Contribution of XPF functional domains to the 5′ and 3′ incisions produced at the site of a psoralen interstrand cross-link
Contribution of XPF functional domains to the 5′ and 3′ incisions produced at the site of a psoralen interstrand cross-link
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DOI：
10.1021/bi011614z
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发表时间：
2002-01-22
期刊：
影响因子：
2.9
通讯作者：
Lambert, MW
中科院分区：
文献类型：
--
作者：
Kumaresan, KR;Hwang, M;Lambert, MW
XPF与ERCC1形成异二聚体复合体，是修复DNA链间交联链所必需的。它与ERCC1一起参与了补骨脂素链间交联处5‘切口处和3’切口处的生产。本研究旨在确定XPF的功能结构域，这些功能结构域在补骨脂素链间交联点的5‘和3’切面的产生中起重要作用。利用针对XPF多肽片段产生的单抗(MAbs)与XPF的特定区域进行亲和映射。检测了这些单抗对含有XPF和ERCC1的正常人类染色质相关蛋白提取物在DNA中产生双切口的不同抑制能力。这些研究表明，XPF的两个区域，一个来自氨基酸12-166的N-末端区域和一个来自氨基酸702-854的C-末端区域，在5‘切口处的产生中是最重要的。同样的N-末端区域和氨基酸702-916的C-末端区域也参与了3‘切开，尽管程度要小得多。由于这一C-末端区域对应于ERCC1与XPF相互作用的位置，这些结果表明ERCC1与XPF的结合对于其在链间交联点产生5‘和3’切割的能力是至关重要的。可能通过激活或调节XPF的N-末端结构域的内切核溶活性。
XPF forms a heterodimeric complex with ERCC1 and is required for the repair of DNA interstrand cross-links. In association with ERCC1, it is involved in production of the 5' incision at the site of a psoralen interstrand cross-link as well as the 3' incision. The present study was carried out to determine the functional domains of XPF that are important in the production of the 5' and 3' incisions that occur at a site of a psoralen interstrand cross-link. Monoclonal antibodies (mAbs) were utilized that had been generated against polypeptide fragments of XPF and affinity-mapped to specific regions of XPF. These mAbs were examined for their ability to differentially inhibit production of dual incisions in DNA by normal human chromatin-associated protein extracts that contain XPF and ERCC1. These studies show that two regions of XPF, one N-terminal region from amino acids 12-166 and one C-terminal region from amino acids 702-854, are the most important in the production of the 5' incision. The same N-terminal region and the C-terminal region from amino acids 702-916 are also involved in the 3' incision, though to a much lesser extent. Since this C-terminal region corresponds to the proposed site of interaction of ERCC1 with XPF, these results suggest that binding of ERCC1 to XPF is critical for its ability to produce the 5' and 3' incisions at the site of an interstrand cross-link. Possibly through activation or regulation of the endonucleolytic activity of the N-terminal domain of XPF.