Ku70/Ku80 protein complex inhibits the binding of nucleotide excision repair proteins on linear DNA in vitro.
We have previously reported that the incision efficiency of the nucleotide excision repair (NER) reaction measured in vitro with cell-free human protein extracts was reduced by up to 80% on a linearized damaged plasmid DNA substrate when compared to supercoiled damaged DNA. The inhibition stemed from the presence of the DNA-end binding Ku70/Ku80 heterodimer which is the regulatory subunit of the DNA-dependent protein kinase (DNA-PK). Here, the origin of the repair inhibition was assessed by a new in vitro assay in which circular or linear plasmid DNA, damaged or undamaged, was quantitatively adsorbed on sensitized microplate wells. The binding of two NER proteins, XPA and p62-TFIIH, indispensable for the incision step of the reaction, was quantified either directly in an ELISA-like reaction in the wells with specific antibodies or in Western blotting experiments on the DNA-bound fraction. We report a dramatic inhibition of XPA and p62-TFIIH association with UVC photoproducts on linear DNA. XPA and p62-TFIIH binding to DNA damage was regained when the reaction was performed with extracts lacking Ku activity (extracts from xrs6 rodent cells) whereas addition of purified human Ku complex to these extracts restored the inhibition. Despite the fact that DNA-PK was active during the NER reaction, the mechanism of inhibition relied on the sole Ku complex, since mutant protein extracts lacking the catalytic DNA-PK subunit (extracts from the human M059J glioma cells) exhibited a strong binding inhibition of XPA and p62-TFIIH proteins on linear damaged DNA, identical to the inhibition observed with the DNA-PK+ control extracts (from M059K cells).
Frit
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Institut de Pharmacologie et de Biologie Structurale, CNRS UPR 9062, 205 route de Narbonne, Toulouse, 31077, France.
Calsou
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P
Chen
D J
DJ
Salles
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B
D000818:Animals; D034961:Antigens, Nuclear; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004249:DNA Damage; D004265:DNA Helicases; D004260:DNA Repair; D004278:DNA, Superhelical / Q000737:chemistry / Q000378:metabolism; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D006801:Humans; D000072200:Ku Autoantigen; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D035381:TATA-Binding Protein Associated Factors; D035362:Transcription Factor TFIID; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000378:metabolism; D019473:Transcription Factors, TFII; D051760:Xeroderma Pigmentosum Group A Protein
D034961:Antigens, Nuclear; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; C490039:TAF6 protein, S cerevisiae; D035381:TATA-Binding Protein Associated Factors; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D019473:Transcription Factors, TFII; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; C081647:high affinity DNA-binding factor, S cerevisiae; D051758:Transcription Factor TFIIH; D004247:DNA; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen