Department of Pathology and Laboratory Medicine, New Jersey Medical School and Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103, USA.
Biochemistry. 2010 Jul 6;49(26):5560-9. doi: 10.1021/bi100584c.
There is evidence that Fanconi anemia (FA) proteins play an important role in the repair of DNA interstrand cross-links (ICLs), but the precise mechanism by which this occurs is not clear. One of the critical steps in the ICL repair process involves unhooking of the cross-link from DNA by incisions on one strand on either side of the ICL and its subsequent removal. The ERCC1-XPF endonuclease is involved in this unhooking step and in the removal of the cross-link. We have previously shown that several of the FA proteins are needed to produce incisions created by ERCC1-XPF at sites of ICLs. To more clearly establish a link between FA proteins and the incision step(s) mediated by ERCC1-XPF, we undertook yeast two-hybrid analysis to determine whether FANCA, FANCC, FANCF, and FANCG directly interact with ERCC1 and XPF and, if so, to determine the sites of interaction. One of these FA proteins, FANCG, was found to have a strong affinity for ERCC1 and a moderate affinity for XPF. FANCG has been shown to contain seven tetratricopeptide repeat (TPR) motifs, which are motifs that mediate protein-protein interactions. Mapping the sites of interaction of FANCG with ERCC1, using site-directed mutagenesis, demonstrated that TPRs 1, 3, 5, and 6 are needed for binding of FANCG to ERCC1. ERCC1, in turn, was shown to interact with FANCG via its central domain, which is different from the region of ERCC1 that binds to XPF. This binding between FANCG and the ERCC1-XPF endonuclease, combined with our previous studies which show that FANCG is involved in the incision step mediated by ERCC1-XPF, establishes a link between an FA protein and the critical unhooking step of the ICL repair process.
有证据表明，范可尼贫血（FA）蛋白在修复 DNA 链间交联（ICLs）方面发挥着重要作用，但具体机制尚不清楚。ICL 修复过程中的关键步骤之一涉及通过 ICL 两侧的一条链上的切口解开交联，并随后将其去除。ERCC1-XPF 内切酶参与此解开步骤以及交联的去除。我们之前已经表明，几种 FA 蛋白对于在 ICL 部位由 ERCC1-XPF 产生的切口是必需的。为了更清楚地确定 FA 蛋白与 ERCC1-XPF 介导的切口步骤之间的联系，我们进行了酵母双杂交分析，以确定 FANCA、FANCC、FANCF 和 FANCG 是否直接与 ERCC1 和 XPF 相互作用，如果是，确定相互作用的位点。这些 FA 蛋白之一，FANCG，被发现与 ERCC1 具有很强的亲和力，与 XPF 具有中等亲和力。已经表明，FANCG 含有七个四肽重复（TPR）基序，这些基序介导蛋白-蛋白相互作用。使用定点突变，对 FANCG 与 ERCC1 的相互作用位点进行作图，表明 TPRs 1、3、5 和 6 对于 FANCG 与 ERCC1 的结合是必需的。反过来，ERCC1 被证明通过其中心结构域与 FANCG 相互作用，该中心结构域与与 XPF 结合的 ERCC1 区域不同。这种 FANCG 与 ERCC1-XPF 内切酶之间的结合，结合我们之前的研究表明 FANCG 参与 ERCC1-XPF 介导的切口步骤，在 FA 蛋白和 ICL 修复过程的关键解开步骤之间建立了联系。
