title: Involvement of SLX4 in interstrand cross-link repair is regulated by the Fanconi anemia pathway.
abstract: Interstrand cross-links (ICLs) block replication and transcription and thus are highly cytotoxic. In higher eukaryotes, ICLs processing involves the Fanconi anemia (FA) pathway and homologous recombination. Stalled replication forks activate the eight-subunit FA core complex, which ubiquitylates FANCD2-FANCI. Once it is posttranslationally modified, this heterodimer recruits downstream members of the ICL repairosome, including the FAN1 nuclease. However, ICL processing has been shown to also involve MUS81-EME1 and XPF-ERCC1, nucleases known to interact with SLX4, a docking protein that also can bind another nuclease, SLX1. To investigate the role of SLX4 more closely, we disrupted the SLX4 gene in avian DT40 cells. SLX4 deficiency caused cell death associated with extensive chromosomal aberrations, including a significant fraction of isochromatid-type breaks, with sister chromatids broken at the same site. SLX4 thus appears to play an essential role in cell proliferation, probably by promoting the resolution of interchromatid homologous recombination intermediates. Because ubiquitylation plays a key role in the FA pathway, and because the N-terminal region of SLX4 contains a ubiquitin-binding zinc finger (UBZ) domain, we asked whether this domain is required for ICL processing. We found that SLX4(-/-) cells expressing UBZ-deficient SLX4 were selectively sensitive to ICL-inducing agents, and that the UBZ domain was required for interaction of SLX4 with ubiquitylated FANCD2 and for its recruitment to DNA-damage foci generated by ICL-inducing agents. Our findings thus suggest that ubiquitylated FANCD2 recruits SLX4 to DNA damage sites, where it mediates the resolution of recombination intermediates generated during the processing of ICLs.
authors.affiliation: Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
authors.forename: Kimiyo N
authors.initials: KN
authors.lastname: Yamamoto
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Kobayashi
authors.forename: Masataka
authors.initials: M
authors.lastname: Tsuda
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Kurumizaka
authors.forename: Minoru
authors.initials: M
authors.lastname: Takata
authors.forename: Koichi
authors.initials: K
authors.lastname: Kono
authors.forename: Josef
authors.initials: J
authors.lastname: Jiricny
authors.forename: Shunichi
authors.initials: S
authors.lastname: Takeda
authors.forename: Kouji
authors.initials: K
authors.lastname: Hirota
mesh_terms: D000818:Animals; D016923:Cell Death / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002645:Chickens; D002842:Chromatids / Q000235:genetics / Q000378:metabolism; D002869:Chromosome Aberrations / Q000187:drug effects; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004249:DNA Damage / Q000187:drug effects / Q000502:physiology; D004260:DNA Repair / Q000187:drug effects / Q000502:physiology*; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D052236:Fanconi Anemia Complementation Group D2 Protein / Q000235:genetics / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D045522:Recombinases / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic / Q000187:drug effects / Q000502:physiology*; D054875:Ubiquitination / Q000187:drug effects / Q000502:physiology*; D016335:Zinc Fingers
chemical_list: D003432:Cross-Linking Reagents; D052236:Fanconi Anemia Complementation Group D2 Protein; D046912:Multiprotein Complexes; D045522:Recombinases; D004720:Endonucleases