RPA activates the XPF-ERCC1 endonuclease to initiate processing of DNA interstrand crosslinks

Ummi B Abdullah, Joanna F McGouran, Sanja Brolih, Denis Ptchelkine, Afaf H El‐Sagheer, Tom Brown, Peter J McHugh

Research output: Contribution to journalArticlepeer-review

44 Scopus citations

Abstract

During replication-coupled DNA interstrand crosslink (ICL) repair, the XPF-ERCC1 endonuclease is required for the incisions that release, or “unhook”, ICLs, but the mechanism of ICL unhooking remains largely unknown. Incisions are triggered when the nascent leading strand of a replication fork strikes the ICL. Here, we report that while purified XPF-ERCC1 incises simple ICL-containing model replication fork structures, the presence of a nascent leading strand, modelling the effects of replication arrest, inhibits this activity. Strikingly, the addition of the single-stranded DNA (ssDNA)-binding replication protein A (RPA) selectively restores XPF-ERCC1 endonuclease activity on this structure. The 5′–3′ exonuclease SNM1A can load from the XPF-ERCC1-RPA-induced incisions and digest past the crosslink to quantitatively complete the unhooking reaction. We postulate that these collaborative activities of XPF-ERCC1, RPA and SNM1A might explain how ICL unhooking is achieved in vivo.
Original languageEnglish (US)
Pages (from-to)2047-2060
Number of pages14
JournalThe EMBO Journal
Volume36
Issue number14
DOIs
StatePublished - Jun 12 2017
Externally publishedYes

Bibliographical note

KAUST Repository Item: Exported on 2020-10-01
Acknowledgements: We thank our laboratory member Sook Y. Lee and Opher Gileadi (Structural Genomics Consortium, Oxford) for the recombinant SNM1A; Fumiko Esashi (Dunn School of Pathology, University of Oxford) and Marc Wold (Iowa State University, USA) for the RPA and RPA70ΔC442, respectively; Hannah Baddock (WIMM), Hazel Aitkenhead and Joseph A. Newman (Structural Genomics Consortium, University of Oxford) for assistance with fluorescence anisotropy. P.J.M. was supported by MRC (Medical Research Council, grant MR/L007665/1). A.H.E.S. and T.B. were supported by BBSRC (Biotechnology and Biological Sciences Research Council, grant BB/J001694/1) BBSRC: sLOLA grant BB/J001694/1 “Extending the boundaries of nucleic acid chemistry” J.F.M. was supported by King Abdullah University of Science and Technology (KAUST) for a grant awarded to B. Nordén. U.B.A. was supported by the Malaysia's King Scholarship (Biasiswa Yang Di-Pertuan Agong).
This publication acknowledges KAUST support, but has no KAUST affiliated authors.

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