Motif WFYY of human PrimPol is crucial to stabilize the incoming 3′-nucleotide during replication fork restart

Author:

Calvo Patricia A1,Martínez-Jiménez María I1,Díaz Marcos2,Stojkovic Gorazd3,Kasho Kazutoshi34,Guerra Susana1,Wanrooij Sjoerd3ORCID,Méndez Juan2ORCID,Blanco Luis1ORCID

Affiliation:

1. Centro de Biología Molecular Severo Ochoa, CSIC-UAM, 28049, Madrid, Spain

2. Spanish National Cancer Research Centre (CNIO), 28029, Madrid, Spain

3. Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden

4. Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan

Abstract

Abstract PrimPol is the second primase in human cells, the first with the ability to start DNA chains with dNTPs. PrimPol contributes to DNA damage tolerance by restarting DNA synthesis beyond stalling lesions, acting as a TLS primase. Multiple alignment of eukaryotic PrimPols allowed us to identify a highly conserved motif, WxxY near the invariant motif A, which contains two active site metal ligands in all members of the archeo-eukaryotic primase (AEP) superfamily. In vivo and in vitro analysis of single variants of the WFYY motif of human PrimPol demonstrated that the invariant Trp87 and Tyr90 residues are essential for both primase and polymerase activities, mainly due to their crucial role in binding incoming nucleotides. Accordingly, the human variant F88L, altering the WFYY motif, displayed reduced binding of incoming nucleotides, affecting its primase/polymerase activities especially during TLS reactions on UV-damaged DNA. Conversely, the Y89D mutation initially associated with High Myopia did not affect the ability to rescue stalled replication forks in human cells. Collectively, our data suggest that the WFYY motif has a fundamental role in stabilizing the incoming 3′-nucleotide, an essential requisite for both its primase and TLS abilities during replication fork restart.

Funder

MINECO

MCI

AEI

FEDER

Kempe JCK

Knut och Alice Wallenbergs Foundation

Fundación Ramón Areces

Banco de Santander to the Centro de Biología Molecular Severo Ochoa

Ministry of Economy and Competitiveness

Publisher

Oxford University Press (OUP)

Subject

Genetics

Reference36 articles.

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2. Eukaryotic DNA primase;Arezi;Trends Biochem. Sci.,2000

3. Repriming of DNA synthesis at stalled replication forks by human PrimPol;Mouron;Nat. Struct. Mol. Biol.,2013

4. PrimPol, an archaic primase/polymerase operating in human cells;Garcia-Gomez;Mol. Cell,2013

5. PrimPol is required for replication reinitiation after mtDNA damage;Torregrosa-Muñumer;Proc. Natl. Acad. Sci. U.S.A.,2017

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