Point mutations V546E and D547H of the RBM-B motif does not affect the binding of PrimPol to RPA and DNA

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Abstract

The human primase-polymerase PrimPol is a key participant of the mechanism of DNA synthesis restart during replication fork stalling at DNA damaged sites. PrimPol has a DNA primase activity and synthesizes DNA primers that are used by processive DNA polymerases to continue replication. Recruitment of PrimPol to the sites of DNA damage, as well as catalytic activity stimulation depends on interaction with the replicative protein RPA, which binds single-stranded DNA. The C-terminal domain of PrimPol contains a negatively charged RPA-binding motif (RBM), which mutations disrupt the interaction between two proteins. The RBM motif also plays a role in the negative regulation of PrimPol interaction with DNA. Deletion of RBM dramatically increases PrimPol affinity to DNA and stimulates PrimPol activity. The mechanism of RBM-mediated regulation of PrimPol activity is unclear. The relatively strong negative charge of RBM potentially may contribute to the interaction of PrimPol with RPA and DNA. RBM contains two negatively charged regions RBM-A and RBM-B. In this work, we additionally added (substitution V546E) or decreased (substitution D547H) the negative charge in RBM-B PrimPol and characterized these mutant variants biochemically. It was shown that the local change of RBM-B charge has no effect on the interaction of PrimPol with DNA and RPA, as well as the catalytic activity of the enzyme.

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About the authors

A. A. Manukyan

National Research Center “Kurchatov Institute”

Email: lizaboldinova@yandex.ru
Russian Federation, Moscow, 123182

A. V. Makarova

National Research Center “Kurchatov Institute”; Institute of Gene Biology, Russian Academy of Sciences

Email: lizaboldinova@yandex.ru
Russian Federation, Moscow, 123182; Moscow, 119334

E. O. Boldinova

National Research Center “Kurchatov Institute”; Institute of Gene Biology, Russian Academy of Sciences

Author for correspondence.
Email: lizaboldinova@yandex.ru
Russian Federation, Moscow, 123182; Moscow, 119334

References

  1. García-Gómez S., Reyes A., Martínez-Jiménez M.I., Chocrón S., Mourón S., Terrados G., Powell C., Salido E., Méndez J., Holt I.J., Blanco L. (2013) PrimPol, an archaic primase/polymerase operating in human cells. Mol. Cell. 52, 541–553.
  2. Bianchi J., Rudd S.G., Jozwiakowski S.K., Bailey L.J., Soura V., Taylor E., Stevanovic I., Green A.J., Stracker T.H., Lindsay H.D., Doherty A.J. (2013) Primpol bypasses UV photoproducts during eukaryotic chromosomal DNA replication. Mol. Cell. 52, 566–573.
  3. Wan L., Lou J., Xia Y., Su B., Liu T., Cui J., Sun Y., Lou H., Huang J. (2013) HPrimpol1/CCDC111 is a human DNA primase-polymerase required for the maintenance of genome integrity. EMBO Rep. 14, 1104–1112.
  4. Iyer L.M., Koonin E.V., Leipe D.D., Aravind L. (2005) Origin and evolution of the archaeo-eukaryotic primase superfamily and related palm-domain proteins: structural insights and new members. Nucl. Acids Res. 33, 3875–3896.
  5. Martínez-Jiménez M.I., Calvo P.A., García-Gómez S., Guerra-González S., Blanco L. (2018) The Zn-finger domain of human PrimPol is required to stabilize the initiating nucleotide during DNA priming. Nucl. Acids Res. 46, 4138–4151.
  6. Schiavone D., Jozwiakowski S.K., Romanello M., Guilbaud G., Guilliam T.A., Bailey L.J., Sale J.E., Doherty A.J. (2016) PrimPol is required for replicative tolerance of G quadruplexes in vertebrate cells. Mol. Cell. 61, 161–169.
  7. Mehta K.P.M., Thada V., Zhao R., Krishnamoorthy A., Leser M., Rose K.L., Cortez D. (2022) CHK1 phosphorylates PRIMPOL to promote replication stress tolerance. Sci. Adv. 8, eabm0314.
  8. Guilliam T.A., Jozwiakowski S.K., Ehlinger A., Barnes R.P., Rudd S.G., Bailey L.J., Skehel J.M., Eckert K.A., Chazin W.J., Doherty A.J. (2015) Human PrimPol is a highly error-prone polymerase regulated by single-stranded DNA binding proteins. Nucl. Acids Res. 43, 1056–1068.
  9. Guilliam T.A., Brissett N.C., Ehlinger A., Keen B.A., Kolesar P., Taylor E., Bailey L.J., Lindsay H.D., Chazin W.J., Doherty A.J. (2017) Molecular basis for PrimPol recruitment to replication forks by RPA. Nat. Commun. 8, 15222.
  10. Martínez-Jiménez M.I., Lahera A., Blanco L. (2017) Human PrimPol activity is enhanced by RPA. Sci. Rep. 7, 783.
  11. Bailey L.J., Bianchi J., Hégarat N., Hochegger H., Doherty A.J. (2016) PrimPol-deficient cells exhibit a pronounced G2 checkpoint response following UV damage. Cell Cycle. 15, 908–918.
  12. Bailey L.J., Bianchi J., Doherty A.J. (2019) PrimPol is required for the maintenance of efficient nuclear and mitochondrial DNA replication in human cells. Nucl. Acids Res. 47, 4026–4038.
  13. Kobayashi K., Guilliam T.A., Tsuda M., Yamamoto J., Bailey L.J., Iwai S., Takeda S., Doherty A.J., Hirota K. (2016) Repriming by PrimPol is critical for DNA replication restart downstream of lesions and chain-terminating nucleosides. Cell Cycle. 15, 1997–2008.
  14. Mourón S., Rodriguez-Acebes S., Martínez-Jiménez M.I., García-Gómez S., Chocrón S., Blanco L., Méndez J. (2013) Repriming of DNA synthesis at stalled replication forks by human PrimPol. Nat. Struct. Mol. Biol. 20, 1383–1389.
  15. Zhao F., Wu J., Xue A., Su Y., Wang X., Lu X., Zhou Z., Qu J., Zhou X. (2013) Exome sequencing reveals CCDC111 mutation associated with high myopia. Hum. Genet. 132, 913–921.
  16. Keen B.A., Bailey L.J., Jozwiakowski S.K., Doherty A.J. (2014) Human PrimPol mutation associated with high myopia has a DNA replication defect. Nucl. Acids Res. 42, 12102–12111.
  17. Kasamo K., Nakamura M., Daimou Y., Sano A. (2020) A PRIMPOL mutation and variants in multiple genes may contribute to phenotypes in a familial case with chronic progressive external ophthalmoplegia symptoms. Neurosci. Res. 157, 58–63.
  18. Yuan H., Wang Q., Li Y., Cheng S., Liu J., Liu Y. (2020) Concurrent pathogenic variants in SLC6A1/NOTCH1/PRIMPOL genes in a Chinese patient with myoclonic-atonic epilepsy, mild aortic valve stenosis and high myopia. BMC Med. Genet. 21, 93.
  19. Duong V.N., Zhou L., Martínez-Jiménez M.I., He L., Cosme M., Blanco L., Paintsil E., Anderson K.S. (2020) Identifying the role of PrimPol in TDF-induced toxicity and implications of its loss of function mutation in an HIV+patient. Sci. Rep. 10, 9343.
  20. Díaz-Talavera A., Calvo P.A., González-Acosta D., Díaz M., Sastre-Moreno G., Blanco-Franco L., Guerra S., Martínez-Jiménez M.I., Méndez J., Blanco L. (2019) A cancer-associated point mutation disables the steric gate of human PrimPol. Sci. Rep. 9, 1121.
  21. Quinet A., Tirman S., Jackson J., Šviković S., Lemaçon D., Carvajal-Maldonado D., González-Acosta D., Vessoni A.T., Cybulla E., Wood M., Tavis S., Batista L.F.Z., Méndez J., Sale J.E., Vindigni A. (2019) PRIMPOL-mediated adaptive response suppresses replication fork reversal in BRCA-deficient сells. Mol. Cell 77, 461–474.
  22. Keen B.A., Jozwiakowski S.K., Bailey L.J., Bianchi J., Doherty A.J. (2014) Molecular dissection of the domain architecture and catalytic activities of human PrimPol. Nucl. Acids Res. 42, 5830–5845.
  23. Rechkoblit O., Gupta Y.K., Malik R., Rajashankar K.R., Johnson R.E., Prakash L., Prakash S., Aggarwal A.K. (2016) Structure and mechanism of human PrimPol, a DNA polymerase with primase activity. Sci. Adv. 2, e1601317.
  24. Boldinova E.O., Baranovskiy A.G., Gagarinskaya D.I., Manukyan А.А., Makarova A.V., Tahirov T.H. (2023) The role of catalytic and regulatory domains of human PrimPol in DNA binding and synthesis. Nucl. Acids Res. 51, 7541–7551.
  25. Boldinova E.O., Stojkovic G., Khairullin R., Wanrooij S., Makarova A.V. (2017) Optimization of the expression, purification and polymerase activity reaction conditions of recombinant human PrimPol. PLoS One. 12, e0184489.
  26. Binz S.K., Dickson A.M., Haring S.J., Wold M.S. (2006) Functional assays for replication protein A (RPA). Methods Enzymol. 409, 11–18.
  27. Болдинова Е.О., Макарова А.В. (2023) Регуляция ДНК-праймазы-полимеразы PrimPol человека. Биохимия. 88, 1392–1411.
  28. Boldinova E.O., Belousova E.A., Gagarinskaya D.I., Maltseva E.A., Khodyreva S.N., Lavrik O.I., Makarova A.V. (2020) Strand displacement activity of primpol. Int. J. Mol. Sci. 21, 9027.
  29. Boldinova E.O., Ghodke P.P., Sudhakar S., Mishra V.K., Manukyan A.A., Miropolskaya N., Pradeepkumar P.I., Makarova A.V. (2022) Translesion synthesis across the N2-ethyl-deoxyguanosine adduct by human PrimPol. ACS Chem. Biol. 17, 3238–3250.
  30. Liu H., Naismith J.H. (2008) An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnol. 8, 91.

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3. Fig. 1. Comparison of the catalytic activity of wild-type PrimPol and the V546E and D547H variants. a – Alignment of the amino acid sequences of RBM-A and RBM-B PrimPol from organisms of different taxa. Negatively charged Asp and Glu residues are marked in red. Orange stars indicate the PrimPol residues that form bonds with the RPA70 subunit, according to the structure [9]. б – DNA polymerase activity of PrimPol and variants with V546E and D547H substitutions. Reactions were carried out in the presence of 20/50/100/200/400/800 nM PrimPol for 10 min. в – DNA polymerase activity of PrimPol and its variants depending on the reaction time, as well as the kobs parameter value. Reactions were carried out in the presence of 200 nM PrimPol for 2/5/10/30/60/120 min. г – Total DNA primase activity of PrimPol and variants with V546E and D547H substitutions. Reactions were carried out in the presence of [γ-32P]ATP, ATP, dGTP and dTTP for 30/60/90 min. д – Formation of dinucleotide of wild-type PrimPol and variants V546E and D547H. Reactions were carried out in the presence of [γ-32P]ATP, ATP and dGTP for 30 min.

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4. Fig. 2. Shift analysis of the PrimPol complex of wild type and variants with V546E and D547H substitutions with DNA in a native gel. Mean values ​​and standard errors are shown.

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5. Fig. 3. Analysis of the effect of V546E and D547H substitutions on the interaction of PrimPol with the replicative protein RPA. a – Formation of the ternary RPA:DNA:PrimPol complex in 5% native gel. б – Electropherogram of the primer extension reactions. DNA polymerase reactions were carried out in the presence of 100 nM PrimPol or its variants for 5/10/30 min. c – Graphs reflecting the efficiency of primer extension depending on the reaction time. Mean values ​​and standard errors are shown. г – Coprecipitation of wild-type PrimPol and the V546E and D547H variants with the RPA protein. The red arrow indicates the position of the band of the RPA70 subunit that formed a complex with GST-PrimPol.

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