Double-strand breaks induce homologous recombinational repair of interstrand cross-links via cooperation of MSH2, ERCC1-XPF, REV3, and the Fanconi anemia pathway.
DNA interstrand cross-linking agents have been widely used in chemotherapeutic treatment of cancer. The majority of interstrand cross-links (ICLs) in mammalian cells are removed via a complex process that involves the formation of double-strand breaks at replication forks, incision of the ICL, and subsequent error-free repair by homologous recombination. How double-strand breaks effect the removal of ICLs and the downstream homologous recombination process is not clear. Here, we describe a plasmid-based recombination assay in which one copy of the CFP gene is inactivated by a site-specific psoralen ICL and can be repaired by gene conversion with a mutated homologous donor sequence. We found that the homology-dependent recombination (HDR) is inhibited by the ICL. However, when we introduced a double-strand break adjacent to the site of the ICL, the removal of the ICL was enhanced and the substrate was funneled into a HDR repair pathway. This process was not dependent on the nucleotide excision repair pathway, but did require the ERCC1-XPF endonuclease and REV3. In addition, both the Fanconi anemia pathway and the mismatch repair protein MSH2 were required for the recombinational repair processing of the ICL. These results suggest that the juxtaposition of an ICL and a DSB stimulates repair of ICLs through a process requiring components of mismatch repair, ERCC1-XPF, REV3, Fanconi anemia proteins, and homologous recombination repair factors.
Department of Cancer Genetics, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, United States.
Nianxiang
N
Zhang
Xiuping
X
Liu
Lei
L
Li
Randy
R
Legerski
D000818:Animals; D002478:Cells, Cultured; D006224:Cricetinae; D003432:Cross-Linking Reagents / Q000633:toxicity*; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism; D005199:Fanconi Anemia / Q000378:metabolism; D051856:Fanconi Anemia Complementation Group Proteins / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D051718:MutS Homolog 2 Protein / Q000378:metabolism; D011995:Recombination, Genetic; D015398:Signal Transduction
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D051856:Fanconi Anemia Complementation Group Proteins; C095888:xeroderma pigmentosum group F protein; D004259:DNA-Directed DNA Polymerase; C118285:REV3L protein, human; C071447:ERCC1 protein, human; D004720:Endonucleases; C497172:MSH2 protein, human; D051718:MutS Homolog 2 Protein