Recruitment and positioning determine the specific role of the XPF-ERCC1 endonuclease in interstrand crosslink repair

TY - JOUR

T1 - Recruitment and positioning determine the specific role of the XPF-ERCC1 endonuclease in interstrand crosslink repair

AU - Klein Douwel, Daisy

AU - Hoogenboom, Wouter S

AU - Boonen, Rick Acm

AU - Knipscheer, Puck

N1 - © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

PY - 2017/7/14

Y1 - 2017/7/14

N2 - 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.

AB - 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.

KW - Animals

KW - DNA Repair

KW - DNA Repair Enzymes

KW - Models, Biological

KW - Mutant Proteins

KW - Mutation

KW - Xenopus

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.15252/embj.201695223

DO - 10.15252/embj.201695223

M3 - Article

C2 - 28292785

SN - 0261-4189

VL - 36

SP - 2034

EP - 2046

JO - EMBO Journal

JF - EMBO Journal

IS - 14

ER -