Isolation of human complexes proficient in nucleotide excision repair.
More than 20 polypeptides are required for the process of nucleotide excision repair (NER) in both human and yeast cells. This pathway of excision repair has most often been viewed as an ordered multi-step process involving steps of damage recognition, incision/excision and finally repair DNA synthesis. Here we present evidence for the existence of a complex of human NER proteins pre-assembled in the absence of damaged DNA. This multi-protein complex was initially isolated from HeLa cell extracts by affinity chromatography on a matrix containing the damage recognition protein XPA. Subsequent co-immunoprecipitation and gel filtration experiments demonstrated that a significant portion of the human NER proteins was present in the form of a high molecular weight complex and that these complexes, or repairosomes, were capable of performing all steps of NER in vitro . Consistent with studies indicating that DNA polymerasesdeltaandstraightepsiloncan both function in NER, these two polymerases are found in these repairosome complexes.
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Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada.
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D002460:Cell Line; D002846:Chromatography, Affinity; D004257:DNA Polymerase II; D004258:DNA Polymerase III; D004260:DNA Repair; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification* / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000302:isolation & purification*; D029968:Escherichia coli Proteins; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D008970:Molecular Weight; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D051760:Xeroderma Pigmentosum Group A Protein
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; D004257:DNA Polymerase II; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; D004706:Endodeoxyribonucleases; C036385:endodeoxyribonuclease uvrABC