RPA activates the XPF ‐ ERCC 1 endonuclease to initiate processing of DNA interstrand crosslinks

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.

A hydroxamic-acid-containing nucleoside inhibits DNA repair nuclease SNM1A

“…SNM1A is a DNA damage repair enzyme implicated in interstrand crosslink (ICL) repair. 1 It digests DNA with a 5′-to-3′ polarity past lesions by hydrolysing the phosphodiester backbone of DNA, producing predominantly mononucleotide products. 2 ICL repair factors are associated with the ageing process, 1 certain genetic diseases, 3 and resistance to cancer therapy.…”

ERCC1–XPF targeting to psoralen–DNA crosslinks depends on XPA and FANCD2

“…Moreover, we were unable to co-immunoprecipitate FANCD2 with XPF, suggesting that these proteins do not interact directly. Because in vitro ERCC1-XPF activity in ICLR is stimulated by RPA [27,57], we also tested whether RPA directly interacts with wild-type or R689S or S786F mutant XPF. However, GFP-tagged XPF was unable to co-immunoprecipitate RPA70, the largest subunit of the heterotrimeric RPA complex, suggesting that stimulation of XPF activity by RPA is not via a direct physical interaction between the proteins.…”

“…Experiments using Xenopus laevis egg extract and in vitro-modeled ICLcontaining replication structures suggest that ERCC1-XPF is then recruited, depending on its interaction with the scaffold protein SLX4 that stimulates its function [2,25,26]. ERCC1-XPF incises the lagging strand to unhook the ICL, possibly together with another endonuclease or together with the exonuclease SNM1A that digests past the ICL [27]. The resulting single-stranded gap is filled by TLS and is used as homology template for repair of the remaining doublestrand break by HR [28,29].…”

XPF–ERCC1: Linchpin of DNA crosslink repair

“…These might involve alternative initiating endonucleases (MUS81 or SLX1, for example [23]) leading to homologous recombination steps requiring XPF-ERCC1 for their successful completion. Moreover, the FAN1 (Fanconi-associated nuclease 1) nuclease, mismatch repair system, and the SNM1A (sensitive to nitrogen mustard 1A) exonuclease have all been implicated in ICL recognition and processing [24][25][26] and are candidates for mediating any XPF-ERCC1-dependent pathway, noting that the cell-cycle phase might be critical (the study of ICL repair outside the synthesis phase [S-phase] has been relatively neglected). Fortunately, the last decade has seen the development of elegant cell-free ICL-repair systems using Xenopus cell extracts [26,27] and structural-and biochemical-reconstitution approaches that will help accelerate our mechanistic understanding of new pathways as they are revealed.…”