DNA structural elements required for ERCC1-XPF endonuclease activity

被引:185
作者
de Laat, WL [1 ]
Appeldoorn, E [1 ]
Jaspers, NGJ [1 ]
Hoeijmakers, JHJ [1 ]
机构
[1] Erasmus Univ, Dept Cell Biol & Genet, Ctr Med Genet, NL-3000 DR Rotterdam, Netherlands
关键词
D O I
10.1074/jbc.273.14.7835
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
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收藏
页码:7835 / 7842
页数:8
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