Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Molecular basis for the initiation of DNA primer synthesis

Abstract

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 immunity1,2,3. 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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: MpCAPP’s PP domain is polymerase and primase proficient.
Fig. 2: Structure of a post-ternary complex of PP bound to dsDNA.
Fig. 3: Structure–function analyses of MsCAPP PP primer synthesis and extension activities.
Fig. 4: The PP domains of eukaryotic replicative primases are primase proficient.

Similar content being viewed by others

Data availability

The coordinates and crystallographic structure factors for MsCAPP have been deposited at PDB under accession codes 7NQD (apo), 7NQE (dGTP complex), 7NQF (postcatalytic complex), 7P9J (primer initiation complex) and 7QAZ (primer initiation complex (alternate model)). All other data needed to evaluate the conclusions in this study are present in the manuscript and/or its supplementary information and tables. Uncropped versions of all gels are provided in Supplementary Fig. 1. Source data for graphs (gel-based assays and FP assays) can be found in Supplementary Table 2.

References

  1. Guilliam, T. A., Keen, B. A., Brissett, N. C. & Doherty, A. J. Primase-polymerases are a functionally diverse superfamily of replication and repair enzymes. Nucleic Acids Res. 43, 6651–6664 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Iyer, L. M., Koonin, E. V., Leipe, D. D. & 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Zabrady, K., Zabrady, M., Kolesar, P., Li, A. W. H. & Doherty, A. J. CRISPR-associated primase-polymerases are implicated in prokaryotic CRISPR–Cas adaptation. Nat. Commun. 12, 3690 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  4. Bouché, J. P., 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).

    Article  PubMed  Google Scholar 

  5. Aravind, L., Leipe, D. D. & Koonin, E. V. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

  7. Pitcher, R. S., Brissett, N. C. & Doherty, A. J. Nonhomologous end-joining in bacteria: a microbial perspective. Annu. Rev. Microbiol. 61, 259–282 (2007).

    Article  CAS  PubMed  Google Scholar 

  8. Bainbridge, L. J., Teague, R. & Doherty, A. J. Repriming DNA synthesis: an intrinsic restart pathway that maintains efficient genome replication. Nucleic Acids Res. 49, 4831–4847 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  10. Bell, S. D. Initiating DNA replication: a matter of prime importance. Biochem. Soc. Trans. 47, 351–356 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Keen, B. A., Jozwiakowski, S. K., Bailey, L. J., Bianchi, J. & Doherty, A. J. Molecular dissection of the domain architecture and catalytic activities of human PrimPol. Nucleic Acids Res. 42, 5830–5845 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

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

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  16. Martínez-Jiménez, M. I., Calvo, P. A., 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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

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

    Article  ADS  CAS  PubMed  Google Scholar 

  19. 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).

    Article  CAS  PubMed  Google Scholar 

  20. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  22. 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 

  23. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Steitz, T. A. & Steitz, J. A. A general two-metal-ion mechanism for catalytic RNA. Proc. Natl Acad. Sci. USA 90, 6498–6502 (1993).

    Article  ADS  CAS  PubMed  PubMed Central  MATH  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Butcher, S. J., Grimes, J. M., Makeyev, E. V., Bamford, D. H. & Stuart, D. I. A mechanism for initiating RNA-dependent RNA polymerization. Nature 410, 235–240 (2001).

    Article  ADS  CAS  PubMed  Google Scholar 

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

    Article  ADS  CAS  PubMed  Google Scholar 

  28. 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 

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

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

  30. Copeland, W. C. & Wang, T. S. Enzymatic characterization of the individual mammalian primase subunits reveals a biphasic mechanism for initiation of DNA replication. J. Biol. Chem. 268, 26179–26189 (1993).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

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

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D 68, 352–367 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  44. 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).

    Google Scholar 

  45. Parker, T. M., Burns, L. A., Parrish, R. M., Ryno, A. G. & Sherrill, C. D. Levels of symmetry adapted perturbation theory (SAPT). I. Efficiency and performance for interaction energies. J. Chem. Phys. 140, 094106 (2014).

    Article  ADS  PubMed  CAS  Google Scholar 

  46. Naseem-Khan, S., Gresh, N., Misquitta, A. J. & 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

Download references

Acknowledgements

The laboratory of A.J.D. was supported by grants from the Biotechnology and Biological Sciences Research Council (BB/S008691/1 and BB/P007031/1). L.J.B. was supported by a PhD studentship funded by an Institutional Strategic Support Fund 2 grant from the Wellcome Trust (204833/Z/16/Z). The computational simulations were funded by NIH grant R01GM108583. Computational time was provided by the University of North Texas CASCaMs CRUNTCh3 high-performance cluster partially supported by NSF grant CHE-1531468 and XSEDE supported by project TG-CHE160044. We thank M. Roe for assistance with phasing, Diamond Light Source for beamtime (proposal MX20145) and the staff of beamlines I03, I04 and I04-1 for assistance with data collection.

Author information

Authors and Affiliations

Authors

Contributions

A.J.D. designed the project and directed the experimental work, and co-wrote the manuscript with A.W.H.L., K.Z. and L.J.B. A.W.H.L., K.Z., L.J.B., M.Z. and P.K. contributed to project design and performed and analysed the experiments: A.W.H.L. performed all crystallographic experiments; K.Z. performed MpCAPP, MsCAPP and HsPri1 polymerase and primase assays, MsCAPP FP assays and MpCAPP iron-binding assays; L.J.B. performed primase assays with eukaryotic PrimPol proteins; and M.Z. performed initial primase assays with MsCAPP and HsPrimPol PPs. P.K. performed initial activity assays with MsCAPP. S.N.-K., M.B.B. and G.A.C. designed, performed and analysed the in silico molecular modelling experiments.

Corresponding author

Correspondence to Aidan J. Doherty.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature thanks Adele Williamson and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 CAPP’s Prim-Pol domain alone is sufficient for polymerase and primase synthesis.

a. Alignment of the C-terminal domains (CTD) of different primases or CAPPs. Sc: Saccharomyces cerevisiae PriL (Pri2p), Ls: Lokiarchaeum sp. GC14_75 PriL, Mp: Marinitoga piezophila CAPP, Tp: Thermotoga profunda CAPP, Fg: Fervidobacterium gondwanense CAPP. Conserved motifs: cysteines – red; basic residues – blue; hydrophobic residues – green; prolines – yellow. b. Visualisation of MpCAPP full length wild-type (WT) and C462S, C464S (CC) proteins after two-step purification. c. UV-visible absorption spectrum of MpCAPP WT and CC. CAPP WT, but not CC, exhibited a major absorbance peak at 412 nm. d. Comparison of three different MpCAPP constructs using an Iron assay kit (MAK025, Sigma). MpCAPP FL WT sample had a significantly higher concentration of iron than the MpCAPP CC mutant or CAPP ΔCTD fragment (aa 1-360). Data are the mean of four measurements, except reduced FL WT and FL CC – three measurements. Error bars indicate the mean ± standard deviation. Individual values are presented as black dots. Reduced – sample after treatment with an iron reducing agent; Non-reduced – sample without treatment. e. MpCAPP CC mutant has similar polymerase activity as WT. 1, 5, 25 and 125 nM MpCAPP FL WT (lanes 2-5) or FL CC (lanes 6-9) was added into 30 nM DNA substrate (DNA template – oPK404 + FAM-labelled DNA primer – oPK405) and 100 µM dNTPs. Reactions were incubated at 37 °C for 30 min. f. MpCAPP CC mutant has comparable priming activity as WT. 0.5, 1, 2 and 4 µM MpCAPP FL WT (lanes 2-5) or FL CC (lanes 6-9) was added into reactions containing 1 µM ssDNA template (oKZ388), 2.5 µM non-labelled dATP, dTTP, dGTP, 2.5 µM FAM-dCTP (dCTP) and 100 µM GTP. The reactions were incubated at 50 °C for 30 min. The products were resolved on 20% urea-PAGE gel. g. MpCAPP PP domain exhibits efficient polymerase activity. 1, 5, 25 and 125 nM MpCAPP full length wild-type (FL) (lanes 2-5) and its fragments (lanes 6-17) or 125 nM D177A, D179A full-length mutant (FL AxA) (lane 18) were tested as described in panel e. h. MpCAPP PP domain exhibits strong primase activity. 0.25, 0.5, 1 and 2 µM of MpCAPP FL (lanes 2-5) and its fragments (lanes 6-17) or 2 µM FL AxA (lane 18) were tested as described in panel f. ‘C’ indicates a control reaction without protein. Oligonucleotide (Nts) length markers are shown on the left of the gel. Results shown in panels e-h are representative of three independent repeats, except PP and FL AxA in polymerase assay – four independent repeats.

Extended Data Fig. 2 MsCAPP PP structure and its primase activity.

a. Structure of MsCAPP PP domain in cartoon representation showing the apo protein (left) and the dGTP complex with Mn(II) ions (right). Protein – grey, dGTP – orange, Mn(II) – purple spheres. b. Protein sequence alignment of MpCAPP111-328 (MpPP) and Marinitoga sp. 1137 CAPP111-328 (MsPP). Conserved amino acids mutated in this study are shown in yellow. Non-conserved amino acids are shown in red. Conserved catalytic motifs I, II and III are in black rectangles as indicated. c. MsPP possesses priming activity. 0.5, 1, 2 and 4 µM MpCAPP full-length (MpFL) (lanes 2-5) or MsCAPP111-328 (MsPP) (lanes 6-9) was added into the reaction containing 1 µM ssDNA mixed sequence template (oKZ388), 100 µM non-labelled dNTP mix and 10 µM FAM-γGTP (γGTP). The reactions were incubated at 50 °C for 30 min. ‘C’ indicates a control reaction without protein. Oligonucleotide (Nts) length markers are shown on the left of the gel. Results shown are representative of three independent repeats.

Extended Data Fig. 3 MsCAPP PP domain structure analyses and comparison.

a. Overall structure of dGTP-complexed MsCAPP PP domain, with N-terminal α/β domain coloured in green and RRM-like domain coloured in yellow (left), and a close-up view showing dGTP (orange), Mn(II) ions (spheres) and residues lining the active site pocket. b. Architecture of MsCAPP PP domain showing the secondary structural elements within the α/β domain (aa 111-164; aa 274–278) in green and the RRM-like domain (aa 169–262; aa 291–328) in yellow. c. Side by side comparison of PP domains from various Prim-Pols with a single nucleotide (ball-and-stick model) bound to the elongation site. N-terminal RRM-like domain (yellow), α/β domain (green) and helical domain (red). d. Simulated annealing Fo-Fc omit map of Co(II) ions in the active site of MsCAPP (contoured at 5 σ-level at 1.90 Å resolution), along with GTP (blue), dATP (orange), and acidic residues D177, D179 and E260 in stick representation. e. Close-up view of Mn(II) bound in the A site of the MsCAPP dGTP complex, showing octahedral coordination to dGTP, ethylene glycol and surrounding acidic residues with distances labelled in Å (left). Close-up view of Mn(II) bound in the B site of MsCAPP dGTP complex, showing octahedral coordination to dGTP, DxD motif and a water molecule, with distances labelled in Å (right). Mn(II) – purple spheres. f. Overlay of Region 1 (aa 130-142) (left), Region 2 (aa 263–274) (middle) and residues around Region 3 (aa 280-289) (right) from the structures of MsCAPP apo (green) and primer initiation complex (grey). Residues 283–286 in Region 3 are flexible and not resolved in the apo structure. g. MsCAPP active site with surface coloured according to hydrophobicity, with regions of high hydrophobicity coloured in red. Three core residues that form part of the active site hydrophobic pocket (L275, I276 and F262) are shown in stick representation (red). I-site GTP (blue) and E-site dATP (orange) are shown in stick representation. Templating DNA (pink) is shown in cartoon representation. h. Comparison of CAPP primer initiation complex active site. Figure on the left – CAPP primer initiation complex shown in the main figures (PDB: 7P6J). Figure on the right shows the structure of an alternative CAPP primer initiation complex (PDB: 7QAZ), where the phosphate tail (red) of I-site GTP adopts a different conformation and coordinate to an extra Co(II) ion. The rest of the GTP molecule is shown in blue and dATP in orange. Templating DNA in pink, surface of α/β domain is coloured in green and RRM-like domain is shown in yellow.

Extended Data Fig. 4 Intermolecular interaction analyses of the active site of the primer initiation complex.

a. Non-Covalent Interaction (NCI) analysis of the active site (isosurface = 0.35, cut-off = 8 Å). b. Representation of the active site used for Symmetry-Adapted Perturbation Theory (SAPT0) calculations. For dA, dC, dT, Y138 and R223, only the side chains have been considered. c. Results from SAPT0 calculations for the indicated pairs using the def2-SV(P) basis set in kcal/mol. The final row indicates the SAPT0/def2-SV(P) calculation for GTP interacting with all other fragments in the system (dATP, Y138, dT and both Mg(II) ions).

Extended Data Fig. 5 Comparison of the different crystal structures of the MsCAPP PP domain.

From left to right, top to bottom; apo, dGTP-bound, primer initiation complex, primer initiation complex (alternative conformation) and post-ternary complex. Protein – grey cartoon, deoxyribonucleotide (dGTP / dATP) – orange sticks, ribonucleotide (GTP) blue sticks, templating DNA strand – pink cartoon, primer DNA strand – orange cartoon, Mn(II) – purple spheres, Co(II) – pink spheres.

Extended Data Fig. 6 Structure-function and binding studies on the PP domain of MsCAPP.

a. Effect of mutations of MpCAPP100-360 (MpPP) on its polymerase activity. 50 nM MpPP wild-type (WT) (lane 2) or its mutated variants (lanes 3-18) were added to 50 nM DNA substrate (DNA template – oNB1 + FAM-labelled DNA primer – oNB2) and 100 µM dNTPs. The reactions were incubated at 37 °C for 15 min. b. Primase activity of MpPP WT and its mutants. 2 µM MpPP WT protein (lane 2) or its mutants (lanes 3-18) were added to reactions containing 1 µM DNA substrate (oKZ388), 100 µM dNTP mix and 10 µM FAM-labelled GTP (fused via γ-phosphate) (γGTP). The reactions were incubated at 50 °C for 30 min. The products were resolved on 20% urea-PAGE gel. ‘C’ indicates a control reaction without protein. AxA – D177A, D179A, RR – R142A, R143A, KK – K181A, K182A, KQN – K264A, Q265A, N274A. Results are representative of five (panel a) and four (panel b) independent repeats. Oligonucleotide length marker (Nts) is shown on the left of the gel. ‘C’ indicates control without protein. c. Fluorescence polarization assays (FP) reveal that the presence of dinucleotide (rG-dA) does not stimulate binding of MsCAPP111-328 (MsPP) to template. FP: 0–80 µM 5’-3prG-dA-3’ (ATDBio) dinucleotide was added to 5 µM MsPP and 50 nM FAM-DNA ( DNA, oKZ409) in presence or absence of 0.1 mM dTTP. d. Stimulation of PP DNA binding affinity in presence of nucleotides is dependent on the template sequence. FP: 0–20 µM MsPP was added to 50 nM DNA templates (oKZ409, oKZ413, oKZ414 and oKZ416) ± 1 mM GTP and 0.1 mM dATP. e. The efficiency of PP dinucleotide formation is dependent on the −2 base on the template. 1 µM MsPP was added into the reaction containing 1 µM template (lane 2 - oKZ435, lane 3 - oKZ447, lane 4 - oKZ449, lane 5 - oKZ450, lane 6 – oKZ448), 100 µM dATP and 10 µM γGTP. The reactions were incubated at 50 °C for 30 min. The gel is representative of three independent repeats (left). Signal of synthetized dinucleotides were normalized to signal of dinucleotide in presence of 3’-AAACTAAA-5’ ssDNA template (100%). Data represent the mean ± standard deviation from three independent experiments. Black dots – individual values. ‘C’ indicates control reaction without protein. f. Affinity of PP to template increases with the template length. FP – 0–20 µM MsPP was added to 50 nM DNA templates (oKZ408–oKZ412). Data representing the mean ± standard deviation from four independent experiments (Panels c, d and f). The mean values were used to calculate the dissociation constants (Kd) shown on the right (Panel d, f); SD – standard deviation of calculated Kd; ND – not determined (Panels d, f).

Extended Data Fig. 7 Comparison of MsCAPP and PP domains of human Prim-Pols.

a. Overlay of HsPrimPol (pink) and MsCAPP (grey) PP domains (left), and of HsPri1 (orange) and MsCAPP (grey) PP domains (right). Protein and DNA strands are displayed in cartoon representation, nucleic acids are displayed in stick representation, and metal ions are displayed as spheres. b. Close-up view of the E-site nucleotide, amino acid residues close to the 2’ position of the ribose ring, and Region 1 of MsCAPP (left), HsPrimPol (middle) and HsPri1 (right). Interaction between D79 of HsPri1 and 2’-OH is displayed with a dashed line.

Extended Data Fig. 8 Analyses of PrimPol mutations and how incoming nucleotides influence the primase activities of different Prim-Pols.

a. The primase activity of HsPrimPol1-354 (PP) is reduced compared to the full-length enzyme (WT) at lower concentrations. HsPrimPol1-354 D114A, E116A (AxA) (1 µM) exhibits no primase activity. Quantification of primase assays represented in Fig. 4c. Data represent the mean ± standard deviation from four independent experiments. b. Efficiency of priming by HsPrimPol1-354 is dependent on FAM-γGTP (γGTP) concentration. Primase reactions contained varying concentrations of γGTP (0.02, 0.1, 0.5 and 2.5 μM), 100 µM dNTP mix and 1 µM ssDNA template (oKZ388). c. GTP is outcompeted by high concentrations of dATP from MsCAPP’s active site. FP – 10 µM MsCAPP111-328 (MsPP) was added to 100 nM γGTP ± 1 µM DNA template (oKZ435) with increasing concentrations of dATP (0.1 µM – 10 mM). d. The effect of different concentration of GTP and dATP on MsPP affinity to DNA. FP – 5 µM MsCAPP111-328 (MsPP) was added to 50 µM DNA template (oKZ409) in presence of 1 mM GTP/dATP and increasing concentrations (x mM) of dATP/GTP (15.625 µM – 1 mM). Data were obtained from four independent repeats (Panels c and d). Error bars in the graphs show the mean ± standard deviation. IC50 and EC50 values were calculated as described in materials and methods. e. Structurally equivalent residues of MpCAPP and HsPrimPol. f. Polymerase activity of HsPrimPol1-354 is severely disrupted by point mutations in key catalytic residues. AxA – D114A, E116A, RNR – R286A, N287R, R288A. 200 nM protein was incubated with 50 nM DNA substrate (oNB1 + oNB2) for 30 min at 37 °C. g. Primase activity of HsPrimPol1-354 is significantly disrupted by point mutations in key catalytic residues. The reactions contained 4 μM protein, 10 μM γGTP, 100 μM dNTP mix and 1 μM DNA template (oKZ388). h. HsPrimPol preferentially utilizes GTP to initiate primer synthesis. Primase assay reaction contained 1 μM HsPrimPol1-354 (HsPP), 1 μM DNA template (oKZ388), 2.5 μM FAM-dATP (dATP), 2.5 μM dCTP, dGTP, and dTTP and 100 μM individual NTPs. i. HsPrimPol preferentially initiates primer synthesis with GTP over ATP. Primase assay reaction contained 1 μM HsPP, 1 μM DNA template (oKZ388), 100 μM dNTPs and 2.5 μM FAM-γATP (γATP) or γGTP. j. Human Pri1 prefers GTP over ATP as the primer initiation base. Reactions containing 1, 2, 4 and 8 μM protein were incubated with 1 μM DNA template (oKZ388), 10 μM γGTP or γATP and 100 μM CTP, ATP and UTP or 100 μM CTP, GTP and UTP, respectively, at 25 °C for 30 min. Results shown in panel b and f-j are representative of three independent repeats. ‘C’ indicates control reaction without protein. Oligonucleotide (Nts) length marker is shown on the left of the gels.

Extended Data Fig. 9 Qualitative gel-based analysis of purified proteins.

a. MpCAPP fragments and FL mutants. 1 µg of each purified MpCAPP variant was resolved on 12% SDS-PAGE and Coomassie stained. Note: FL WT, FL CC and ΔTPR fragment are fused to MBP. FL WT – full-length wild type WT, FL AxA – full-length D177A, D179A, ΔCTD – aa1-360, ΔTPR – aa100-546, PP – aa100-360, FL CC – full-length C462S, C464S. b. Mutants of MpCAPP PP domain. 1 µg of each purified MpCAPP PP mutant was resolved using 12% SDS-PAGE and Coomassie stained. AxA – D177A, D179A, RR – R142A, R143A, KK – K181A, K182A, KQN – K264A, Q265A, N274A. c. MsCAPP fragments. 2 µg of purified fragments of MsCAPP100-359 and MsCAPPaa111-328 were resolved using 12% SDS-PAGE and Coomassie stained. d. HsPrimPol full-length (HsFL) and HsPrimPol1-354 fragment (HsPP) and mutants. 2 µg of each purified variant was resolved using 12% SDS-PAGE and Coomassie stained. e. HsPP WT and mutants. 2 µg of each purified mutant was resolved on 12% SDS-PAGE and Coomassie stained. Note: AxA – D114A, E116A, RNR – R286A, N287A, R288A. f. Eukaryotic PrimPols. 2 µg of each purified PP was resolved using 15% SDS-PAGE and Coomassie stained. g. HsPri1. 1 µg of HsPri1 wild-type (HsPri1) or HsPri1 D109A, D111A, D306A (HsPri1AAA) was resolved using 12% SDS-PAGE and Coomassie stained.

Extended Data Table 1 X-Ray Data Collection and Structure Refinement Statistics

Supplementary information

Supplementary Information

This file contains Supplementary Fig. 1 (uncropped gels used in the main figures and extended data figures), Supplementary Note 1 (gBlock sequences of MsCAPP100–359 and HsPri1) and Supplementary Note 2 (protein sequence of the X. tropicalis PrimPol used in this study).

Reporting Summary

Supplementary Table 1

Constructs and oligonucleotide revision.

Supplementary Table 2

Raw data used in graphs.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, A.W.H., Zabrady, K., Bainbridge, L.J. et al. Molecular basis for the initiation of DNA primer synthesis. Nature 605, 767–773 (2022). https://doi.org/10.1038/s41586-022-04695-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41586-022-04695-0

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing