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Human PrimPol is a highly error-prone polymerase regulated by single-stranded DNA binding proteins

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posted on 2023-06-08, 20:27 authored by Thomas A Guilliam, Stanislaw K Jozwiakowski, Aaron Ehlinger, Ryan P Barnes, Sean G Rudd, Laura BaileyLaura Bailey, J Mark Skehel, Kristin A Eckert, Walter J Chazin, Aidan DohertyAidan Doherty
PrimPol is a recently identified polymerase involved in eukaryotic DNA damage tolerance, employed in both re-priming and translesion synthesis mechanisms to bypass nuclear and mitochondrial DNA lesions. In this report, we investigate how the enzymatic activities of human PrimPol are regulated. We show that, unlike other TLS polymerases, PrimPol is not stimulated by PCNA and does not interact with it in vivo. We identify that PrimPol interacts with both of the major single-strand binding proteins, RPA and mtSSB in vivo. Using NMR spectroscopy, we characterize the domains responsible for the PrimPol-RPA interaction, revealing that PrimPol binds directly to the N-terminal domain of RPA70. In contrast to the established role of SSBs in stimulating replicative polymerases, we find that SSBs significantly limit the primase and polymerase activities of PrimPol. To identify the requirement for this regulation, we employed two forward mutation assays to characterize PrimPol's replication fidelity. We find that PrimPol is a mutagenic polymerase, with a unique error specificity that is highly biased towards insertion-deletion errors. Given the error-prone disposition of PrimPol, we propose a mechanism whereby SSBs greatly restrict the contribution of this enzyme to DNA replication at stalled forks, thus reducing the mutagenic potential of PrimPol during genome replication.

Funding

Molecular basis for repairing DNA double-strand breaks by non homologous end-joining; G0887; BBSRC-BIOTECHNOLOGY & BIOLOGICAL SCIENCES RESEARCH COUNCIL; BB/J018643/1

Cell cycle regulation of the NHEJ DNA double-strand break repair pathway in eukaryotes; G1554; BBSRC; BB/M004236/1

History

Publication status

  • Published

File Version

  • Published version

Journal

Nucleic Acids Research

ISSN

0305-1048

Publisher

Oxford University Press

Issue

2

Volume

43

Page range

1056-1068

Department affiliated with

  • Sussex Centre for Genome Damage Stability Publications

Full text available

  • Yes

Peer reviewed?

  • Yes

Legacy Posted Date

2015-03-27

First Open Access (FOA) Date

2015-03-27

First Compliant Deposit (FCD) Date

2015-03-27

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