Co-correction of the ERCC1, ERCC4 and xeroderma pigmentosum group F DNA repair defects in vitro.
The mammalian ERCC1-encoded polypeptide is required for nucleotide excision repair of damaged DNA and is homologous to Saccharomyces cerevisiae RAD10, which functions in repair and mitotic intrachromosomal recombination. Rodent cells representing repair complementation group 1 have nonfunctional ERCC1. We report that repair of UV-irradiated DNA can be reconstituted by combining rodent group 1 cell extracts with correcting protein from HeLa cells. Background repair was minimized by employing fractionated rodent cell extracts supplemented with human replication proteins RPA and PCNA. Group 1-correcting activity has a native molecular mass of 100 kDa and contains the 33 kDa ERCC1 polypeptide, as well as complementing activities for extracts from rodent group 4 and xeroderma pigmentosum group F (XP-F) cells. Extracts of group 1, group 4 or XP-F cells do not complement one another in vitro, although they complement extracts from other groups. The amount of ERCC1 detectable by immunoblotting is reduced in group 1, group 4 and XP-F extracts. Recombinant ERCC1 from Escherichia coli only weakly corrected the group 1 defect. The data suggest that ERCC1 is part of a functional protein complex with group 4 and XP-F correcting activities. The latter two may be equivalent to one another and analogous to S. cerevisiae RAD1.
Biggerstaff
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Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts, UK.
Szymkowski
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Wood
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D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004247:DNA / Q000528:radiation effects*; D004249:DNA Damage; D017931:DNA Primers; D004260:DNA Repair; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004720:Endonucleases; D004926:Escherichia coli; D005038:Ethylnitrosourea / Q000633:toxicity; D005656:Fungal Proteins / Q000378:metabolism; D015723:Gene Library; D005816:Genetic Complementation Test; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D015719:Single-Strand Specific DNA and RNA Endonucleases; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000235:genetics*
D000906:Antibodies; D017931:DNA Primers; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D010455:Peptides; D011506:Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C095888:xeroderma pigmentosum group F protein; D004247:DNA; C071447:ERCC1 protein, human; D004720:Endonucleases; C064162:RAD10 protein, S cerevisiae; D015719:Single-Strand Specific DNA and RNA Endonucleases; D005038:Ethylnitrosourea