Interactions of the transcription/DNA repair factor TFIIH and XP repair proteins with DNA lesions in a cell-free repair assay.
We have studied the interactions between DNA damage and human proteins involved in the early steps of nucleotide excision repair (NER) reaction under in vitro conditions with human protein extracts. By using a new assay, we have detected a long-lived DNA/protein complex involving XPA and TFIIH in the course of the NER process. The formation of this complex is exclusively limited to DNA lesions that are substrates of the human excinuclease. We show that, while XPA binding to damaged DNA is ATP-independent, stable association of TFIIH with DNA lesions is promoted by ATP hydrolysis and is dependent on the integrity of XPA and XPC proteins in the cell extract. In addition, XPC is necessary to promote a stable binding of XPA to UV-irradiated DNA. Finally, the co-binding of XPA and TFIIH to DNA damage is correlated to a dose-dependent titration of TFIIH and not XPA from the free protein fraction.
Li
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Société Française de Recherches et d'Investissements (SFRI), Berganton, Saint Jean d'Illac, 33127, France.
Calsou
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P
Jones
C J
CJ
Salles
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B
D000255:Adenosine Triphosphate / Q000378:metabolism; D002457:Cell Extracts; D002474:Cell-Free System; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D015151:Immunoblotting; D008163:Luminescent Measurements; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000378:metabolism*; D019473:Transcription Factors, TFII; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000235:genetics; D051760:Xeroderma Pigmentosum Group A Protein
D002457:Cell Extracts; D004268:DNA-Binding Proteins; D014157:Transcription Factors; D019473:Transcription Factors, TFII; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; D051758:Transcription Factor TFIIH; C086984:XPC protein, human; D000255:Adenosine Triphosphate; D004247:DNA