Recent studies have revealed that the general transcription factor TFIIH is also a general excision repair factor which, along with several other proteins, is required for transcription-independent excision reaction. As a general transcription factor, TFIIH is recruited to RNA polymerase II-promoter complex by another general transcription factor called TFIIE. We were interested in knowing whether TFIIE is also involved in recruiting TFIIH to the excision repair complex. We found that cell-free extract depleted of TFIIE carried out excision repair at a normal rate, leading us to conclude that TFIIE is not involved in recruiting TFIIH to the damage site and has no role in general excision repair. In contrast, the human damage recognition protein XPA specifically binds to TFIIH and apparently recruits it to the damage site. The carboxyl-terminal half of XPA is responsible for specific interaction with TFIIH. The C261S/C264S mutant of XPA bound the ERCC1-XPF complex normally, but failed to bind TFIIH and failed to complement an XP-A mutant cell-free extract indicating that the XPA-TFIIH interaction is essential to effecting the excision reaction. Interestingly, XPA also binds to the p34 subunit of TFIIE specifically and in competition with the p56 subunit of TFIIE. This latter interaction has no apparent role in general excision repair but may be relevant in the transcription-coupled repair reaction.
A complex, which consists of ERCC1 (38 kDa) and a 112-kDa protein, was purified from HeLa cells to homogeneity. This complex complemented the nucleotide excision repair defects of rodent ERCC-1, ERCC-4, and human XP-F mutant cell-free extracts, indicating that the 112-kDa protein is XPF/ERCC4 and providing direct biochemical evidence that XPF and ERCC4 are identical. The XPF/ERCC4-ERCC1 complex has an endonuclease activity with preference for single-stranded DNA and a single-stranded region of duplex DNA with a "bubble" structure. This complex also nicks supercoiled DNA weakly, and this nicking activity is stimulated by human replication protein A when the DNA contains UV damage.Xeroderma pigmentosum (XP) 1 is a human disease characterized by a high incidence of actinic cancers and in some cases neurological abnormalities; it is caused by a defect in excision repair as a result of mutations in one of seven genes, XPA through XPG (1). In recent years all of the XP genes required for excision, with the exception of XPF, have been cloned and sequenced (2, 3). Previously it was found that cell-free extracts (CFEs) from human XP-F and rodent ERCC-4 mutants did not complement each other in vitro, raising the possibility that these two complementation groups contained mutations in the human and rodent homologs of the XPF gene (4, 5). In addition, it was found that the complementing activity of XP-F and ERCC-4 mutant CFEs was tightly associated with the ERCC1 protein (6 -8). Here we report the purification of the XPF/ ERCC4 protein in complex with ERCC1 to homogeneity, providing direct evidence that XPF and ERCC4 are identical genes. The tight complex of XPF/ERCC4 and ERCC1 is a single-stranded DNA-specific endonuclease with weak endonucleolytic activity on supercoiled DNA. The nicking of supercoiled DNA is stimulated by human replication protein A (RPA) when the DNA contains UV damage. MATERIALS AND METHODS Purification of the Complex of ERCC1 and XPF/ERCC4 from HeLaCells-All purification procedures were done at 0 -4°C. HeLa whole cell extracts were prepared from a 400-liter culture containing approximately 2.5 ϫ 10 11 cells (about 35 g of protein) as described previously (7). ERCC1-containing fractions were detected by immunoblotting using polyclonal antibodies raised against maltose-binding protein-ERCC1 fusion protein expressed in Escherichia coli (6). The HeLa cell extracts were applied to a DE52 column (5 ϫ 21 cm, Whatman) as described (7). The ERCC1 protein was in the flow-through, which was applied to an Affi-Gel Blue column (5 ϫ 18 cm, Bio-Rad) pre-equilibrated with buffer A (25 mM HEPES-KOH, pH 7.9, 100 mM KCl, 12 mM MgCl 2 , 0.5 mM EDTA, 2 mM dithiothreitol, 15.5% glycerol). The bound proteins were eluted with a linear 800-ml gradient of 0.1-0.7 M KCl, with 1.2 liters of buffer A plus 0.8 M KCl, and then with 500 ml of buffer A plus 1.5 M NaSCN. Fractions containing ERCC1 protein were pooled, and ammonium sulfate was added to 0.6 g/ml. Precipitated proteins were pelleted by centrifugation at 15,000 ϫ g for ...
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