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Molecular basis for PrimPol recruitment to replication forks by RPA

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Version 2 2023-06-12, 08:41
Version 1 2023-06-09, 06:21
journal contribution
posted on 2023-06-12, 08:41 authored by Thomas A Guilliam, Nigel Brissett, Aaron Ehlinger, Benjamin A Keen, Peter Kolesar, Elaine Taylor, Laura BaileyLaura Bailey, Howard Lindsay, Walter J Chazin, Aidan DohertyAidan Doherty
DNA damage and secondary structures can stall the replication machinery. Cells possess numerous tolerance mechanisms to complete genome duplication in the presence of such impediments. In addition to translesion synthesis (TLS) polymerases, most eukaryotic cells contain a multi-functional replicative enzyme called Primase-Polymerase (PrimPol) that is capable of directly bypassing DNA damage by TLS, as well as repriming replication downstream of impediments. Here, we report that PrimPol is recruited to reprime through its interaction with RPA. Using biophysical and crystallographic approaches, we identify that PrimPol possesses two RPA-binding motifs and ascertained the key residues required for these interactions. We demonstrate that one of these motifs is critical for PrimPol's recruitment to stalled replication forks in vivo. In addition, biochemical analysis reveals that RPA serves to stimulate the primase activity of PrimPol. Together, these findings provide significant molecular insights into PrimPol's mode of recruitment to stalled forks to facilitate repriming and restart.

Funding

The role of a novel family of eukaryotic DNA polymerases in mitochondrial DNA replication; G0207; BBSRC-BIOTECHNOLOGY & BIOLOGICAL SCIENCES RESEARCH COUNCIL; BB/H019723/1

Cell cycle regulation of the NHEJ DNA double-strand break repair pathway in eukaryotes; G1554; BBSRC-BIOTECHNOLOGY & BIOLOGICAL SCIENCES RESEARCH COUNCIL; BB/M004236/1

Understanding the role of PrimPol in damage tolerance during genome replication in eukaryotic cells; G1621; BBSRC-BIOTECHNOLOGY & BIOLOGICAL SCIENCES RESEARCH COUNCIL; BB/M008800/1

History

Publication status

  • Published

File Version

  • Published version

Journal

Nature Communications

ISSN

2041-1723

Publisher

Nature Publishing Group

Volume

8

Article number

a15222

Department affiliated with

  • Sussex Centre for Genome Damage Stability Publications

Research groups affiliated with

  • Genome Damage and Stability Centre Publications

Full text available

  • Yes

Peer reviewed?

  • Yes

Legacy Posted Date

2017-05-22

First Open Access (FOA) Date

2017-06-14

First Compliant Deposit (FCD) Date

2017-05-22

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