During the initiation of DNA replication, oligonucleotide primers are synthesized de novo by primases and are subsequently extended by replicative polymerases to complete genome duplication. The primase-polymerase (Prim-Pol) superfamily is a diverse grouping of primases, which includes replicative primases and CRISPR-associated primase-polymerases (CAPPs) involved in adaptive immunity1-3. Although much is known about the activities of these enzymes, the precise mechanism used by primases to initiate primer synthesis has not been elucidated. Here we identify the molecular bases for the initiation of primer synthesis by CAPP and show that this mechanism is also conserved in replicative primases. The crystal structure of a primer initiation complex reveals how the incoming nucleotides are positioned within the active site, adjacent to metal cofactors and paired to the templating single-stranded DNA strand, before synthesis of the first phosphodiester bond. Furthermore, the structure of a Prim-Pol complex with double-stranded DNA shows how the enzyme subsequently extends primers in a processive polymerase mode. The structural and mechanistic studies presented here establish how Prim-Pol proteins instigate primer synthesis, revealing the requisite molecular determinants for primer synthesis within the catalytic domain. This work also establishes that the catalytic domain of Prim-Pol enzymes, including replicative primases, is sufficient to catalyse primer formation.

Department of Chemistry University of North Texas Denton TX USA

Department of Physics and Department of Chemistry and Biochemistry University of Texas at Dallas Richardson TX USA

Genome Damage and Stability Centre School of Life Sciences University of Sussex Brighton UK

National Centre for Biomolecular Research Masaryk University Brno Czech Republic

See more in PubMed

Guilliam TA, Keen BA, Brissett NC & Doherty AJ Primase-polymerases are a functionally diverse superfamily of replication and repair enzymes. Nucleic Acids Res. 43, 6651–6664 (2015). PubMed PMC

Iyer LM, Koonin EV, Leipe DD & Aravind L Origin and evolution of the archaeo-eukaryotic primase superfamily and related palm-domain proteins: structural insights and new members. Nucleic Acids Res. 33, 3875–3896 (2005). PubMed PMC

Zabrady K, Zabrady M, Kolesar P, Li AWH & Doherty AJ CRISPR-associated primase-polymerases are implicated in prokaryotic CRISPR–Cas adaptation. Nat. Commun 12, 3690 (2021). PubMed PMC

Bouché JP, Zechel K & Kornberg A dnaG gene product, a rifampicin-resistant RNA polymerase, initiates the conversion of a single-stranded coliphage DNA to its duplex replicative form. J. Biol. Chem 250, 5995–6001 (1975). PubMed

Aravind L, Leipe DD & Koonin EV Toprim—a conserved catalytic domain in type IA and II topoisomerases, DnaG-type primases, OLD family nucleases and RecR proteins. Nucleic Acids Res. 26, 4205–4213 (1998). PubMed PMC

Płociński P et al. DNA ligase C and Prim–PolC participate in base excision repair in mycobacteria. Nat. Commun 8, 1251 (2017). PubMed PMC

Pitcher RS, Brissett NC & Doherty AJ Nonhomologous end-joining in bacteria: a microbial perspective. Annu. Rev. Microbiol 61, 259–282 (2007). PubMed

Bainbridge LJ, Teague R & Doherty AJ Repriming DNA synthesis: an intrinsic restart pathway that maintains efficient genome replication. Nucleic Acids Res. 49, 4831–4847 (2021). PubMed PMC

Brissett NC et al. Molecular basis for DNA repair synthesis on short gaps by mycobacterial primase–polymerase C. Nat. Commun 11, 4196 (2020). PubMed PMC

Bell SD Initiating DNA replication: a matter of prime importance. Biochem. Soc. Trans 47, 351–356 (2019). PubMed PMC

Boudet J, Devillier J-C, Allain FH-T & Lipps G Structures to complement the archaeo-eukaryotic primases catalytic cycle description: what’s next? Comput. Struct. Biotechnol. J 13, 339–351 (2015). PubMed PMC

Bianchi J et al. PrimPol bypasses UV photoproducts during eukaryotic chromosomal DNA replication. Mol. Cell 52, 566–573 (2013). PubMed PMC

Keen BA, Jozwiakowski SK, Bailey LJ, Bianchi J & Doherty AJ Molecular dissection of the domain architecture and catalytic activities of human PrimPol. Nucleic Acids Res. 42, 5830–5845 (2014). PubMed PMC

Rechkoblit O et al. Structure and mechanism of human PrimPol, a DNA polymerase with primase activity. Sci. Adv 2, e1601317 (2016). PubMed PMC

Rechkoblit O et al. Structural basis of DNA synthesis opposite 8-oxoguanine by human PrimPol primase-polymerase. Nat. Commun 12, 4020 (2021). PubMed PMC

Martínez-Jiménez MI, Calvo PA, García-Gómez S, Guerra-González S & Blanco L The Zn-finger domain of human PrimPol is required to stabilize the initiating nucleotide during DNA priming. Nucleic Acids Res. 46, 4138–4151 (2018). PubMed PMC

Holzer S, Yan J, Kilkenny ML, Bell SD & Pellegrini L Primer synthesis by a eukaryotic-like archaeal primase is independent of its Fe–S cluster. Nat. Commun 8, 1718 (2017). PubMed PMC

Liu B et al. A primase subunit essential for efficient primer synthesis by an archaeal eukaryotic-type primase. Nat. Commun 6, 7300 (2015). PubMed PMC

Boudet J et al. A small helical bundle prepares primer synthesis by binding two nucleotides that enhance sequence-specific recognition of the DNA template. Cell 176, 154–166 (2019). PubMed

Beck K, Vannini A, Cramer P & Lipps G The archaeo-eukaryotic primase of plasmid pRN1 requires a helix bundle domain for faithful primer synthesis. Nucleic Acids Res. 38, 6707–6718 (2010). PubMed PMC

Baranovskiy AG et al. Crystal structure of the human primase. J. Biol. Chem 290, 5635–5646 (2015). PubMed PMC

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

Holzer S et al. Structural basis for inhibition of human primase by arabinofuranosyl nucleoside analogues fludarabine and vidarabine. ACS Chem. Biol 14, 1904–1912 (2019). PubMed PMC

Steitz TA & Steitz JA A general two-metal-ion mechanism for catalytic RNA. Proc. Natl Acad. Sci. USA 90, 6498–6502 (1993). PubMed PMC

Basu RS et al. Structural basis of transcription initiation by bacterial RNA polymerase holoenzyme. J. Biol. Chem 289, 24549–24559 (2014). PubMed PMC

Butcher SJ, Grimes JM, Makeyev EV, Bamford DH & Stuart DI A mechanism for initiating RNA-dependent RNA polymerization. Nature 410, 235–240 (2001). PubMed

Appleby TC et al. Structural basis for RNA replication by the hepatitis C virus polymerase. Science 347, 771–775 (2015). PubMed

Sheaff RJ & Kuchta RD Mechanism of calf thymus DNA primase: slow initiation, rapid polymerization, and intelligent termination. Biochemistry 32, 3027–3037 (1993). PubMed

Díaz-Talavera A et al. A cancer-associated point mutation disables the steric gate of human PrimPol. Sci. Rep 9, 1121 (2019). PubMed PMC

Copeland WC & Wang TS Enzymatic characterization of the individual mammalian primase subunits reveals a biphasic mechanism for initiation of DNA replication. J. Biol. Chem 268, 26179–26189 (1993). PubMed

Lee J-G et al. Structural and biochemical insights into inhibition of human primase by citrate. Biochem. Biophys. Res. Commun 507, 383–388 (2018). PubMed

Copeland WC Expression, purification, and characterization of the two human primase subunits and truncated complexes from Escherichia coli. Protein Expr. Purif 9, 1–9 (1997). PubMed

Lee S-J, Zhu B, Hamdan SM & Richardson CC Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7. Nucleic Acids Res. 38, 4372–4383 (2010). PubMed PMC

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

Berrow NS et al. A versatile ligation-independent cloning method suitable for high-throughput expression screening applications. Nucleic Acids Res. 35, e45 (2007). PubMed PMC

Sheldrick GM Experimental phasing with SHELXC/D/E: combining chain tracing with density modification. Acta Crystallogr. D 66, 479–485 (2010). PubMed PMC

Langer G, Cohen SX, Lamzin VS & Perrakis A Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7. Nat. Protoc 3, 1171–1179 (2008). PubMed PMC

Emsley P, Lohkamp B, Scott WG & Cowtan K Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010). PubMed PMC

Afonine PV et al. Towards automated crystallographic structure refinement with phenix. refine. Acta Crystallogr. D 68, 352–367 (2012). PubMed PMC

McCoy AJ et al. Phaser crystallographic software. J. Appl. Crystallogr 40, 658–674 (2007). PubMed PMC

Eisenberg D, Schwarz E, Komaromy M & Wall R Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J. Mol. Biol 179, 125–142 (1984). PubMed

Contreras-García J et al. NCIPLOT: a program for plotting noncovalent interaction regions. J. Chem. Theory Comput 7, 625–632 (2011). PubMed PMC

Humphrey W, Dalke A & Schulten K VMD: visual molecular dynamics. J. Mol. Graph 14, 33–38 (1996). PubMed

Jeziorski B et al. SAPT: a program for many-body symmetry-adapted perturbation theory calculations of intermolecular interaction energies. Methods Tech. Comput. Chem B, 79–129 (1993).

Parker TM, Burns LA, Parrish RM, Ryno AG & Sherrill CD Levels of symmetry adapted perturbation theory (SAPT). I. Efficiency and performance for interaction energies. J. Chem. Phys 140, 094106 (2014). PubMed

Naseem-Khan S, Gresh N, Misquitta AJ & Piquemal J-P Assessment of SAPT and supermolecular EDA approaches for the development of separable and polarizable force fields. J. Chem. Theory Comput 17, 2759–2774 (2021). PubMed

Turney JM et al. Psi4: an open-source ab initio electronic structure program. WIREs Comput. Mol. Sci 2, 556–565 (2012).

Stone AJ & Misquitta AJ Charge-transfer in symmetry-adapted perturbation theory. Chem. Phys. Lett 473, 201–205 (2009).