DNA structural elements required for ERCC1-XPF endonuclease activity
DNA structural elements required for ERCC1-XPF endonuclease activity
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DOI：
10.1074/jbc.273.14.7835
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发表时间：
1998-04-03
影响因子：
4.8
通讯作者：
Hoeijmakers, JHJ
中科院分区：
文献类型：
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作者：
de Laat, WL;Appeldoorn, E;Hoeijmakers, JHJ
异二聚体复合物ERCC1-XPF是哺乳动物核苷酸切除修复（NER）过程中负责5'切口的结构特异性内切酶。此外，ERCC1-XPF被认为在修复链间DNA交联中起作用，并类似于酿酒酵母中的同源Rad1-Rad10复合体，在直接重复DNA序列之间进行重组。为了深入了解ERCC1-XPF在这种重组过程和NER反应中的作用，我们详细研究了ERCC1-XPF内溶活性所需的DNA结构元件，从昆虫细胞中纯化的重组ERCC1-XPF被发现在没有其他蛋白质如复制蛋白A的情况下，在DNA连接处切割茎环底物，表明结构特异性内切酶活性是复合物固有的。切割依赖于二价阳离子的存在，并且在低Mn2+浓度（0.2 mM）下是最佳的。ERCC1-XPF的切口至少需要4-8个未配对的核苷酸。ERCC1-XPF也切割了三叉臂和皮瓣底物，导致3‘突出的单链臂被去除，所有切口都发生在与单链DNA连接的5’侧的一条双链DNA上，准确的切割位置从2到8个核苷酸的距离，在3‘或5’方向上突出的单链臂是ERCC1-XPF正确定位切口所必需的和足够的。我们的数据明确了ERCC1-XPF在NER中的作用，并允许更直接地搜索其在重组中的特定作用。
The heterodimeric complex ERCC1-XPF is a structure-specific endonuclease responsible for the 5' incision during mammalian nucleotide excision repair (NER). Additionally, ERCC1-XPF is thought to function in the repair of interstrand DNA cross-links and, by analogy to the homologous Rad1-Rad10 complex in Saccharomyces cerevisiae, in recombination between direct repeated DNA sequences. To gain insight into the role of ERCC1-XPF in such recombinational processes and in the NER reaction, we studied in detail the DNA structural elements required for ERCC1-XPF endonucleolytic activity, Recombinant ERCC1-XPF, purified from insect cells, was found to cleave stem-loop substrates at the DNA junction in the absence of other proteins like replication protein A, showing that the structure-specific endonuclease activity is intrinsic to the complex, Cleavage depended on the presence of divalent cations and was optimal in low Mn2+ concentrations (0.2 mM). A minimum of 4-8 unpaired nucleotides was required for incisions by ERCC1-XPF. Splayed arm and flap substrates were also cut by ERCC1-XPF, resulting in the removal of 3' protruding single-stranded arms, All incisions occurred in one strand of duplex DNA at the 5' side of a junction with single-stranded DNA, The exact cleavage position varied from 2 to 8 nucleotides away from the junction, One single-stranded arm, protruding either in the 3' or 5' direction, was necessary and sufficient for correct positioning of incisions by ERCC1-XPF, Our data specify the engagement of ERCC1-XPF in NER and allow a more direct search for its specific role in recombination.