Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
Proc Natl Acad Sci U S A. 2011 Apr 19;108(16):6492-6. doi: 10.1073/pnas.1018487108. Epub 2011 Apr 4.
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.
链间交联（ICLs）会阻断复制和转录，因此具有高度细胞毒性。在高等真核生物中，ICLs 的处理涉及范可尼贫血（FA）途径和同源重组。停滞的复制叉激活了包含 8 个亚基的 FA 核心复合物，该复合物使 FANCD2-FANCI 泛素化。一旦发生翻译后修饰，这种异二聚体就会招募 ICL 修复体的下游成员，包括 FAN1 核酸内切酶。然而，已经表明 ICL 处理还涉及 MUS81-EME1 和 XPF-ERCC1 核酸内切酶，这些核酸内切酶已知与 SLX4 相互作用，SLX4 是一种对接蛋白，也可以与另一种核酸内切酶 SLX1 结合。为了更深入地研究 SLX4 的作用，我们在禽类 DT40 细胞中破坏了 SLX4 基因。SLX4 缺陷导致与广泛染色体异常相关的细胞死亡，包括相当一部分等臂型断裂，姐妹染色单体在同一部位断裂。因此，SLX4 似乎在细胞增殖中发挥着至关重要的作用，可能是通过促进染色体内同源重组中间体的解决。由于泛素化在 FA 途径中起着关键作用，并且 SLX4 的 N 端区域包含一个泛素结合锌指（UBZ）结构域，我们询问这个结构域是否是 ICL 处理所必需的。我们发现表达缺乏 UBZ 的 SLX4 的 SLX4(-/-)细胞对 ICL 诱导剂具有选择性敏感性，并且 UBZ 结构域对于 SLX4 与泛素化的 FANCD2 的相互作用以及其招募到 ICL 诱导剂产生的 DNA 损伤焦点都是必需的。因此，我们的研究结果表明，泛素化的 FANCD2 招募 SLX4 到 DNA 损伤部位，在那里它介导 ICL 处理过程中产生的重组中间体的解决。
