title: Reaction mechanism of human DNA repair excision nuclease.
abstract: Nucleotide excision repair consists of removal of the damaged nucleotide(s) from DNA by dual incision of the damaged strand on both sides of the lesion, followed by filling of the resulting gap and ligation. In humans, 14-16 polypeptides are required for the dual incision step. We have purified the required proteins to homogeneity and reconstituted the dual incision activity (excision nuclease) in a defined enzyme/substrate system. The system was highly efficient, removing >30% of the thymine dimers under optimal conditions. All of the six fractions that constitute the excision nuclease were required for dual incision of the thymine dimer substrate. However, when a cholesterol-substituted oligonucleotide was used as substrate, excision occurred in the absence of the XPC-HHR23B complex, reminiscent of transcription-coupled repair in the XP-C mutant cell line. Replication protein A is absolutely required for both incisions. The XPG subunit is essential to the formation of the preincision complex, but the repair complex can assemble and produce normal levels of 3'-incision in the absence of XPF-ERCC1. Kinetic experiments revealed that the 3'-incision precedes the 5'-incision. Consistent with the kinetic data, uncoupled 5'-incision was never observed in the reconstituted system. Two forms of TFIIH were used in the reconstitution reaction, one containing the CDK7-cyclin H pair and one lacking it. Both forms were equally active in excision. The excised oligomer dissociated from the gapped DNA in a nucleoprotein complex. In total, these results provide a detailed account of the reactions occurring during damage removal by human excision nuclease.
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, 27599-7260, USA.
authors.forename: D
authors.initials: D
authors.lastname: Mu
authors.forename: D S
authors.initials: DS
authors.lastname: Hsu
authors.forename: A
authors.initials: A
authors.lastname: Sancar
mesh_terms: D002784:Cholesterol / Q000737:chemistry; D018844:Cyclin-Dependent Kinases; D004249:DNA Damage; D004260:DNA Repair; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004720:Endonucleases; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D011740:Pyrimidine Dimers / Q000737:chemistry; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000378:metabolism; D019473:Transcription Factors, TFII; D000095043:Cyclin-Dependent Kinase-Activating Kinase
chemical_list: D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011506:Proteins; D011740:Pyrimidine Dimers; C513287:RAD23B protein, human; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D051758:Transcription Factor TFIIH; C086985:RAD23A protein, human; C086984:XPC protein, human; D002784:Cholesterol; D017346:Protein Serine-Threonine Kinases; D018844:Cyclin-Dependent Kinases; C071447:ERCC1 protein, human; D004706:Endodeoxyribonucleases; D004720:Endonucleases; D045643:DNA Repair Enzymes; D000095043:Cyclin-Dependent Kinase-Activating Kinase; C000722416:CDK7 protein, human