Assembly, subunit composition, and footprint of human DNA repair excision nuclease.
The assembly and composition of human excision nuclease were investigated by electrophoretic mobility shift assay and DNase I footprinting. Individual repair factors or any combination of up to four repair factors failed to form DNA-protein complexes of high specificity and stability. A stable complex of high specificity can be detected only when XPA/RPA, transcription factor IIH, XPC.HHR23B, and XPG and ATP are present in the reaction mixture. The XPF.ERCC1 heterodimer changes the electrophoretic mobility of the DNA-protein complex formed with the other five repair factors, but it does not confer additional specificity. By using proteins with peptide tags or antibodies to the repair factors in electrophoretic mobility shift assays, it was found that XPA, replication protein A, transcription factor IIH, XPG, and XPF.excision repair cross-complementing 1 but not XPC.HHR23B were present in the penultimate and ultimate dual incision complexes. Thus, it appears that XPC.HHR23B is a molecular matchmaker that participates in the assembly of the excision nuclease but is not present in the ultimate dual incision complex. The excision nuclease makes an assymmetric DNase I footprint of approximately 30 bp around the damage and increases the DNase I sensitivity of the DNA on both sides of the footprint.
Wakasugi
M
M
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA.
Sancar
A
A
D001483:Base Sequence; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism; D004720:Endonucleases; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D051716:Replication Protein A; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism; D019473:Transcription Factors, TFII; D051760:Xeroderma Pigmentosum Group A Protein; D000099250:DNA Excision Repair Protein ERCC-5
D004268:DNA-Binding Proteins; D004706:Endodeoxyribonucleases; D004720:Endonucleases; D009687:Nuclear Proteins; D051716:Replication Protein A; D051758:Transcription Factor TFIIH; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D051760:Xeroderma Pigmentosum Group A Protein; D000099250:DNA Excision Repair Protein ERCC-5; C497167:RPA1 protein, human; C497314:XPA protein, human