Mapping of interaction domains between human repair proteins ERCC1 and XPF.

ERCC1-XPF is a heterodimeric protein complexinvolved in nucleotide excision repair and recombinational processes. Like its homologous complex in Saccharomyces cerevisiae , Rad10-Rad1, it acts as a structure-specific DNA endonuclease, cleaving at duplex-single-stranded DNA junctions. In repair, ERCC1-XPF and Rad10-Rad1 make an incision on the the 5'-side of the lesion. No humans with a defect in the ERCC1 subunit of this protein complex have been identified and ERCC1-deficient mice suffer from severe developmental problems and signs of premature aging on top of a repair-deficient phenotype. Xeroderma pigmentosum group F patients carry mutations in the XPF subunit and generally show the clinical symptoms of mild DNA repair deficiency. All XP-F patients examined demonstrate reduced levels of XPF and ERCC1 protein, suggesting that proper complex formation is required for stability of the two proteins. To better understand the molecular and clinical consequences of mutations in the ERCC1-XPF complex, we decided to map the interaction domains between the two subunits. The XPF-binding domain comprises C-terminal residues 224-297 of ERCC1. Intriguingly, this domain resides outside the region of homology with its yeast Rad10 counterpart. The ERCC1-binding domain in XPF maps to C-terminal residues 814-905. ERCC1-XPF complex formation is established by a direct interaction between these two binding domains. A mutation from an XP-F patient that alters the ERCC1-binding domain in XPF indeed affects complex formation with ERCC1.ERCC1-XPF是一种异二聚体蛋白质复合物，参与核苷酸切除修复（nucleotide excision repair）与重组过程。与酿酒酵母（Saccharomyces cerevisiae）中的同源复合物Rad10-Rad1类似，该复合物属于结构特异性DNA核酸内切酶（structure-specific DNA endonuclease），可在双链-单链DNA连接处进行切割。在修复过程中，ERCC1-XPF与Rad10-Rad1会在损伤位点的5'侧进行切口切割。目前尚未发现携带该复合物ERCC1亚基缺陷的人类个体，而ERCC1缺陷小鼠除表现出修复缺陷表型外，还会出现严重的发育异常与早衰征象。着色性干皮病F组（Xeroderma pigmentosum group F，XP-F）患者携带XPF亚基突变，通常会出现轻度DNA修复缺陷的临床症状。所有被检测的XP-F患者均呈现XPF与ERCC1蛋白水平降低的现象，这提示复合物的正确组装是维持两种蛋白稳定性的必要条件。为了更深入地解析ERCC1-XPF复合物突变所带来的分子与临床后果，我们决定定位两个亚基之间的相互作用结构域。XPF的结合结构域位于ERCC1的C端224-297位残基。值得注意的是，该结构域位于其与酵母Rad10的同源区域之外。XPF中结合ERCC1的结构域则定位于C端814-905位残基。ERCC1-XPF复合物的形成依赖于这两个结合结构域之间的直接相互作用。一名XP-F患者的突变（该突变改变了XPF中的ERCC1结合结构域）确实会影响其与ERCC1的复合物形成。