title: Recruitment and positioning determine the specific role of the XPF-ERCC1 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 usedXenopusegg 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.
authors.affiliation: Hubrecht Institute - KNAW, University Medical Center Utrecht & Cancer GenomiCs Netherlands, Utrecht, The Netherlands.
authors.forename: Daisy
authors.initials: D
authors.lastname: Klein Douwel
authors.affiliation: Hubrecht Institute - KNAW, University Medical Center Utrecht & Cancer GenomiCs Netherlands, Utrecht, The Netherlands.
authors.forename: Wouter S
authors.initials: WS
authors.lastname: Hoogenboom
authors.affiliation: Hubrecht Institute - KNAW, University Medical Center Utrecht & Cancer GenomiCs Netherlands, Utrecht, The Netherlands.
authors.forename: Rick Acm
authors.initials: RA
authors.lastname: Boonen
authors.affiliation: Hubrecht Institute - KNAW, University Medical Center Utrecht & Cancer GenomiCs Netherlands, Utrecht, The Netherlands p.knipscheer@hubrecht.eu.
authors.forename: Puck
authors.identifier: 0000-0003-4198-0132
authors.initials: P
authors.lastname: Knipscheer
mesh_terms: D000818:Animals; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000235:genetics* / Q000378:metabolism*; D008954:Models, Biological; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D014981:Xenopus
chemical_list: D050505:Mutant Proteins; D045643:DNA Repair Enzymes
keywords: Fanconi anemia; XPF‐ERCC1; Xenopus egg extract; interstrand crosslink repair; nucleotide excision repair