Open complex formation around a lesion during nucleotide excision repair provides a structure for cleavage by human XPG protein.
Human XPG nuclease makes the 3' incision during nucleotide excision repair of DNA. The enzyme cleaves model DNA bubble structures specifically near the junction of unpaired DNA with a duplex region. It is not yet known, however, whether an unpaired structure is an intermediate during actual DNA repair. We find here that XPG requires opening of >5 bp for efficient cleavage. To seek direct evidence for formation of an open structure around a lesion in DNA during a nucleotide excision repair reaction in vitro, KMnO4 footprinting experiments were performed on a damaged DNA molecule bearing a uniquely placed cisplatin adduct. An unwound open complex spanning approximately 25 nucleotides was observed that extended to the positions of 5' and 3' incision sites and was dependent on XPA protein and on ATP. Opening during repair occurred prior to strand incision by XPG.
Evans
E
E
Imperial Cancer Research Fund, Clare Hall Laboratories, London, UK.
Fellows
J
J
Coffer
A
A
Wood
R D
RD
D000255:Adenosine Triphosphate / Q000378:metabolism; D000970:Antineoplastic Agents / Q000378:metabolism; D001483:Base Sequence; D002945:Cisplatin / Q000378:metabolism; D004249:DNA Damage / Q000235:genetics; D018983:DNA Footprinting; D004260:DNA Repair / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004720:Endonucleases; D006801:Humans; D007364:Intercalating Agents / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D014157:Transcription Factors; D051760:Xeroderma Pigmentosum Group A Protein; D000099250:DNA Excision Repair Protein ERCC-5
D000255:Adenosine Triphosphate; D000970:Antineoplastic Agents; D002945:Cisplatin; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D004720:Endonucleases; D007364:Intercalating Agents; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D018809:Proliferating Cell Nuclear Antigen; D011994:Recombinant Proteins; D015719:Single-Strand Specific DNA and RNA Endonucleases; D014157:Transcription Factors; D051760:Xeroderma Pigmentosum Group A Protein; D000099250:DNA Excision Repair Protein ERCC-5; C497314:XPA protein, human