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A mechanistic model of primer synthesis from catalytic structures of DNA polymerase α–primase

Nature Structural & Molecular Biology (2024)Cite this article

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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.

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Fig. 1: Conformational and configurational dynamics.
Fig. 2: Configurational rearrangements during DNA initiation and DNA elongation.
Fig. 3: Primer–template binding and elongation.
Fig. 4: Substrate recognition and duplex remodeling.
Fig. 5: Configurational rearrangements upon DNA termination.
Fig. 6: Structural rearrangements during chimeric primer synthesis.

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Data availability

Structures were deposited in the Protein Data Bank under accession codes 8G99 (AI complex, partial),8G9F (AI complex, complete), 8V5M (TC subcomplex, conformation 1), 8V5N (TC subcomplex, conformation 2), 8V5O (TC subcomplex, conformation 3), 8G9L (DI subcomplex), 8V6G (DI complex, configuration 1), 8V6H (DI complex, configuration 2), 8G9N (DE subcomplex, partial), 8G9O (DE subcomplex, complete), 8V6I (DE complex, configuration 1), 8V6J (DE complex, configuration 2), 8UCU (DT subcomplex, partial), 8UCV (DT subcomplex 1, complete) and 8UCW (DT subcomplex 2, complete). Maps were deposited in the Electron Microscopy Data Bank under accession codes EMD-29862 (AI complex, partial), EMD-29864 (AI complex, complete), EMD-29888 (TC subcomplex, conformation 1), EMD-29889 (TC subcomplex, conformation 2), EMD-29891 (TC subcomplex, conformation 3), EMD-29871 (DI subcomplex), EMD-42990 (DI complex, configuration 1), EMD-42991 (DI complex, configuration 2), EMD-29872 (DE subcomplex, partial), EMD-29873 (DE subcomplex, complete), EMD-42992 (DE complex, configuration 1), EMD-42993 (DE complex, configuration 2), EMD-42140 (DT subcomplex, partial), EMD-42141 (DT subcomplex 1, complete) and EMD-42142 (DT subcomplex 2, complete). Source data are provided with this paper.

References

  1. Nethanel, T., Reisfeld, S., Dinter-Gottlieb, G. & Kaufmann, G. An Okazaki piece of simian virus 40 may be synthesized by ligation of shorter precursor chains. J. Virol. 62, 2867–2873 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Bullock, P. A., Seo, Y. S. & Hurwitz, J. Initiation of simian virus 40 DNA synthesis in vitro. Mol. Cell. Biol. 11, 2350–2361 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Eki, T., Matsumoto, T., Murakami, Y. & Hurwitz, J. The replication of DNA containing the simian virus 40 origin by the monopolymerase and dipolymerase systems. J. Biol. Chem. 267, 7284–7294 (1992).

    Article  CAS  PubMed  Google Scholar 

  4. Garg, P. & Burgers, P. M. DNA polymerases that propagate the eukaryotic DNA replication fork. Crit. Rev. Biochem. Mol. Biol. 40, 115–128 (2005).

    Article  CAS  PubMed  Google Scholar 

  5. Stodola, J. L. & Burgers, P. M. Mechanism of lagging-strand DNA replication in eukaryotes. Adv. Exp. Med. Biol. 1042, 117–133 (2017).

    Article  CAS  PubMed  Google Scholar 

  6. Fukuda, M., Taguchi, T. & Ohashi, M. Age-dependent changes in DNA polymerase fidelity and proofreading activity during cellular aging. Mech. Ageing Dev. 109, 141–151 (1999).

    Article  CAS  PubMed  Google Scholar 

  7. Levy, N. et al. XRCC1 interacts with the p58 subunit of DNA pol α-primase and may coordinate DNA repair and replication during S phase. Nucleic Acids Res. 37, 3177–3188 (2009).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  8. Srivastava, V. K. & Busbee, D. L. Replicative enzymes and ageing: importance of DNA polymerase alpha function to the events of cellular ageing. Ageing Res. Rev. 1, 443–463 (2002).

    Article  CAS  PubMed  Google Scholar 

  9. Taricani, L., Shanahan, F. & Parry, D. Replication stress activates DNA polymerase alpha-associated Chk1. Cell Cycle 8, 482–489 (2009).

    Article  CAS  PubMed  Google Scholar 

  10. Yan, S. & Michael, W. M. 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. Cell Cycle 8, 2877–2884 (2009).

    Article  CAS  PubMed  Google Scholar 

  11. Kuchta, R. D., Reid, B. & Chang, L. M. DNA primase. Processivity and the primase to polymerase α activity switch. J. Biol. Chem. 265, 16158–16165 (1990).

    Article  CAS  PubMed  Google Scholar 

  12. Sheaff, R. J. & Kuchta, R. D. Mechanism of calf thymus DNA primase: slow initiation, rapid polymerization, and intelligent termination. Biochemistry 32, 3027–3037 (1993).

    Article  CAS  PubMed  Google Scholar 

  13. Thomas, D. C. et al. Fidelity of mammalian DNA replication and replicative DNA polymerases. Biochemistry 30, 11751–11759 (1991).

    Article  CAS  PubMed  Google Scholar 

  14. Maga, G. et al. Okazaki fragment processing: modulation of the strand displacement activity of DNA polymerase δ by the concerted action of replication protein A, proliferating cell nuclear antigen, and flap endonuclease-1. Proc. Natl Acad. Sci. USA 98, 14298–14303 (2001).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  15. Pavlov, Y. I. et al. Evidence that errors made by DNA polymerase α are corrected by DNA polymerase δ. Curr. Biol. 16, 202–207 (2006).

    Article  CAS  PubMed  Google Scholar 

  16. Rossi, M. L. & Bambara, R. A. Reconstituted Okazaki fragment processing indicates two pathways of primer removal. J. Biol. Chem. 281, 26051–26061 (2006).

    Article  CAS  PubMed  Google Scholar 

  17. Stith, C. M., Sterling, J., Resnick, M. A., Gordenin, D. A. & Burgers, P. M. Flexibility of eukaryotic Okazaki fragment maturation through regulated strand displacement synthesis. J. Biol. Chem. 283, 34129–34140 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Williams, J. S. & Kunkel, T. A. Ribonucleotides in DNA: origins, repair and consequences. DNA Repair 19, 27–37 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Burgers, P. M. J. & Kunkel, T. A. Eukaryotic DNA replication fork. Annu. Rev. Biochem. 86, 417–438 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Pellegrini, L. The pol α–primase complex. Subcell. Biochem. 62, 157–169 (2012).

    Article  CAS  PubMed  Google Scholar 

  21. Zerbe, L. K. & Kuchta, R. D. The p58 subunit of human DNA primase is important for primer initiation, elongation, and counting. Biochemistry 41, 4891–4900 (2002).

    Article  CAS  PubMed  Google Scholar 

  22. Lao-Sirieix, S. H., Nookala, R. K., Roversi, P., Bell, S. D. & Pellegrini, L. Structure of the heterodimeric core primase. Nat. Struct. Mol. Biol. 12, 1137–1144 (2005).

    Article  CAS  PubMed  Google Scholar 

  23. Vaithiyalingam, S., Warren, E. M., Eichman, B. F. & Chazin, W. J. 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. USA 107, 13684–13689 (2010).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  24. Sauguet, L., Klinge, S., Perera, R. L., Maman, J. D. & Pellegrini, L. Shared active site architecture between the large subunit of eukaryotic primase and DNA photolyase. PLoS ONE 5, e10083 (2010).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  25. Agarkar, V. B., Babayeva, N. D., Pavlov, Y. I. & Tahirov, T. H. Crystal structure of the C-terminal domain of human DNA primase large subunit: implications for the mechanism of the primase-polymerase α switch. Cell Cycle 10, 926–931 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Nunez-Ramirez, R. et al. Flexible tethering of primase and DNA pol α in the eukaryotic primosome. Nucleic Acids Res. 39, 8187–8199 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Kilkenny, M. L., Longo, M. A., Perera, R. L. & Pellegrini, L. Structures of human primase reveal design of nucleotide elongation site and mode of pol α tethering. Proc. Natl Acad. Sci. USA 110, 15961–15966 (2013).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  28. Vaithiyalingam, S. et al. Insights into eukaryotic primer synthesis from structures of the p48 subunit of human DNA primase. J. Mol. Biol. 426, 558–569 (2014).

    Article  CAS  PubMed  Google Scholar 

  29. Baranovskiy, A. G. et al. Crystal structure of the human primase. J. Biol. Chem. 290, 5635–5646 (2015).

    Article  CAS  PubMed  Google Scholar 

  30. Klinge, S., Nunez-Ramirez, R., Llorca, O. & Pellegrini, L. 3D architecture of DNA pol α reveals the functional core of multi-subunit replicative polymerases. EMBO J. 28, 1978–1987 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Kilkenny, M. L., De Piccoli, G., Perera, R. L., Labib, K. & Pellegrini, L. A conserved motif in the C-terminal tail of DNA polymerase α tethers primase to the eukaryotic replisome. J. Biol. Chem. 287, 23740–23747 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Perera, R. L. et al. Mechanism for priming DNA synthesis by yeast DNA polymerase α. eLife 2, e00482 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Attali, I., Botchan, M. R. & Berger, J. M. Structural mechanisms for replicating DNA in eukaryotes. Annu. Rev. Biochem. 90, 77–106 (2021).

    Article  CAS  PubMed  Google Scholar 

  34. Baranovskiy, A. G. et al. Mechanism of concerted RNA–DNA primer synthesis by the human primosome. J. Biol. Chem. 291, 10006–10020 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Kilkenny, M. L. et al. Structural basis for the interaction of SARS-CoV-2 virulence factor nsp1 with DNA polymerase α–primase. Protein Sci. 31, 333–344 (2022).

    Article  CAS  PubMed  Google Scholar 

  36. Jones, M. L., Aria, V., Baris, Y. & Yeeles, J. T. P. How pol α-primase is targeted to replisomes to prime eukaryotic DNA replication. Mol. Cell 83, 2911–2924 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. He, Q. et al. Structures of the human CST-polα–primase complex bound to telomere templates. Nature 608, 826–832 (2022).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  38. Klinge, S., Hirst, J., Maman, J. D., Krude, T. & Pellegrini, L. An iron-sulfur domain of the eukaryotic primase is essential for RNA primer synthesis. Nat. Struct. Mol. Biol. 14, 875–877 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Weiner, B. E. et al. An iron-sulfur cluster in the C-terminal domain of the p58 subunit of human DNA primase. J. Biol. Chem. 282, 33444–33451 (2007).

    Article  CAS  PubMed  Google Scholar 

  40. Arezi, B., Kirk, B. W., Copeland, W. C. & Kuchta, R. D. Interactions of DNA with human DNA primase monitored with photoactivatable cross-linking agents: implications for the role of the p58 subunit. Biochemistry 38, 12899–12907 (1999).

    Article  CAS  PubMed  Google Scholar 

  41. Baranovskiy, A. G. et al. Insight into the human DNA primase interaction with template-primer. J. Biol. Chem. 291, 4793–4802 (2016).

    Article  CAS  PubMed  Google Scholar 

  42. Xie, P. A model for dynamics of primer extension by eukaryotic DNA primase. Eur. Biophys. J. 40, 1157–1165 (2011).

    Article  CAS  PubMed  Google Scholar 

  43. Thompson, H. C., Sheaff, R. J. & Kuchta, R. D. Interactions of calf thymus DNA polymerase α with primer/templates. Nucleic Acids Res. 23, 4109–4115 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Baranovskiy, A. G., Lisova, A. E., Morstadt, L. M., Babayeva, N. D. & Tahirov, T. H. Insight into RNA–DNA primer length counting by human primosome. Nucleic Acids Res. 50, 6264–6270 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Coloma, J., Johnson, R. E., Prakash, L., Prakash, S. & Aggarwal, A. K. Human DNA polymerase α in binary complex with a DNA:DNA template–primer. Sci. Rep. 6, 23784 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  46. He, Q. et al. Structures of human primosome elongation complexes. Nat. Struct. Mol. Biol. 30, 579–583 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Yuan, Z., Georgescu, R., Li, H. & O’Donnell, M. E. Molecular choreography of primer synthesis by the eukaryotic pol α–primase. Nat. Commun. 14, 3697 (2023).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  48. Cai, S. W. et al. Cryo-EM structure of the human CST–polα/primase complex in a recruitment state. Nat. Struct. Mol. Biol. 29, 813–819 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. He, Y. et al. Structure of Tetrahymena telomerase-bound CST with polymerase α-primase. Nature 608, 813–818 (2022).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  50. Zhang, Y., Baranovskiy, A. G., Tahirov, T. H. & Pavlov, Y. I. The C-terminal domain of the DNA polymerase catalytic subunit regulates the primase and polymerase activities of the human DNA polymerase α-primase complex. J. Biol. Chem. 289, 22021–22034 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Zhang, Y. et al. Divalent ions attenuate DNA synthesis by human DNA polymerase α by changing the structure of the template/primer or by perturbing the polymerase reaction. DNA Repair 43, 24–33 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Swan, M. K., Johnson, R. E., Prakash, L., Prakash, S. & Aggarwal, A. K. Structural basis of high-fidelity DNA synthesis by yeast DNA polymerase δ. Nat. Struct. Mol. Biol. 16, 979–986 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Hogg, M. et al. Structural basis for processive DNA synthesis by yeast DNA polymerase ε. Nat. Struct. Mol. Biol. 21, 49–55 (2014).

    Article  CAS  PubMed  Google Scholar 

  54. El Hassan, M. A. & Calladine, C. R. Conformational characteristics of DNA: empirical classifications and a hypothesis for the conformational behaviour of dinucleotide steps. Philos. Trans. A Math. Phys. Eng. Sci. 355, 43–100 (1997).

    Article  ADS  CAS  Google Scholar 

  55. Lu, X. J. & Olson, W. K. 3DNA: a software package for the analysis, rebuilding and visualization of three-dimensional nucleic acid structures. Nucleic Acids Res. 31, 5108–5121 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Neidle, S. Nucleic Acid Structure and Recognition (Oxford University Press, 2002).

  57. Lu, X. J., Shakked, Z. & Olson, W. K. A-form conformational motifs in ligand-bound DNA structures. J. Mol. Biol. 300, 819–840 (2000).

    Article  CAS  PubMed  Google Scholar 

  58. Schneider, A. et al. Primase activity of human DNA polymerase α-primase. Divalent cations stabilize the enzyme activity of the p48 subunit. J. Biol. Chem. 273, 21608–21615 (1998).

    Article  CAS  PubMed  Google Scholar 

  59. Cordoba, J. J., Mullins, E. A., Salay, L. E., Eichman, B. F. & Chazin, W. J. Flexibility and distributive synthesis regulate RNA priming and handoff in human DNA polymerase α-primase. J. Mol. Biol. 435, 168330 (2023).

    Article  CAS  PubMed  Google Scholar 

  60. Salay, L. E., Cordoba, J. J. & Chazin, W. J. in Encyclopedia of Biological Chemistry 3rd edn 431–444 (Elsevier, 2021).

  61. Evrin, C., Maman, J. D., Diamante, A., Pellegrini, L. & Labib, K. Histone H2A-H2B binding by pol α in the eukaryotic replisome contributes to the maintenance of repressive chromatin. EMBO J. 37, e99021 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  62. Li, Z. et al. DNA polymerase α interacts with H3–H4 and facilitates the transfer of parental histones to lagging strands. Sci. Adv. 6, eabb5820 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  63. Sun, J. et al. The architecture of a eukaryotic replisome. Nat. Struct. Mol. Biol. 22, 976–982 (2015).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  64. Yuan, Z., Georgescu, R., Schauer, G. D., O’Donnell, M. E. & Li, H. Structure of the polymerase ε holoenzyme and atomic model of the leading strand replisome. Nat. Commun. 11, 3156 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  65. Lim, C. J. et al. The structure of human CST reveals a decameric assembly bound to telomeric DNA. Science 368, 1081–1085 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  66. Braun, K. A., Lao, Y., He, Z., Ingles, C. J. & Wold, M. S. Role of protein–protein interactions in the function of replication protein A (RPA): RPA modulates the activity of DNA polymerase α by multiple mechanisms. Biochemistry 36, 8443–8454 (1997).

    Article  CAS  PubMed  Google Scholar 

  67. Abad, M. G., Rao, B. S. & Jackman, J. E. Template-dependent 3′–5′ nucleotide addition is a shared feature of tRNAHis guanylyltransferase enzymes from multiple domains of life. Proc. Natl Acad. Sci. USA 107, 674–679 (2010).

    Article  ADS  CAS  PubMed  Google Scholar 

  68. Abad, M. G. et al. A role for tRNAHis guanylyltransferase (Thg1)-like proteins from Dictyostelium discoideum in mitochondrial 5′-tRNA editing. RNA 17, 613–623 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Rao, B. S., Mohammad, F., Gray, M. W. & Jackman, J. E. Absence of a universal element for tRNAHis identity in Acanthamoeba castellanii. Nucleic Acids Res. 41, 1885–1894 (2013).

    Article  CAS  PubMed  Google Scholar 

  70. Smith, B. A. & Jackman, J. E. Saccharomyces cerevisiae Thg1 uses 5′-pyrophosphate removal to control addition of nucleotides to tRNAHis. Biochemistry 53, 1380–1391 (2014).

    Article  CAS  PubMed  Google Scholar 

  71. Mullins, E. A. et al. An HPLC-tandem mass spectrometry method for simultaneous detection of alkylated base excision repair products. Methods 64, 59–66 (2013).

    Article  CAS  PubMed  Google Scholar 

  72. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    Article  PubMed  Google Scholar 

  73. Suloway, C. et al. Automated molecular microscopy: the new Leginon system. J. Struct. Biol. 151, 41–60 (2005).

    Article  CAS  PubMed  Google Scholar 

  74. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  PubMed  Google Scholar 

  76. Bepler, T. et al. Positive-unlabeled convolutional neural networks for particle picking in cryo-electron micrographs. Nat. Methods 16, 1153–1160 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  78. Sanchez-Garcia, R. et al. DeepEMhancer: a deep learning solution for cryo-EM volume post-processing. Commun. Biol. 4, 874 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  79. Waterhouse, A. et al. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 46, W296–W303 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  81. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  82. Davis, I. W. et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383 (2007).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  83. Goddard, T. D. et al. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018).

    Article  CAS  PubMed  Google Scholar 

  84. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  85. Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was funded by National Institutes of Health grants R35GM136401 (B.F.E.), R35GM118089 (W.J.C.), R01GM087543 (J.E.J.) and PO1CA92584. We thank M.-S. Tsai and members of the Expression and Molecular Biology Core of the Structural Cell Biology of DNA Repair Machines Program Project for baculovirus production and insect cell protein expression, and members of the Eichman and Chazin research groups for helpful discussions. Cryo-EM data were collected at the Center for Structural Biology Cryo-EM Facility at Vanderbilt University and the Life Sciences Institute Cryo-EM Facility at the University of Michigan. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Author information

Author notes

  1. Lauren E. Salay

    Present address: Department of Biochemistry, University of Washington, Seattle, WA, USA

  2. Clarissa L. Durie

    Present address: Department of Biochemistry, University of Missouri, Columbia, MO, USA

Authors and Affiliations

  1. Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA

    Elwood A. Mullins, Noah P. Bradley & Brandt F. Eichman

  2. Center for Structural Biology, Vanderbilt University, Nashville, TN, USA

    Elwood A. Mullins, Lauren E. Salay, Noah P. Bradley, Walter J. Chazin & Brandt F. Eichman

  3. Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA

    Lauren E. Salay, Walter J. Chazin & Brandt F. Eichman

  4. Department of Cell and Developmental Biology, University of Michigan School of Medicine, Ann Arbor, MI, USA

    Clarissa L. Durie & Melanie D. Ohi

  5. Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA

    Clarissa L. Durie & Melanie D. Ohi

  6. Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, USA

    Jane E. Jackman

  7. Center for RNA Biology, The Ohio State University, Columbus, OH, USA

    Jane E. Jackman

  8. Department of Chemistry, Vanderbilt University, Nashville, TN, USA

    Walter J. Chazin

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Contributions

B.F.E. conceived the study; B.F.E. and W.J.C. oversaw the study; E.A.M., L.E.S., N.P.B. and B.F.E. designed experiments; J.E.J. provided reagents; E.A.M., L.E.S., C.L.D. and N.P.B. collected data; E.A.M., L.E.S., N.P.B., M.D.O., W.J.C. and B.F.E. analyzed data; E.A.M., L.E.S., N.P.B., W.J.C. and B.F.E. wrote the manuscript.

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Correspondence to Walter J. Chazin or Brandt F. Eichman.

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Extended data

Extended Data Fig. 1 Preparation of 5′-triphosphorylated RNA and RNA-DNA primers.

a, Chemical reaction of AcaTLP2. b, Comparison of AcaTLP2 substrates (S) and products (P) for the DI, DE, and DT primers. Oligonucleotides were separated by denaturing urea-PAGE and visualized by UV-shadowing. Monophosphorylated oligonucleotide lengths (number of nucleotides) are indicated beside the gel. Phosphate groups are depicted as brown circles. The experiment was performed in singlicate. Source data are provided as a Source Data file.

Source data

Extended Data Fig. 2 Structural basis for DNA initiation with variable-length RNA primers.

a, DI subcomplex showing surface representations of POLA1cat and PRIM2C bound to a 9-mer RNA primer, a DNA template, and an incoming dGTP. b, Subunit interface between POLA1cat (blue) and PRIM2C (green) in the DI subcomplex. All residues within 6 Å are shown. Only POLA1 Ser1183 and PRIM2 Gly363 are within van der Waals contact distance. c, Overlay of the DI subcomplex and hypothetical DI models containing 8-mer, 7-mer, or 6-mer RNA primers, DNA template, and incoming dGTP, aligned on POLA1cat. Models were constructed by serially removing the second base pair from the 5′-end of the primer and then manually repositioning the first base pair, the template 3′-overhang, and PRIM2C as a rigid body to reform the phosphodiester bonds. As expected for an A-form to intermediate AB-form duplex, reformation of the bonds required the rigid body to be rotated by ~30° and translated by ~3 Å after deletion of each base pair. d, Comparison of the subunit interface between POLA1cat and PRIM2C in the DI subcomplex (left) and the hypothetical DI models containing 8-mer (middle left), 7-mer (middle right), or 6-mer (right) RNA primers, aligned on PRIM2C. The asterisks denote a common point on POLA1cat in all complexes. Residues in POLA1cat that sterically clash with PRIM2C are colored red. The extensive steric clashes in the 6-mer model suggest DNA initiation is unlikely with RNA primers ≤ 6 nucleotides in length. However, the minor steric clashes—involving only side chains—in the 7-mer and 8-mer models suggest DNA initiation is possible with RNA primers ≥ 7 nucleotides in length.

Extended Data Fig. 3 Comparison of RNA handoff, DNA initiation, and early DNA elongation complexes.

A comparison of homologous polα–primase structures representing RNA handoff (top left), DNA initiation (top middle, bottom left), and early DNA elongation states (bottom middle, bottom right) reveals substantial configurational heterogeneity. All structures are aligned on POLA1cat. Nucleic acid substrates in each complex are depicted beside the structures. In both Saccharomyces cerevisiae complexes, the nucleic acid substrates lack incoming nucleotide and the 5′-triphosphate group on the primer. With respect to the overall configuration of the complexes, only configuration DI-2 of the Xenopus laevis DNA initiation complex and the Homo sapiens early DNA elongation complex are similar. The most notable difference between the two complexes is the position of PRIM2C, which is determined by the differing lengths of the primer/template duplexes in the two structures. The X. laevis complexes and the H. sapiens complex are also similar with respect to the arrangement of primer/template and incoming dNTP. Conversely, recognition of primer/template in the S. cerevisiae early DNA elongation complex is distinct, both from the ternary structures shown here and from the binary structures shown in Extended Data Fig. 6. The reason for this difference is unclear, as is the reason for the primer/template being bound outside the active site of POLA1cat in the S. cerevisiae RNA handoff complex.

Extended Data Fig. 4 Control experiments for the primer elongation assays.

a, Selective incorporation of NTPs and dNTPs by polα (POLA1cat) and primase (ΔPOLA1cat). Polα failed to incorporate NTPs at detectable levels. However, primase readily incorporated both NTPs and dNTPs, producing intermediate and doublet bands in reactions with both substrates. Because polα is unable to incorporate NTPs, all DNA elongation products in the reactions containing both POLA1cat and ΔPOLA1cat but lacking dATP were initially extended by primase and then transferred to polα via an intermolecular handoff. b, Denaturation of the primer/template duplex. Identical aliquots from the primer elongation assays were worked up with (+) and without (−) a competitor oligonucleotide prior to denaturing gel electrophoresis. In the absence of competitor, incomplete denaturation and/or partial re-annealing of the primer/template caused smearing of elongation products ≥ 15 nucleotides in length. In all control experiments, reactions were incubated at 22 °C for 15 min. Primer lengths (number of nucleotides) are indicated beside the gels. Experiments were performed in duplicate. Source data are provided as a Source Data file.

Source data

Extended Data Fig. 5 Substrate-dependent conformational changes in POLA1cat and PRIM2C.

a,b, Comparison of POLA1cat (a) and PRIM2C (b) from the AI, DI, and DE (sub)complexes. Recognition of an RNA/DNA or DNA/DNA duplex by POLA1cat causes conformational changes in the thumb and the palm that in turn form the dNTP binding site. Subsequent recognition of an incoming dNTP results in closure of the fingers, creating a catalytically competent conformation poised for phosphodiester bond formation. Recognition of an RNA/DNA duplex by PRIM2C requires only conformational changes in the template-binding loop. c, Selected 2D class average showing POLA1cat from the substrate-free dataset. d–f, Substrate-binding surfaces in the substrate-free AI complex. Images are oriented as in Fig. 4. d, Unformed dNTP-binding pocket in the open conformation of POLA1cat. e, Thumb domain in the open conformation of POLA1cat (blue). The thumb forms protein-protein contacts with POLA1C (blue) and POLA2C (cyan) that sterically occlude substrate binding by POLA1cat. f, Substrate-binding surface of PRIM2C (green). Helices α17 (residues 344–349) and α18 (residues 352–358) and the template-binding loop (residues 359–372) form protein-protein contacts with POLA1cat (blue) and POLA1C (blue) that sterically block substrate binding by PRIM2C.

Extended Data Fig. 6 Substrate recognition in POLA1cat crystal structures.

a, Comparison of DNA initiation complexes containing RNA/DNA duplexes. The Xenopus laevis (top left) and Homo sapiens (PDB accession 4QCL, top right) ternary complexes are fully closed on the primer/template and the incoming dNTP, while the Saccharomyces cerevisiae binary complex (PDB accession 4FXD, bottom left) is partially open, owing to the absence of an incoming dNTP to induce closure of the fingers. POLA1cat and PRIM2C are colored blue and green, respectively. b, Comparison of DNA elongation complexes containing RNA-DNA/DNA or DNA/DNA duplexes. Only the X. laevis (top left) and S. cerevisiae (PDB accession 4FYD, bottom left) ternary complexes are fully closed. As expected, in the absence of an incoming dNTP, the H. sapiens binary complex (PDB accession 5IUD, bottom right) is partially open. However, unexpectedly, the H. sapiens ternary complex (PDB accession 6AS7, top right) is also partially open. Due to crystallographic lattice contacts, the fingers are not closed on the incoming dCTP.

Extended Data Fig. 7 Substrate recognition by polα, polδ, and polε.

a, Comparison of ternary RNA/DNA (DIS, top left) and RNA-DNA/DNA (DES, top right) complexes of Xenopus laevis POLA1cat and ternary DNA/DNA complexes of Saccharomcyes cerevisiae POLD1cat (PDB accession 3IAY, bottom left) and POLE1cat (PDB accession 4M8O, bottom right). POLA1cat and PRIM2C are colored blue and green, respectively. b, Comparison of oligonucleotide duplexes extracted from the structures shown in panel a. Ideal B-form DNA/DNA and A-form RNA/RNA duplexes are shown to the left. Major (M) and minor (m) grooves are indicated. Numbers to the left of the duplexes indicate the base step relative to the 3′-end of the primers. c, Analysis of duplex conformation in the structures shown in panels a and b. ZP is the interstrand phosphate-phosphate distance after projection onto the helical (Z) axis. d, Superposition of the structures shown in panel a. Structural differences in the thumb domain of POLA1cat create a wider substrate-binding cleft for preferential recognition of wider intermediate AB-form duplexes. Source data are provided as a Source Data file.

Source data

Extended Data Fig. 8 Substrate recognition in PRIM2C crystal structures.

a, Comparison of Xenopus laevis PRIM2C bound to RNA/DNA (DIS, left) and RNA-DNA/DNA (DES, middle left) duplexes and Homo sapiens PRIM2C bound to an RNA/DNA duplex (PDB accession 5F0Q, middle right, right). The asymmetric unit (ASU) of the HsPRIM2C crystal structure contains two crystallographically unique complexes, ASU-1 (middle right) and ASU-2 (right). POLA1cat and PRIM2C are colored blue and green, respectively. b,c, Schematic (b) and molecular (c) depictions of substrate-binding interactions with HsPRIM2C (ASU-2). Due to crystallographic lattice contacts, interactions with the last three nucleotides (A10–A12) of the DNA template differ between the two HsPRIM2C complexes. d, Analysis of duplex conformation in the crystal structure of HsPRIM2C bound to RNA/DNA (ASU-2). ZP is the interstrand phosphate-phosphate distance after projection onto the helical (Z) axis. e, Comparison of oligonucleotide duplexes extracted from the structures shown in panel a. Ideal B-form DNA/DNA and A-form RNA/RNA duplexes are shown to the left. Major (M) and minor (m) grooves are indicated. Numbers to the left of the RNA/DNA duplex from the HsPRIM2C complex (ASU-2) indicate the base step relative to the 5′-end of the primer. Source data are provided as a Source Data file.

Source data

Extended Data Fig. 9 Conformational changes in the DNA synthesis assembly.

a, Domain separation during DNA synthesis. As the length of the primer increases by 57 Å, the distance between POLA1cat and PRIM2C (measured at the attachment points of the interdomain linkers) increases by 55 Å. b, Conformational differences in the RNA-DNA/DNA duplexes from the DT subcomplexes. In the absence of PRIM2C, the base pair inclination of the DTS-2 substrate is reduced in the RNA/DNA region of the duplex. c, Substrate recognition by PRIM2C. The conformation of PRIM2C remains unchanged as the conformation of the substrates varies. In the DI subcomplex, duplex remodeling by POLA1cat substantially alters interactions between PRIM2C and the DNA template. For clarity, POLA1cat is omitted. d, Substrate recognition by POLA1cat. POLA1cat remodels all substrates to achieve similar (near) intermediate AB-form conformations. Modest narrowing of the minor groove (DIS, 15.1 Å; DES, 13.5 Å; DTS-1, 12.9 Å; DTS-2, 13.4 Å) in the DNA/DNA region of the DE and DT substrates is correlated with conformational changes in the adaptive duplex-binding loop. For clarity, PRIM2C is omitted. e,f, Conformational analysis of the DI, DE, and DT substrates. ZP is the interstrand phosphate-phosphate distance after projection onto the helical (Z) axis. The base step indicated in the plots is relative to either the 5′-end (e) or 3′-end (f) of the primer. The regions of the duplexes bound by POLA1cat (blue) and PRIM2C (green) are shown below the plots. Source data are provided as a Source Data file.

Source data

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Source Data Figs. 3–5 and Extended Data Figs 7–9

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Source Data Extended Data Fig. 1

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Mullins, E.A., Salay, L.E., Durie, C.L. et al. A mechanistic model of primer synthesis from catalytic structures of DNA polymerase α–primase. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01227-4

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