Klein Douwel Daisy, Hoogenboom Wouter S, Boonen Rick Acm, Knipscheer Puck
Hubrecht Institute - KNAW, University Medical Center Utrecht & Cancer GenomiCs Netherlands, Utrecht, The Netherlands.
Hubrecht Institute - KNAW, University Medical Center Utrecht & Cancer GenomiCs Netherlands, Utrecht, The Netherlands
EMBO J. 2017 Jul 14;36(14):2034-2046. doi: 10.15252/embj.201695223. Epub 2017 Mar 14.
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 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.
XPF-ERCC1是一种对多种DNA修复途径至关重要的结构特异性核酸内切酶，发生突变时可引发多种疾病。尽管人们认为疾病特异性突变会影响不同的DNA修复途径，但其分子基础尚不清楚。在此，我们研究了XPF-ERCC1在DNA链间交联（ICL）修复中的功能。我们使用卵提取物来测量ICL修复和核苷酸切除修复，并鉴定出在ICL修复中存在特异性缺陷的突变。这些功能分离突变之一位于XPF的解旋酶样结构域中，破坏了与SLX4的结合以及向ICL的募集。同一结构域中的一个小缺失支持XPF向ICL的募集，但很可能通过破坏与SLX4的第二种瞬时相互作用而抑制了脱钩切口。最后，核酸酶结构域中残基的突变并不影响XPF-ERCC1在ICL上的定位，但确实阻止了ICL底物上的切口。我们的数据支持一种模型，其中XPF-ERCC1的ICL修复特异性功能取决于募集、定位和底物识别。
