DNA structural elements required for ERCC1-XPF endonuclease activity.
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.
|Keywords||0 (Cross-Linking Reagents), 0 (DNA, Single-Stranded), 0 (DNA-Binding Proteins), 0 (ERCC4 protein), 0 (Proteins), 0 (Recombinant Fusion Proteins), 9007-49-2 (dna), Animals, Base Sequence, Binding Sites/genetics, Cell Line, Cross-Linking Reagents, DNA, Single-Stranded/genetics/metabolism, DNA-Binding Proteins/*metabolism, DNA/genetics/*metabolism, EC 3.1.- (ERCC-1 protein, human), EC 3.1.- (Endonucleases), Endonucleases/*metabolism, Enzyme Activation, Molecular Sequence Data, Proteins/*metabolism, Recombinant Fusion Proteins/genetics/metabolism, Support, Non-U.S. Gov't|
|Persistent URL||dx.doi.org/10.1074/jbc.273.14.7835, hdl.handle.net/1765/3134|
de Laat, W.L., Appeldoorn, E., Jaspers, N.G.J., & Hoeijmakers, J.H.J.. (1998). DNA structural elements required for ERCC1-XPF endonuclease activity.. Journal of Biological Chemistry, 273(14), 7835–7842. doi:10.1074/jbc.273.14.7835