Recruitment and positioning determine the specific role of the XPF ‐ ERCC 1 endonuclease in interstrand crosslink repair

Abstract

XPF‐ERCC1 is a structure‐specific endonuclease pivotal for several DNA repair pathways and, when mutated, can cause multiple diseases. Although the disease‐specific mutations are thought to affect different DNA repair pathways, the molecular basis for this is unknown. Here we examine the function of XPF‐ERCC1 in DNA interstrand crosslink (ICL) repair. We used Xenopus egg extracts to measure both ICL and nucleotide excision repair, and we identified mutations that are specifically defective in ICL repair. One of these separation‐of‐function mutations resides in the helicase‐like domain of XPF and disrupts binding to SLX4 and recruitment to the ICL. A small deletion in the same domain supports recruitment of XPF to the ICL, but inhibited the unhooking incisions most likely by disrupting a second, transient interaction with SLX4. Finally, mutation of residues in the nuclease domain did not affect localization of XPF‐ERCC1 to the ICL but did prevent incisions on the ICL substrate. Our data support a model in which the ICL repair‐specific function of XPF‐ERCC1 is dependent on recruitment, positioning and substrate recognition.

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

“…Furthermore, the Xenopus cell-free replication-coupled repair system demonstrated that ICL unhooking is abolished when XPF is immunodepleted, but not MUS81 or FAN1 (Klein Douwel et al, 2014). Additionally, in vitro reconstitution assays have shown that XPF-ERCC1 alone is able to perform dual incisions flanking an ICL located near the ssDNA/dsDNA junction of a splayed arm "forklike" (simple fork) DNA substrate, or in conjunction with the nuclease scaffold protein SLX4 (Kuraoka et al, 2000;Hodskinson et al, 2014;Klein Douwel et al, 2014), where SLX4 plays a key role in recruiting and positioning XPF-ERCC1 for incision (Klein Douwel et al, 2017). Finally, in the context of double-stranded DNA substrates, XPF-ERCC1 has been reported to act in an 3 0 -to-5 0 exonuclease-like fashion, digesting past a site-specific ICL (Mu et al, 2000).…”

Cryo-EM structures of the XPF-ERCC1 endonuclease reveal how DNA-junction engagement disrupts an auto-inhibited conformation

“…In this context XPF-ERCC1 nicks the damaged DNA strand 5′ of the lesion at the ds/ss junction of an NER repair bubble. It is also required for interstrand cross-link repair (ICLR), some double-stranded break repair processes, base excision repair, Holliday junction resolution, gene-conversion and telomere maintenance [4][5][6][7][8][9][10] . Mutations in XPF and ERCC1 genes are associated with genetic disorders exhibiting diverse phenotypes.…”

HSF2BP Negatively Regulates Homologous Recombination in DNA Interstrand Crosslink Repair in Human Cells by Direct Interaction With BRCA2

“…Incision assay was performed as described previously (Klein Douwel et al, 2014; 2017). Briefly, pICL (225 ng) and pQuant (11.3 ng) were incubated with 1.5 units NB-BSR DI enzyme (NEB) in 1× NEBuffer 2.1 for 30 min at room temperature.…”