Order of assembly of human DNA repair excision nuclease.
Human excision nuclease removes DNA damage by concerted dual incisions bracketing the lesion. The dual incisions are accomplished by sequential and partly overlapping actions of six repair factors, RPA, XPA, XPC, TFIIH, XPG, and XPF.ERCC1. Of these, RPA, XPA, and XPC have specific binding affinity for damaged DNA. To learn about the role of these three proteins in damage recognition and the order of assembly of the excision nuclease, we measured the binding affinities of XPA, RPA, and XPC to a DNA fragment containing a single (6-4) photoproduct and determined the rate of damage excision under a variety of reaction conditions. We found that XPC has the highest affinity to DNA and that RPA has the highest selectivity for damaged DNA. Under experimental conditions conducive to binding of either XPA + RPA or XPC to damaged DNA, the rate of damage removal was about 5-fold faster for reactions in which XPA + RPA was the first damage recognition factor presented to DNA compared with reactions in which XPC was the first protein that had the opportunity to bind to DNA. We conclude that RPA and XPA are the initial damage sensing factors of human excision nuclease.
Wakasugi
M
M
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7260, USA.
Sancar
A
A
D004249:DNA Damage; D018983:DNA Footprinting; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D046911:Macromolecular Substances; D016601:RNA-Binding Proteins / Q000378:metabolism*; D051716:Replication Protein A; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000378:metabolism; D019473:Transcription Factors, TFII; D051760:Xeroderma Pigmentosum Group A Protein
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D016601:RNA-Binding Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; 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