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 ubiquitinbinding 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 DNAdamage 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.endonuclease | mitomycin C | cisplatin | DNA repair I nterstrand cross-links (ICLs) inhibit transcription and replication. Their considerable cytotoxicity has been ascribed primarily to their blockage of replication forks, and this phenomenon is believed to be responsible for the success of ICL-inducing agents, such as cisplatin and mitomycin-C (MMC), in cancer chemotherapy (1). ICL processing is complex, involving proteins from several distinct pathways of DNA metabolism. In higher organisms, ICL processing is orchestrated by the Fanconi anemia (FA) pathway (2, 3). Collision of replication forks with ICLs activates the ATR kinase, which in turn licenses the FANCL ubiquitin ligase subunit of the FA core complex (composed of FANCA, B, C, E, F, G, L and M proteins) to modify the FANCD2-FANCI heterodimer (2, 4-6). The monoubiquitylated FANCD2-FANCI complex is then targeted to chromatin (7,8), where it recruits downstream components of the repairosome, including the structure-specific nuclease FAN1 (9-12). However, ICL processing also requires other enzymes, such as the nucleases MUS81-EME1 and XPF-ERCC1, and how these are recruited to sites of damage in ICL repair is not known.The structure-specific endonucleases XPF-ERCC1 and MUS81-EME1, which are implicated in ICL repair and in the res...

The Fanconi anemia ID2 complex: Dueling saxes at the crossroads

“…[156][157][158][159][160] Similar to FAN1 and FANCD2, FANCP/SLX4 and FANCD2 co-localize in nuclear foci and co-immunoprecipitate. 154 While Yamamoto and colleagues established that the FANCP/SLX4 UBZ domain and the monoubiquitination of FANCD2 are required for FANCP/SLX4 targeting to nuclear foci, a recent report has contradicted these findings and shown that SLX4 recruitment occurs independent of FANCD2 monoubiquitination. 154,161 Taken together, these findings suggest that one major function for chromatin-bound monoubiquitinated FANCD2 may be the recruitment of structure-specific endonucleases that mediate key incision steps during the process of ICL repair.…”

“…145 While FAN1 mutations have yet to be uncovered in FA patients, mutations in FAN1 have been identified as a cause of karyomegalic interstitial nephritis, a chronic kidney disease. 151 Mutations in the SLX4 gene were recently uncovered in FA complementation group P. [152][153][154] FANCP/SLX4 is a »200 kDa multidomain protein that, similar to FAN1, harbors an aminoterminal UBZ domain.…”

The Fanconi anaemia pathway orchestrates incisions at sites of crosslinked DNA

“…Disruption of Slx4 in chicken cells has revealed it to be cellularly essential-at odds with yeast, mouse and human systems [18,56,57,128,129]. This may be a peculiarity of chicken, due to its abnormal complement of nucleases-as it lacks both Mus81 and Ercc1 but has orthologues of Xpf and Eme1.…”

“…This may be a peculiarity of chicken, due to its abnormal complement of nucleases-as it lacks both Mus81 and Ercc1 but has orthologues of Xpf and Eme1. In this system Xpf is cellularly essential, which is also in contrast to human, mouse and yeast [114,128,130]. This makes the analysis of nucleases involved in crosslinking very difficult in chicken cells.…”

Low prevalence of SLX4 loss-of-function mutations in non-BRCA1/2 breast and/or ovarian cancer families

“…Moreover, this variant targets the UBZ4 domain (Ubiquiting-Binding Zinc finger motif 4) essential for SLX4 interaction with multiple-partner proteins, 14 and recruitment to sites of DNA damage. 16 Indeed, four FA patients harbor SLX4 splicing mutations causing in-frame exon 5 skipping, 8,9 supporting that the UBZ4 domain (encoded by exon 5) is clinically relevant. Other variants do not appear to represent disease-causing mutations, in particular those detected in control samples, but some might represent low-or moderate-risk alleles contributing to the overall cancer risk.…”