to influence [4Fe4S] redox signaling properties. These results suggest that these factors should be considered generally in characterizing signaling networks of large, multisubunit DNA-processing [4Fe4S] enzymes.
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Affiliation(s)
- Elizabeth O'Brien
- Division of Chemistry and Chemical Engineering , California Institute of Technology , Pasadena , California 91125 , United States
| | - Marilyn E Holt
- Departments of Biochemistry and Chemistry, Center for Structural Biology , Vanderbilt University , Nashville , Tennessee 37240 , United States
| | - Lauren E Salay
- Departments of Biochemistry and Chemistry, Center for Structural Biology , Vanderbilt University , Nashville , Tennessee 37240 , United States
| | - Walter J Chazin
- Departments of Biochemistry and Chemistry, Center for Structural Biology , Vanderbilt University , Nashville , Tennessee 37240 , United States
| | - Jacqueline K Barton
- Division of Chemistry and Chemical Engineering , California Institute of Technology , Pasadena , California 91125 , United States
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Ilic S, Cohen S, Singh M, Tam B, Dayan A, Akabayov B. DnaG Primase-A Target for the Development of Novel Antibacterial Agents. Antibiotics (Basel) 2018; 7:E72. [PMID: 30104489 PMCID: PMC6163395 DOI: 10.3390/antibiotics7030072] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2018] [Revised: 08/06/2018] [Accepted: 08/09/2018] [Indexed: 12/21/2022] Open
Abstract
The bacterial primase-an essential component in the replisome-is a promising but underexploited target for novel antibiotic drugs. Bacterial primases have a markedly different structure than the human primase. Inhibition of primase activity is expected to selectively halt bacterial DNA replication. Evidence is growing that halting DNA replication has a bacteriocidal effect. Therefore, inhibitors of DNA primase could provide antibiotic agents. Compounds that inhibit bacterial DnaG primase have been developed using different approaches. In this paper, we provide an overview of the current literature on DNA primases as novel drug targets and the methods used to find their inhibitors. Although few inhibitors have been identified, there are still challenges to develop inhibitors that can efficiently halt DNA replication and may be applied in a clinical setting.
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Affiliation(s)
- Stefan Ilic
- Department of Chemistry, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel.
| | - Shira Cohen
- Department of Chemistry, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel.
| | - Meenakshi Singh
- Department of Chemistry, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel.
| | - Benjamin Tam
- Department of Chemistry, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel.
| | - Adi Dayan
- Department of Chemistry, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel.
| | - Barak Akabayov
- Department of Chemistry, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel.
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Primer synthesis by a eukaryotic-like archaeal primase is independent of its Fe-S cluster. Nat Commun 2017; 8:1718. [PMID: 29167441 PMCID: PMC5700102 DOI: 10.1038/s41467-017-01707-w] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2017] [Accepted: 10/09/2017] [Indexed: 01/11/2023] Open
Abstract
DNA replication depends on primase, the specialised polymerase responsible for synthesis of the RNA primers that are elongated by the replicative DNA polymerases. In eukaryotic and archaeal replication, primase is a heterodimer of two subunits, PriS and PriL. Recently, a third primase subunit named PriX was identified in the archaeon Sulfolobus solfataricus. PriX is essential for primer synthesis and is structurally related to the Fe–S cluster domain of eukaryotic PriL. Here we show that PriX contains a nucleotide-binding site required for primer synthesis, and demonstrate equivalence of nucleotide-binding residues in PriX with eukaryotic PriL residues that are known to be important for primer synthesis. A primase chimera, where PriX is fused to a truncated version of PriL lacking the Fe–S cluster domain retains wild-type levels of primer synthesis. Our evidence shows that PriX has replaced PriL as the subunit that endows primase with the unique ability to initiate nucleic acid synthesis. Importantly, our findings reveal that the Fe–S cluster is not required for primer synthesis. Primase is the specialised DNA-dependent RNA polymerase responsible for the initiation of DNA synthesis during DNA replication. Here the authors use a structural biology approach to identify the initiation site in the S. solfataricus PriSLX primase and to demonstrate that its Fe-S cluster is dispensable for primer synthesis.
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O'Brien E, Holt ME, Thompson MK, Salay LE, Ehlinger AC, Chazin WJ, Barton JK. The [4Fe4S] cluster of human DNA primase functions as a redox switch using DNA charge transport. Science 2017; 355:355/6327/eaag1789. [PMID: 28232525 DOI: 10.1126/science.aag1789] [Citation(s) in RCA: 98] [Impact Index Per Article: 12.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2016] [Accepted: 01/23/2017] [Indexed: 01/05/2023]
Abstract
DNA charge transport chemistry offers a means of long-range, rapid redox signaling. We demonstrate that the [4Fe4S] cluster in human DNA primase can make use of this chemistry to coordinate the first steps of DNA synthesis. Using DNA electrochemistry, we found that a change in oxidation state of the [4Fe4S] cluster acts as a switch for DNA binding. Single-atom mutations that inhibit this charge transfer hinder primase initiation without affecting primase structure or polymerization. Generating a single base mismatch in the growing primer duplex, which attenuates DNA charge transport, inhibits primer truncation. Thus, redox signaling by [4Fe4S] clusters using DNA charge transport regulates primase binding to DNA and illustrates chemistry that may efficiently drive substrate handoff between polymerases during DNA replication.
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Affiliation(s)
- Elizabeth O'Brien
- Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA
| | - Marilyn E Holt
- Departments of Biochemistry and Chemistry, Center for Structural Biology, Vanderbilt University, Nashville, TN 37235, USA
| | - Matthew K Thompson
- Departments of Biochemistry and Chemistry, Center for Structural Biology, Vanderbilt University, Nashville, TN 37235, USA
| | - Lauren E Salay
- Departments of Biochemistry and Chemistry, Center for Structural Biology, Vanderbilt University, Nashville, TN 37235, USA
| | - Aaron C Ehlinger
- Departments of Biochemistry and Chemistry, Center for Structural Biology, Vanderbilt University, Nashville, TN 37235, USA
| | - Walter J Chazin
- Departments of Biochemistry and Chemistry, Center for Structural Biology, Vanderbilt University, Nashville, TN 37235, USA.
| | - Jacqueline K Barton
- Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
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Zanello P. The competition between chemistry and biology in assembling iron–sulfur derivatives. Molecular structures and electrochemistry. Part V. {[Fe4S4](SCysγ)4} proteins. Coord Chem Rev 2017. [DOI: 10.1016/j.ccr.2016.10.003] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
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Liu B, Hu J, Wang J, Kong D. Direct Visualization of RNA-DNA Primer Removal from Okazaki Fragments Provides Support for Flap Cleavage and Exonucleolytic Pathways in Eukaryotic Cells. J Biol Chem 2017; 292:4777-4788. [PMID: 28159842 DOI: 10.1074/jbc.m116.758599] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2016] [Revised: 02/01/2017] [Indexed: 11/06/2022] Open
Abstract
During DNA replication in eukaryotic cells, short single-stranded DNA segments known as Okazaki fragments are first synthesized on the lagging strand. The Okazaki fragments originate from ∼35-nucleotide-long RNA-DNA primers. After Okazaki fragment synthesis, these primers must be removed to allow fragment joining into a continuous lagging strand. To date, the models of enzymatic machinery that removes the RNA-DNA primers have come almost exclusively from biochemical reconstitution studies and some genetic interaction assays, and there is little direct evidence to confirm these models. One obstacle to elucidating Okazaki fragment processing has been the lack of methods that can directly examine primer removal in vivo In this study, we developed an electron microscopy assay that can visualize nucleotide flap structures on DNA replication forks in fission yeast (Schizosaccharomyces pombe). With this assay, we first demonstrated the generation of flap structures during Okazaki fragment processing in vivo The mean and median lengths of the flaps in wild-type cells were ∼51 and ∼41 nucleotides, respectively. We also used yeast mutants to investigate the impact of deleting key DNA replication nucleases on these flap structures. Our results provided direct in vivo evidence for a previously proposed flap cleavage pathway and the critical function of Dna2 and Fen1 in cleaving these flaps. In addition, we found evidence for another previously proposed exonucleolytic pathway involving RNA-DNA primer digestion by exonucleases RNase H2 and Exo1. Taken together, our observations suggest a dual mechanism for Okazaki fragment maturation in lagging strand synthesis and establish a new strategy for interrogation of this fascinating process.
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Affiliation(s)
- Bochao Liu
- From the Peking-Tsinghua Center for Life Sciences, The National Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing 100871, China
| | - Jiazhi Hu
- From the Peking-Tsinghua Center for Life Sciences, The National Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing 100871, China
| | - Jingna Wang
- From the Peking-Tsinghua Center for Life Sciences, The National Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing 100871, China
| | - Daochun Kong
- From the Peking-Tsinghua Center for Life Sciences, The National Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing 100871, China
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Identification of DNA primase inhibitors via a combined fragment-based and virtual screening. Sci Rep 2016; 6:36322. [PMID: 27805033 PMCID: PMC5090872 DOI: 10.1038/srep36322] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2016] [Accepted: 10/14/2016] [Indexed: 11/12/2022] Open
Abstract
The structural differences between bacterial and human primases render the former an excellent target for drug design. Here we describe a technique for selecting small molecule inhibitors of the activity of T7 DNA primase, an ideal model for bacterial primases due to their common structural and functional features. Using NMR screening, fragment molecules that bind T7 primase were identified and then exploited in virtual filtration to select larger molecules from the ZINC database. The molecules were docked to the primase active site using the available primase crystal structure and ranked based on their predicted binding energies to identify the best candidates for functional and structural investigations. Biochemical assays revealed that some of the molecules inhibit T7 primase-dependent DNA replication. The binding mechanism was delineated via NMR spectroscopy. Our approach, which combines fragment based and virtual screening, is rapid and cost effective and can be applied to other targets.
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Coloma J, Johnson RE, Prakash L, Prakash S, Aggarwal AK. Human DNA polymerase α in binary complex with a DNA:DNA template-primer. Sci Rep 2016; 6:23784. [PMID: 27032819 PMCID: PMC4817131 DOI: 10.1038/srep23784] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2015] [Accepted: 03/14/2016] [Indexed: 11/26/2022] Open
Abstract
The Polα/primase complex assembles the short RNA-DNA fragments for priming of lagging and leading strand DNA replication in eukaryotes. As such, the Polα polymerase subunit encounters two types of substrates during primer synthesis: an RNA:DNA helix and a DNA:DNA helix. The engagement of the polymerase subunit with the DNA:DNA helix has been suggested as the of basis for primer termination in eukaryotes. However, there is no structural information on how the Polα polymerase subunit actually engages with a DNA:DNA helix during primer synthesis. We present here the first crystal structure of human Polα polymerase subunit in complex with a DNA:DNA helix. Unexpectedly, we find that portion of the DNA:DNA helix in contact with the polymerase is not in a B-form but in a hybrid A-B form. Almost all of the contacts observed previously with an RNA primer are preserved with a DNA primer – with the same set of polymerase residues tracking the sugar-phosphate backbone of the DNA or RNA primer. Thus, rather than loss of specific contacts, the free energy cost of distorting DNA from B- to hybrid A-B form may augur the termination of primer synthesis in eukaryotes.
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Affiliation(s)
- Javier Coloma
- Department of Structural &Chemical Biology, Mount Sinai School of Medicine, Box 1677, 1425 Madison Avenue, New York, NY 10029, USA
| | - Robert E Johnson
- Department of Biochemistry and Molecular Biology, 301 University Boulevard, University of Texas Medical Branch, Galveston, TX 77755-1061, USA
| | - Louise Prakash
- Department of Biochemistry and Molecular Biology, 301 University Boulevard, University of Texas Medical Branch, Galveston, TX 77755-1061, USA
| | - Satya Prakash
- Department of Biochemistry and Molecular Biology, 301 University Boulevard, University of Texas Medical Branch, Galveston, TX 77755-1061, USA
| | - Aneel K Aggarwal
- Department of Structural &Chemical Biology, Mount Sinai School of Medicine, Box 1677, 1425 Madison Avenue, New York, NY 10029, USA
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Boudet J, Devillier JC, Allain FHT, Lipps G. Structures to complement the archaeo-eukaryotic primases catalytic cycle description: What's next? Comput Struct Biotechnol J 2015; 13:339-51. [PMID: 25987967 PMCID: PMC4434180 DOI: 10.1016/j.csbj.2015.04.006] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2015] [Revised: 04/20/2015] [Accepted: 04/24/2015] [Indexed: 12/25/2022] Open
Abstract
DNA replication is a crucial stage in the transfer of genetic information from parent to daughter cells. This mechanism involves multiple proteins with one key player being the primase. Primases are single-stranded DNA dependent RNA polymerases. On the leading strand, they synthesize the primer once allowing DNA elongation while on the lagging strand primers are generated repeatedly (Okazaki fragments). Primases have the unique ability to create the first phosphodiester bond yielding a dinucleotide which is initially elongated by primases and then by DNA polymerases. Primase activity has been studied in the last decades but the detailed molecular steps explaining some unique features remain unclear. High-resolution structures of free and bound primases domains have brought significant insights in the understanding of the primase reaction cycle. Here, we give a short review of the structural work conducted in the field of archaeo-eukaryotic primases and we underline the missing “pictures” of the active forms of the enzyme which are of major interest. We organized our analysis with respect to the progression through the catalytic pathway.
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Affiliation(s)
- Julien Boudet
- Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zürich, 8093 Zürich, Switzerland
- Corresponding author. Tel.: + 41 446330723; fax: + 41 446331294.
| | - Jean-Christophe Devillier
- University of Applied Sciences and Arts Northwestern Switzerland, Gründenstrasse 40, 4132 Muttenz, Switzerland
| | - Frédéric H.-T. Allain
- Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zürich, 8093 Zürich, Switzerland
| | - Georg Lipps
- University of Applied Sciences and Arts Northwestern Switzerland, Gründenstrasse 40, 4132 Muttenz, Switzerland
- Corresponding author. Tel.: + 41 614674301; fax: + 41 614674701.
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Suwa Y, Gu J, Baranovskiy AG, Babayeva ND, Pavlov YI, Tahirov TH. Crystal Structure of the Human Pol α B Subunit in Complex with the C-terminal Domain of the Catalytic Subunit. J Biol Chem 2015; 290:14328-37. [PMID: 25847248 DOI: 10.1074/jbc.m115.649954] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2015] [Indexed: 11/06/2022] Open
Abstract
In eukaryotic DNA replication, short RNA-DNA hybrid primers synthesized by primase-DNA polymerase α (Prim-Pol α) are needed to start DNA replication by the replicative DNA polymerases, Pol δ and Pol ϵ. The C terminus of the Pol α catalytic subunit (p180C) in complex with the B subunit (p70) regulates the RNA priming and DNA polymerizing activities of Prim-Pol α. It tethers Pol α and primase, facilitating RNA primer handover from primase to Pol α. To understand these regulatory mechanisms and to reveal the details of human Pol α organization, we determined the crystal structure of p70 in complex with p180C. The structured portion of p70 includes a phosphodiesterase (PDE) domain and an oligonucleotide/oligosaccharide binding (OB) domain. The N-terminal domain and the linker connecting it to the PDE domain are disordered in the reported crystal structure. The p180C adopts an elongated asymmetric saddle shape, with a three-helix bundle in the middle and zinc-binding modules (Zn1 and Zn2) on each side. The extensive p180C-p70 interactions involve 20 hydrogen bonds and a number of hydrophobic interactions resulting in an extended buried surface of 4080 Å(2). Importantly, in the structure of the p180C-p70 complex with full-length p70, the residues from the N-terminal to the OB domain contribute to interactions with p180C. The comparative structural analysis revealed both the conserved features and the differences between the human and yeast Pol α complexes.
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Affiliation(s)
- Yoshiaki Suwa
- From the Eppley Institute for Research in Cancer and Allied Diseases and
| | - Jianyou Gu
- From the Eppley Institute for Research in Cancer and Allied Diseases and
| | | | - Nigar D Babayeva
- From the Eppley Institute for Research in Cancer and Allied Diseases and
| | - Youri I Pavlov
- From the Eppley Institute for Research in Cancer and Allied Diseases and the Departments of Biochemistry and Molecular Biology and Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68198
| | - Tahir H Tahirov
- From the Eppley Institute for Research in Cancer and Allied Diseases and
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Liu L, Huang M. Essential role of the iron-sulfur cluster binding domain of the primase regulatory subunit Pri2 in DNA replication initiation. Protein Cell 2015; 6:194-210. [PMID: 25645023 PMCID: PMC4348247 DOI: 10.1007/s13238-015-0134-8] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2014] [Accepted: 01/04/2015] [Indexed: 11/26/2022] Open
Abstract
DNA primase catalyzes de novo synthesis of a short RNA primer that is further extended by replicative DNA polymerases during initiation of DNA replication. The eukaryotic primase is a heterodimeric enzyme comprising a catalytic subunit Pri1 and a regulatory subunit Pri2. Pri2 is responsible for facilitating optimal RNA primer synthesis by Pri1 and mediating interaction between Pri1 and DNA polymerase α for transition from RNA synthesis to DNA elongation. All eukaryotic Pri2 proteins contain a conserved C-terminal iron-sulfur (Fe-S) cluster-binding domain that is critical for primase catalytic activity in vitro. Here we show that mutations at conserved cysteine ligands for the Pri2 Fe-S cluster markedly decrease the protein stability, thereby causing S phase arrest at the restrictive temperature. Furthermore, Pri2 cysteine mutants are defective in loading of the entire DNA pol α-primase complex onto early replication origins resulting in defective initiation. Importantly, assembly of the Fe-S cluster in Pri2 is impaired not only by mutations at the conserved cysteine ligands but also by increased oxidative stress in the sod1Δ mutant lacking the Cu/Zn superoxide dismutase. Together these findings highlight the critical role of Pri2's Fe-S cluster domain in replication initiation in vivo and suggest a molecular basis for how DNA replication can be influenced by changes in cellular redox state.
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Affiliation(s)
- Lili Liu
- Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213 USA
- Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO 80045 USA
| | - Mingxia Huang
- Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO 80045 USA
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You Z, De Falco M, Kamada K, Pisani FM, Masai H. The mini-chromosome maintenance (Mcm) complexes interact with DNA polymerase α-primase and stimulate its ability to synthesize RNA primers. PLoS One 2013; 8:e72408. [PMID: 23977294 PMCID: PMC3748026 DOI: 10.1371/journal.pone.0072408] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2012] [Accepted: 07/16/2013] [Indexed: 01/14/2023] Open
Abstract
The Mini-chromosome maintenance (Mcm) proteins are essential as central components for the DNA unwinding machinery during eukaryotic DNA replication. DNA primase activity is required at the DNA replication fork to synthesize short RNA primers for DNA chain elongation on the lagging strand. Although direct physical and functional interactions between helicase and primase have been known in many prokaryotic and viral systems, potential interactions between helicase and primase have not been explored in eukaryotes. Using purified Mcm and DNA primase complexes, a direct physical interaction is detected in pull-down assays between the Mcm2∼7 complex and the hetero-dimeric DNA primase composed of the p48 and p58 subunits. The Mcm4/6/7 complex co-sediments with the primase and the DNA polymerase α-primase complex in glycerol gradient centrifugation and forms a Mcm4/6/7-primase-DNA ternary complex in gel-shift assays. Both the Mcm4/6/7 and Mcm2∼7 complexes stimulate RNA primer synthesis by DNA primase in vitro. However, primase inhibits the Mcm4/6/7 helicase activity and this inhibition is abolished by the addition of competitor DNA. In contrast, the ATP hydrolysis activity of Mcm4/6/7 complex is not affected by primase. Mcm and primase proteins mutually stimulate their DNA-binding activities. Our findings indicate that a direct physical interaction between primase and Mcm proteins may facilitate priming reaction by the former protein, suggesting that efficient DNA synthesis through helicase-primase interactions may be conserved in eukaryotic chromosomes.
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Affiliation(s)
- Zhiying You
- Genome Dynamics Project, Department of Genome Medicine, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan.
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Pei Y, Fu W, Yang E, Shen A, Chen YC, Gong H, Chen J, Huang J, Xiao G, Liu F. A Hsp40 chaperone protein interacts with and modulates the cellular distribution of the primase protein of human cytomegalovirus. PLoS Pathog 2012; 8:e1002968. [PMID: 23133382 PMCID: PMC3486897 DOI: 10.1371/journal.ppat.1002968] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2012] [Accepted: 08/28/2012] [Indexed: 12/13/2022] Open
Abstract
Genomic DNA replication is a universal and essential process for all herpesvirus including human cytomegalovirus (HCMV). HCMV UL70 protein, which is believed to encode the primase activity of the viral DNA replication machinery and is highly conserved among herpesviruses, needs to be localized in the nucleus, the site of viral DNA synthesis. No host factors that facilitate the nuclear import of UL70 have been reported. In this study, we provided the first direct evidence that UL70 specifically interacts with a highly conserved and ubiquitously expressed member of the heat shock protein Hsp40/DNAJ family, DNAJB6, which is expressed as two isoforms, a and b, as a result of alternative splicing. The interaction of UL70 with a common region of DNAJB6a and b was identified by both a two hybrid screen in yeast and coimmunoprecipitation in human cells. In transfected cells, UL70 was primarily co-localized with DNAJB6a in the nuclei and with DNAJB6b in the cytoplasm, respectively. The nuclear import of UL70 was increased in cells in which DNAJB6a was up-regulated or DNAJB6b was down-regulated, and was reduced in cells in which DNAJB6a was down-regulated or DNAJB6b was up-regulated. Furthermore, the level of viral DNA synthesis and progeny production was increased in cells in which DNAJB6a was up-regulated or DNAJB6b was down-regulated, and was reduced in cells in which DNAJB6a was down-regulated or DNAJB6b was up-regulated. Thus, DNAJB6a and b appear to enhance the nuclear import and cytoplasmic accumulation of UL70, respectively. Our results also suggest that the relative expression levels of DNAJB6 isoforms may play a key role in regulating the cellular localization of UL70, leading to modulation of HCMV DNA synthesis and lytic infection. Genomic DNA replication is highly conserved across all herpesviruses including human cytomegalovirus (HCMV) and is the target for most of the current FDA-approved anti-herpes therapeutic agents. Little is known about how UL70, which is believed to encode the primase activity of the viral DNA replication machinery and is essential for genomic replication, is imported to the nuclei, the site of viral DNA synthesis. In this study, we demonstrated that the HCMV primase interacts with a highly conserved and ubiquitously expressed chaperone protein DNAJB6 that belongs to the heat shock protein 40 (Hsp40) family. As a result of alternative splicing, DNAJB6 is expressed as two isoforms, a and b. While DNAJB6b promotes cytoplasmic accumulation of the viral primase, DNAJB6a enhances its nuclear distribution, representing the first example of a cellular factor involved in facilitating nuclear import of a herpesvirus primase. Our study suggests that the relative expression level of DNAJB6 isoforms may represent a novel mechanism for modulating HCMV lytic replication by regulating the cellular localization of the viral primase. Furthermore, our results raise the possibility of developing new strategies for treating herpesvirus replication by modulating the cellular distribution of the primase with altered expression of a cellular protein.
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Affiliation(s)
- Yonggang Pei
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, China
| | - Wenmin Fu
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, China
| | - Ed Yang
- Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, California, United States of America
| | - Ao Shen
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, China
- Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, California, United States of America
| | - Yuan-Chuan Chen
- Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, California, United States of America
| | - Hao Gong
- Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, California, United States of America
| | - Jun Chen
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, China
| | - Jun Huang
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, China
| | - Gengfu Xiao
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, China
| | - Fenyong Liu
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, China
- Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, California, United States of America
- * E-mail:
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Gloor JW, Balakrishnan L, Campbell JL, Bambara RA. Biochemical analyses indicate that binding and cleavage specificities define the ordered processing of human Okazaki fragments by Dna2 and FEN1. Nucleic Acids Res 2012; 40:6774-86. [PMID: 22570407 PMCID: PMC3413157 DOI: 10.1093/nar/gks388] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
In eukaryotic Okazaki fragment processing, the RNA primer is displaced into a single-stranded flap prior to removal. Evidence suggests that some flaps become long before they are cleaved, and that this cleavage involves the sequential action of two nucleases. Strand displacement characteristics of the polymerase show that a short gap precedes the flap during synthesis. Using biochemical techniques, binding and cleavage assays presented here indicate that when the flap is ∼30 nt long the nuclease Dna2 can bind with high affinity to the flap and downstream double strand and begin cleavage. When the polymerase idles or dissociates the Dna2 can reorient for additional contacts with the upstream primer region, allowing the nuclease to remain stably bound as the flap is further shortened. The DNA can then equilibrate to a double flap that can bind Dna2 and flap endonuclease (FEN1) simultaneously. When Dna2 shortens the flap even more, FEN1 can displace the Dna2 and cleave at the flap base to make a nick for ligation.
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Affiliation(s)
- Jason W Gloor
- Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA
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Vilenchik LZ, Sheth PR, Chuang CC, Le HV. Affinity characterization-mass spectrometry methodology for quantitative analyses of small molecule protein binding in solution. Anal Biochem 2011; 418:10-8. [PMID: 21726521 DOI: 10.1016/j.ab.2011.06.007] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2011] [Revised: 06/03/2011] [Accepted: 06/06/2011] [Indexed: 10/18/2022]
Abstract
Affinity characterization by mass spectrometry (AC-MS) is a novel LC-MS methodology for quantitative determination of small molecule ligand binding to macromolecules. Its most distinguishing feature is the direct determination of all three concentration terms of the equilibrium binding equation, i.e., (M), (L), and (ML), which denote the macromolecule, ligand, and the corresponding complex, respectively. Although it is possible to obtain the dissociation constant from a single mixing experiment, saturation analyses are still valuable for assessing the overall binding phenomenon based on an established formalism. In addition to providing the prerequisite dissociation constant and binding stoichiometry, the technique also provides valuable information about the actual solubility of both macromolecule and ligand upon dilution and mixing in binding buffers. The dissociation constants and binding mode for interactions of DNA primase and thymidylate synthetase (TS) with high and low affinity small molecule ligands were obtained using the AC-MS method. The data were consistent with the expected affinity of TS for these ligands based on dissociation constants determined by alternative thermal-denaturation techniques: TdF or TdCD, and also consistent enzyme inhibition constants reported in the literature. The validity of AC-MS was likewise extended to a larger set of soluble protein-ligand systems. It was established as a valuable resource for counter screen and structure-activity relationship studies in drug discovery, especially when other classical techniques could only provide ambiguous results.
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Affiliation(s)
- Lev Z Vilenchik
- Protein Science, Merck Research Laboratories, Cambridge, MA 02141, USA
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40
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Human cytomegalovirus primase UL70 specifically interacts with cellular factor Snapin. J Virol 2011; 85:11732-41. [PMID: 21917956 DOI: 10.1128/jvi.05357-11] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Genomic DNA synthesis is a universally conserved process for all herpesviruses, including human cytomegalovirus (HCMV). HCMV UL70 is believed to encode the primase of the DNA replication machinery, a function which requires localization in the nucleus, the site of viral DNA synthesis. No host factors that interact with UL70 have been reported. In this study, we provide the first direct evidence that UL70 specifically interacts with Snapin, a human protein that is predominantly localized in the cytoplasm and is associated with cellular vesicles. The interaction between UL70 and Snapin was identified in both the two-hybrid screen in yeast and coimmunoprecipitation in human cells. The nuclear import of UL70 was decreased in cells overexpressing Snapin and increased in cells in which the expression of Snapin was downregulated with anti-Snapin small interfering RNA (siRNA) molecules, respectively. Furthermore, viral DNA synthesis and progeny production were decreased in cells overexpressing Snapin and increased in the anti-Snapin siRNA-treated cells, respectively. In contrast, no significant difference in the nuclear level of UL70, viral DNA synthesis, and progeny production was found among the parental cells and cells that either expressed a control empty vector or were treated with control siRNA molecules that did not recognize any viral or cellular transcripts. Our results suggest that Snapin may play a key role in regulating the cellular localization of UL70 in HCMV, leading to modulation of viral DNA synthesis and progeny production.
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A model for dynamics of primer extension by eukaryotic DNA primase. EUROPEAN BIOPHYSICS JOURNAL: EBJ 2011; 40:1157-65. [PMID: 21898172 DOI: 10.1007/s00249-011-0746-8] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/10/2011] [Accepted: 08/18/2011] [Indexed: 12/23/2022]
Abstract
A mathematical model is proposed for processive primer extension by eukaryotic DNA primase. The model uses available experimental data to predict rate constants for the dynamic behavior of primase activity as a function of NTP concentration. The model also predicts some data such as the binding affinities of the primase for the DNA template and for the RNA primer.
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Lund TJ, Cavanaugh NA, Joubert N, Urban M, Patro JN, Hocek M, Kuchta RD. B family DNA polymerases asymmetrically recognize pyrimidines and purines. Biochemistry 2011; 50:7243-50. [PMID: 21761848 DOI: 10.1021/bi2006916] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
We utilized a series of pyrimidine analogues modified at O(2), N-3, and N(4)/O(4) to determine if two B family DNA polymerases, human DNA polymerase α and herpes simplex virus I DNA polymerase, choose whether to polymerize pyrimidine dNTPs using the same mechanisms they use for purine dNTPs. Removing O(2) of a pyrimidine dNTP vastly decreased the level of incorporation by these enzymes and also compromised fidelity in the case of C analogues, while removing O(2) from the templating base had more modest effects. Removing the Watson-Crick hydrogen bonding groups of N-3 and N(4)/O(4) greatly impaired polymerization, both of the resulting dNTP analogues and of natural dNTPs opposite these pyrimidine analogues when present in the template strand. Thus, the Watson-Crick hydrogen bonding groups of a pyrimidine clearly play an important role in enhancing correct dNTP polymerization but are not essential for preventing misincorporation. These studies also indicate that DNA polymerases recognize bases extremely asymmetrically, both in terms of whether they are a purine or pyrimidine and whether they are in the template or are the incoming dNTP. The mechanistic implications of these results with regard to how polymerases discriminate between right and wrong dNTPs are discussed.
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Affiliation(s)
- Travis J Lund
- Department of Chemistry and Biochemistry, University of Colorado, UCB 215, Boulder, Colorado 80309, USA
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Balakrishnan L, Bambara RA. Eukaryotic lagging strand DNA replication employs a multi-pathway mechanism that protects genome integrity. J Biol Chem 2010; 286:6865-70. [PMID: 21177245 DOI: 10.1074/jbc.r110.209502] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
In eukaryotic nuclear DNA replication, one strand of DNA is synthesized continuously, but the other is made as Okazaki fragments that are later joined. Discontinuous synthesis is inherently more complex, and fragmented intermediates create risks for disruptions of genome integrity. Genetic analyses and biochemical reconstitutions indicate that several parallel pathways evolved to ensure that the fragments are made and joined with integrity. An RNA primer is removed from each fragment before joining by a process involving polymerase-dependent displacement into a single-stranded flap. Evidence in vitro suggests that, with most fragments, short flaps are displaced and efficiently cleaved. Some flaps can become long, but these are also removed to allow joining. Rarely, a flap can form structure, necessitating displacement of the entire fragment. There is now evidence that post-translational protein modification regulates the flow through the pathways to favor protection of genomic information in regions of actively transcribed chromatin.
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Affiliation(s)
- Lata Balakrishnan
- Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA
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Balakrishnan L, Polaczek P, Pokharel S, Campbell JL, Bambara RA. Dna2 exhibits a unique strand end-dependent helicase function. J Biol Chem 2010; 285:38861-8. [PMID: 20929864 DOI: 10.1074/jbc.m110.165191] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Dna2 endonuclease/helicase participates in eukaryotic DNA transactions including cleavage of long flaps generated during Okazaki fragment processing. Its unusual substrate interaction consists of recognition and binding of the flap base, then threading over the 5'-end of the flap, and cleaving periodically to produce a terminal product ∼5 nt in length. Blocking the 5'-end prevents cleavage. The Dna2 ATP-driven 5' to 3' DNA helicase function promotes motion of Dna2 on the flap, presumably aiding its nuclease function. Here we demonstrate using two different nuclease-dead Dna2 mutants that on substrates simulating Okazaki fragments, Dna2 must thread onto an unblocked 5' flap to display helicase activity. This requirement is maintained on substrates with single-stranded regions thousands of nucleotides in length. To our knowledge this is the first description of a eukaryotic helicase that cannot load onto its tracking strand internally but instead must enter from the end. Biologically, the loading requirement likely helps the helicase to coordinate with the Dna2 nuclease function to prevent creation of undesirably long flaps during DNA transactions.
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Affiliation(s)
- Lata Balakrishnan
- Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA
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Insights into eukaryotic DNA priming from the structure and functional interactions of the 4Fe-4S cluster domain of human DNA primase. Proc Natl Acad Sci U S A 2010; 107:13684-9. [PMID: 20643958 DOI: 10.1073/pnas.1002009107] [Citation(s) in RCA: 75] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
DNA replication requires priming of DNA templates by enzymes known as primases. Although DNA primase structures are available from archaea and bacteria, the mechanism of DNA priming in higher eukaryotes remains poorly understood in large part due to the absence of the structure of the unique, highly conserved C-terminal regulatory domain of the large subunit (p58C). Here, we present the structure of this domain determined to 1.7-A resolution by X-ray crystallography. The p58C structure reveals a novel arrangement of an evolutionarily conserved 4Fe-4S cluster buried deeply within the protein core and is not similar to any known protein structure. Analysis of the binding of DNA to p58C by fluorescence anisotropy measurements revealed a strong preference for ss/dsDNA junction substrates. This approach was combined with site-directed mutagenesis to confirm that the binding of DNA occurs to a distinctively basic surface on p58C. A specific interaction of p58C with the C-terminal domain of the intermediate subunit of replication protein A (RPA32C) was identified and characterized by isothermal titration calorimetry and NMR. Restraints from NMR experiments were used to drive computational docking of the two domains and generate a model of the p58C-RPA32C complex. Together, our results explain functional defects in human DNA primase mutants and provide insights into primosome loading on RPA-coated ssDNA and regulation of primase activity.
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Sauguet L, Klinge S, Perera RL, Maman JD, Pellegrini L. Shared active site architecture between the large subunit of eukaryotic primase and DNA photolyase. PLoS One 2010; 5:e10083. [PMID: 20404922 PMCID: PMC2852410 DOI: 10.1371/journal.pone.0010083] [Citation(s) in RCA: 62] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2010] [Accepted: 03/17/2010] [Indexed: 11/19/2022] Open
Abstract
BACKGROUND DNA synthesis during replication relies on RNA primers synthesised by the primase, a specialised DNA-dependent RNA polymerase that can initiate nucleic acid synthesis de novo. In archaeal and eukaryotic organisms, the primase is a heterodimeric enzyme resulting from the constitutive association of a small (PriS) and large (PriL) subunit. The ability of the primase to initiate synthesis of an RNA primer depends on a conserved Fe-S domain at the C-terminus of PriL (PriL-CTD). However, the critical role of the PriL-CTD in the catalytic mechanism of initiation is not understood. METHODOLOGY/PRINCIPAL FINDINGS Here we report the crystal structure of the yeast PriL-CTD at 1.55 A resolution. The structure reveals that the PriL-CTD folds in two largely independent alpha-helical domains joined at their interface by a [4Fe-4S] cluster. The larger N-terminal domain represents the most conserved portion of the PriL-CTD, whereas the smaller C-terminal domain is largely absent in archaeal PriL. Unexpectedly, the N-terminal domain reveals a striking structural similarity with the active site region of the DNA photolyase/cryptochrome family of flavoproteins. The region of similarity includes PriL-CTD residues that are known to be essential for initiation of RNA primer synthesis by the primase. CONCLUSION/SIGNIFICANCE Our study reports the first crystallographic model of the conserved Fe-S domain of the archaeal/eukaryotic primase. The structural comparison with a cryptochrome protein bound to flavin adenine dinucleotide and single-stranded DNA provides important insight into the mechanism of RNA primer synthesis by the primase.
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Affiliation(s)
- Ludovic Sauguet
- Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom
| | - Sebastian Klinge
- Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom
| | - Rajika L. Perera
- Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom
| | - Joseph D. Maman
- Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom
| | - Luca Pellegrini
- Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom
- * E-mail:
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Balakrishnan L, Gloor JW, Bambara RA. Reconstitution of eukaryotic lagging strand DNA replication. Methods 2010; 51:347-57. [PMID: 20178844 DOI: 10.1016/j.ymeth.2010.02.017] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2009] [Revised: 02/15/2010] [Accepted: 02/17/2010] [Indexed: 11/17/2022] Open
Abstract
Eukaryotic DNA replication is a complex process requiring the proper functioning of a multitude of proteins to create error-free daughter DNA strands and maintain genome integrity. Even though synthesis and joining of Okazaki fragments on the lagging strand involves only half the DNA in the nucleus, the complexity associated with processing these fragments is about twice that needed for leading strand synthesis. Flap endonuclease 1 (FEN1) is the central component of the Okazaki fragment maturation pathway. FEN1 cleaves flaps that are displaced by DNA polymerase delta (pol delta), to create a nick that is effectively joined by DNA ligase I. The Pif1 helicase and Dna2 helicase/nuclease contribute to the maturation process by elongating the flap displaced by pol delta. Though the reason for generating long flaps is still a matter of debate, genetic studies have shown that Dna2 and Pif1 are both important components of DNA replication. Our current knowledge of the exact enzymatic steps that govern Okazaki fragment maturation has heavily derived from reconstitution reactions in vitro, which have augmented genetic information, to yield current mechanistic models. In this review, we describe both the design of specific DNA substrates that simulate intermediates of fragment maturation and protocols for reconstituting partial and complete lagging strand replication.
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Affiliation(s)
- Lata Balakrishnan
- Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA
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Urban M, Joubert N, Hocek M, Kuchta RD. Mechanisms by which human DNA primase chooses to polymerize a nucleoside triphosphate. Biochemistry 2010; 49:727-35. [PMID: 20030400 PMCID: PMC2847881 DOI: 10.1021/bi9019516] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Human DNA primase synthesizes short RNA primers that DNA polymerase alpha then elongates during the initiation of all new DNA strands. Even though primase misincorporates NTPs at a relatively high frequency, this likely does not impact the final DNA product since the RNA primer is replaced with DNA. We used an extensive series of purine and pyrimidine analogues to provide further insights into the mechanism by which primase chooses whether or not to polymerize a NTP. Primase readily polymerized a size-expanded cytosine analogue, 1,3-diaza-2-oxophenothiazine NTP, across from a templating G but not across from A. The enzyme did not efficiently polymerize NTPs incapable of forming two Watson-Crick hydrogen bonds with the templating base with the exception of UTP opposite purine deoxyribonucleoside. Likewise, primase did not generate base pairs between two nucleotides with altered Watson-Crick hydrogen-bonding patterns. Examining the mechanism of NTP polymerization revealed that human primase can misincorporate NTPs via both template misreading and a primer-template slippage mechanism. Together, these data demonstrate that human primase strongly depends on Watson-Crick hydrogen bonds for efficient nucleotide polymerization, much more so than the mechanistically related herpes primase, and provide insights into the potential roles of primer-template stability and base tautomerization during misincorporation.
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Affiliation(s)
- Milan Urban
- Department of Chemistry and Biochemistry, University of Colorado, UCB 215, Boulder, CO 80309 and Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Gilead Sciences & IOCB Research Center, Flemingovo nam. 2, CZ-16610 Prague 6, Czech Republic
| | | | | | - Robert D. Kuchta
- Department of Chemistry and Biochemistry, University of Colorado, UCB 215, Boulder, CO 80309 and Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Gilead Sciences & IOCB Research Center, Flemingovo nam. 2, CZ-16610 Prague 6, Czech Republic
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Broderick S, Rehmet K, Concannon C, Nasheuer HP. Eukaryotic single-stranded DNA binding proteins: central factors in genome stability. Subcell Biochem 2010; 50:143-163. [PMID: 20012581 DOI: 10.1007/978-90-481-3471-7_8] [Citation(s) in RCA: 58] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/28/2023]
Abstract
The single-stranded DNA binding proteins (SSBs) are required to maintain the integrity of the genome in all organisms. Replication protein A (RPA) is a nuclear SSB protein found in all eukaryotes and is required for multiple processes in DNA metabolism such as DNA replication, DNA repair, DNA recombination, telomere maintenance and DNA damage signalling. RPA is a heterotrimeric complex, binds ssDNA with high affinity, and interacts specifically with multiple proteins to fulfil its function in eukaryotes. RPA is phosphorylated in a cell cycle and DNA damage-dependent manner with evidence suggesting that phosphorylation has an important function in modulating the cellular DNA damage response. Considering the DNA-binding properties of RPA a mechanism of "molecular counting" to initiate DNA damage-dependent signalling is discussed. Recently a human homologue to the RPA2 subunit, called RPA4, was discovered and RPA4 can substitute for RPA2 in the RPA complex resulting in an "alternative" RPA (aRPA), which can bind to ssDNA with similar affinity as canonical RPA. Additional human SSBs, hSSB1 and hSSB2, were recently identified, with hSSB1 being localized in the nucleus and having implications in DNA repair. Mitochondrial SSBs (mtSSBs) have been found in all eukaryotes studied. mtSSBs are related to prokaryotic SSBs and essential to main the genome stability in eukaryotic mitochondria. Recently human mtSSB was identified as a novel binding partner of p53 and that it is able to stimulate the intrinsic exonuclease activity of p53. These findings and recent results associated with mutations in RPA suggest a link of SSBs to cancer.
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Affiliation(s)
- Sandra Broderick
- Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland, Galway, Galway, Ireland
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