DNA Structural Elements Required for ERCC1-XPF Endonuclease Activity

Wouter L De Laat,Esther Appeldoorn,Nicolaas G.J Jaspers,Jan H.J Hoeijmakers

doi:10.1074/jbc.273.14.7835

Wouter L De Laat, Esther Appeldoorn + Show 2 more

Open Access

https://doi.org/10.1074/jbc.273.14.7835

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Abstract

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.

Highlights

Nucleotide excision repair (NER)1 guards the integrity of the genome by removing bulky adducts and the most prominent UV-induced lesions from the DNA
Incisions made by ERCC1-XPF and XPG during NER co-localize to the borders of the opened DNA intermediate [8] and protein-protein interactions with other NER factors are likely to determine the exact positioning of both nucleases
As only little further purification was obtained with this gradient and no significant differences were detected between the enzymatic activities of Nipurified versus glycerol gradient-purified protein fractions, most experiments were performed with Ni-purified proteins

Summary

EXPERIMENTAL PROCEDURES

Construction of Recombinant Baculoviruses—The construction of a cDNA encoding ERCC1 with a C-terminal 6xHis-tag was described previously [6]. The 3Ј end (NcoI-BamHI) of this cDNA was used to replace the 3Ј end (NcoI-BamHI) of wild-type ERCC1 cDNA in the vector pET3C-ERCC1 which contains a unique NdeI site at the translation initiation site This NdeI site and a ClaI site were used to isolate ERCC1His cDNA, recessive ends were filled in by Klenow fragment, and the cDNA was cloned into the blunt-ended BamHI site of the donor plasmid pFastBacI (BAC-TO-BAC Baculovirus Expression System, Life Technologies, Inc.), resulting in pFastBacI-ERCC1His. To introduce a 6xHis-HA epitope encoding DNA sequence at the 3Ј end of XPF cDNA, first a (blunt) in-frame NruI site was introduced at the 3Ј end of the XPF coding region by means of polymerase chain reaction using the antisense primer 5Ј-CGATCGATTCGGAAGCGCTGCCTCCCTTTTTCCCTTTTCCTTTTGAT-3Ј.

RESULTS

TABLE I Artificial DNA substrates

DISCUSSION