XB-ART-60636
Nat Struct Mol Biol
2024 May 01;315:777-790. doi: 10.1038/s41594-024-01227-4.
Show Gene links
Show Anatomy links
A mechanistic model of primer synthesis from catalytic structures of DNA polymerase α-primase.
Mullins EA
,
Salay LE
,
Durie CL
,
Bradley NP
,
Jackman JE
,
Ohi MD
,
Chazin WJ
,
Eichman BF
.
Abstract
The mechanism by which polymerase α-primase (polα-primase) synthesizes chimeric RNA-DNA primers of defined length and composition, necessary for replication fidelity and genome stability, is unknown. Here, we report cryo-EM structures of Xenopus laevis polα-primase in complex with primed templates representing various stages of DNA synthesis. Our data show how interaction of the primase regulatory subunit with the primer 5' end facilitates handoff of the primer to polα and increases polα processivity, thereby regulating both RNA and DNA composition. The structures detail how flexibility within the heterotetramer enables synthesis across two active sites and provide evidence that termination of DNA synthesis is facilitated by reduction of polα and primase affinities for the varied conformations along the chimeric primer-template duplex. Together, these findings elucidate a critical catalytic step in replication initiation and provide a comprehensive model for primer synthesis by polα-primase.
PubMed ID: 38491139
PMC ID: PMC11102853
Article link: Nat Struct Mol Biol
Grant support: [+]
Species referenced: Xenopus laevis
References [+] :
Abad,
A role for tRNA(His) guanylyltransferase (Thg1)-like proteins from Dictyostelium discoideum in mitochondrial 5'-tRNA editing.
2011, Pubmed
Abad, A role for tRNA(His) guanylyltransferase (Thg1)-like proteins from Dictyostelium discoideum in mitochondrial 5'-tRNA editing. 2011, Pubmed
Abad, Template-dependent 3'-5' nucleotide addition is a shared feature of tRNAHis guanylyltransferase enzymes from multiple domains of life. 2010, Pubmed
Adams, PHENIX: a comprehensive Python-based system for macromolecular structure solution. 2010, Pubmed
Agarkar, Crystal structure of the C-terminal domain of human DNA primase large subunit: implications for the mechanism of the primase-polymerase α switch. 2011, Pubmed
Arezi, Interactions of DNA with human DNA primase monitored with photoactivatable cross-linking agents: implications for the role of the p58 subunit. 1999, Pubmed
Attali, Structural Mechanisms for Replicating DNA in Eukaryotes. 2021, Pubmed
Baranovskiy, Insight into the Human DNA Primase Interaction with Template-Primer. 2016, Pubmed
Baranovskiy, Crystal structure of the human primase. 2015, Pubmed
Baranovskiy, Mechanism of Concerted RNA-DNA Primer Synthesis by the Human Primosome. 2016, Pubmed
Baranovskiy, Insight into RNA-DNA primer length counting by human primosome. 2022, Pubmed
Bepler, Positive-unlabeled convolutional neural networks for particle picking in cryo-electron micrographs. 2019, Pubmed
Braun, Role of protein-protein interactions in the function of replication protein A (RPA): RPA modulates the activity of DNA polymerase alpha by multiple mechanisms. 1997, Pubmed
Bullock, Initiation of simian virus 40 DNA synthesis in vitro. 1991, Pubmed
Burgers, Eukaryotic DNA Replication Fork. 2017, Pubmed
Cai, Cryo-EM structure of the human CST-Polα/primase complex in a recruitment state. 2022, Pubmed
Coloma, Human DNA polymerase α in binary complex with a DNA:DNA template-primer. 2016, Pubmed
Cordoba, Flexibility and Distributive Synthesis Regulate RNA Priming and Handoff in Human DNA Polymerase α-Primase. 2023, Pubmed
Davis, MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. 2007, Pubmed
Eki, The replication of DNA containing the simian virus 40 origin by the monopolymerase and dipolymerase systems. 1992, Pubmed
Emsley, Features and development of Coot. 2010, Pubmed
Evrin, Histone H2A-H2B binding by Pol α in the eukaryotic replisome contributes to the maintenance of repressive chromatin. 2018, Pubmed
Fukuda, Age-dependent changes in DNA polymerase fidelity and proofreading activity during cellular aging. 1999, Pubmed
Garg, DNA polymerases that propagate the eukaryotic DNA replication fork. 2005, Pubmed
Goddard, UCSF ChimeraX: Meeting modern challenges in visualization and analysis. 2018, Pubmed
He, Structure of Tetrahymena telomerase-bound CST with polymerase α-primase. 2022, Pubmed
He, Structures of the human CST-Polα-primase complex bound to telomere templates. 2022, Pubmed
He, Structures of human primosome elongation complexes. 2023, Pubmed
Hogg, Structural basis for processive DNA synthesis by yeast DNA polymerase ɛ. 2014, Pubmed
Jones, How Pol α-primase is targeted to replisomes to prime eukaryotic DNA replication. 2023, Pubmed
Kilkenny, Structural basis for the interaction of SARS-CoV-2 virulence factor nsp1 with DNA polymerase α-primase. 2022, Pubmed
Kilkenny, Structures of human primase reveal design of nucleotide elongation site and mode of Pol α tethering. 2013, Pubmed
Kilkenny, A conserved motif in the C-terminal tail of DNA polymerase α tethers primase to the eukaryotic replisome. 2012, Pubmed
Klinge, An iron-sulfur domain of the eukaryotic primase is essential for RNA primer synthesis. 2007, Pubmed
Klinge, 3D architecture of DNA Pol alpha reveals the functional core of multi-subunit replicative polymerases. 2009, Pubmed
Kuchta, DNA primase. Processivity and the primase to polymerase alpha activity switch. 1990, Pubmed
Lao-Sirieix, Structure of the heterodimeric core primase. 2005, Pubmed
Lévy, XRCC1 interacts with the p58 subunit of DNA Pol alpha-primase and may coordinate DNA repair and replication during S phase. 2009, Pubmed , Xenbase
Li, DNA polymerase α interacts with H3-H4 and facilitates the transfer of parental histones to lagging strands. 2020, Pubmed
Lim, The structure of human CST reveals a decameric assembly bound to telomeric DNA. 2020, Pubmed
Lu, 3DNA: a software package for the analysis, rebuilding and visualization of three-dimensional nucleic acid structures. 2003, Pubmed
Lu, A-form conformational motifs in ligand-bound DNA structures. 2000, Pubmed
Maga, Okazaki fragment processing: modulation of the strand displacement activity of DNA polymerase delta by the concerted action of replication protein A, proliferating cell nuclear antigen, and flap endonuclease-1. 2001, Pubmed
Mastronarde, Automated electron microscope tomography using robust prediction of specimen movements. 2005, Pubmed
Mullins, An HPLC-tandem mass spectrometry method for simultaneous detection of alkylated base excision repair products. 2013, Pubmed
Nethanel, An Okazaki piece of simian virus 40 may be synthesized by ligation of shorter precursor chains. 1988, Pubmed
Núñez-Ramírez, Flexible tethering of primase and DNA Pol α in the eukaryotic primosome. 2011, Pubmed
Pavlov, Evidence that errors made by DNA polymerase alpha are corrected by DNA polymerase delta. 2006, Pubmed
Pellegrini, The Pol α-primase complex. 2012, Pubmed
Perera, Mechanism for priming DNA synthesis by yeast DNA polymerase α. 2013, Pubmed
Pettersen, UCSF Chimera--a visualization system for exploratory research and analysis. 2004, Pubmed
Punjani, cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. 2017, Pubmed
Rao, Absence of a universal element for tRNAHis identity in Acanthamoeba castellanii. 2013, Pubmed
Rossi, Reconstituted Okazaki fragment processing indicates two pathways of primer removal. 2006, Pubmed
Sanchez-Garcia, DeepEMhancer: a deep learning solution for cryo-EM volume post-processing. 2021, Pubmed
Sauguet, Shared active site architecture between the large subunit of eukaryotic primase and DNA photolyase. 2010, Pubmed
Schneider, Primase activity of human DNA polymerase alpha-primase. Divalent cations stabilize the enzyme activity of the p48 subunit. 1998, Pubmed
Schneider, NIH Image to ImageJ: 25 years of image analysis. 2012, Pubmed
Sheaff, Mechanism of calf thymus DNA primase: slow initiation, rapid polymerization, and intelligent termination. 1993, Pubmed
Smith, Saccharomyces cerevisiae Thg1 uses 5'-pyrophosphate removal to control addition of nucleotides to tRNA(His.). 2014, Pubmed
Srivastava, Replicative enzymes and ageing: importance of DNA polymerase alpha function to the events of cellular ageing. 2002, Pubmed
Stith, Flexibility of eukaryotic Okazaki fragment maturation through regulated strand displacement synthesis. 2008, Pubmed
Stodola, Mechanism of Lagging-Strand DNA Replication in Eukaryotes. 2017, Pubmed
Suloway, Automated molecular microscopy: the new Leginon system. 2005, Pubmed
Sun, The architecture of a eukaryotic replisome. 2015, Pubmed
Swan, Structural basis of high-fidelity DNA synthesis by yeast DNA polymerase delta. 2009, Pubmed
Taricani, Replication stress activates DNA polymerase alpha-associated Chk1. 2009, Pubmed
Thomas, Fidelity of mammalian DNA replication and replicative DNA polymerases. 1991, Pubmed
Thompson, Interactions of calf thymus DNA polymerase alpha with primer/templates. 1995, Pubmed
Vaithiyalingam, Insights into eukaryotic DNA priming from the structure and functional interactions of the 4Fe-4S cluster domain of human DNA primase. 2010, Pubmed
Vaithiyalingam, Insights into eukaryotic primer synthesis from structures of the p48 subunit of human DNA primase. 2014, Pubmed
Waterhouse, SWISS-MODEL: homology modelling of protein structures and complexes. 2018, Pubmed
Weiner, An iron-sulfur cluster in the C-terminal domain of the p58 subunit of human DNA primase. 2007, Pubmed
Williams, Ribonucleotides in DNA: origins, repair and consequences. 2014, Pubmed
Xie, A model for dynamics of primer extension by eukaryotic DNA primase. 2011, Pubmed
Yan, TopBP1 and DNA polymerase alpha-mediated recruitment of the 9-1-1 complex to stalled replication forks: implications for a replication restart-based mechanism for ATR checkpoint activation. 2009, Pubmed
Yuan, Molecular choreography of primer synthesis by the eukaryotic Pol α-primase. 2023, Pubmed
Yuan, Structure of the polymerase ε holoenzyme and atomic model of the leading strand replisome. 2020, Pubmed
Zerbe, The p58 subunit of human DNA primase is important for primer initiation, elongation, and counting. 2002, Pubmed
Zhang, Divalent ions attenuate DNA synthesis by human DNA polymerase α by changing the structure of the template/primer or by perturbing the polymerase reaction. 2016, Pubmed
Zhang, The C-terminal domain of the DNA polymerase catalytic subunit regulates the primase and polymerase activities of the human DNA polymerase α-primase complex. 2014, Pubmed
Zheng, MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. 2017, Pubmed
Zivanov, New tools for automated high-resolution cryo-EM structure determination in RELION-3. 2018, Pubmed