title: Coupling of human DNA excision repair and the DNA damage checkpoint in a defined in vitro system.
abstract: DNA repair and DNA damage checkpoints work in concert to help maintain genomic integrity. In vivo data suggest that these two global responses to DNA damage are coupled. It has been proposed that the canonical 30 nucleotide single-stranded DNA gap generated by nucleotide excision repair is the signal that activates the ATR-mediated DNA damage checkpoint response and that the signal is enhanced by gap enlargement by EXO1 (exonuclease 1) 5' to 3' exonuclease activity. Here we have used purified core nucleotide excision repair factors (RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1), core DNA damage checkpoint proteins (ATR-ATRIP, TopBP1, RPA), and DNA damaged by a UV-mimetic agent to analyze the basic steps of DNA damage checkpoint response in a biochemically defined system. We find that checkpoint signaling as measured by phosphorylation of target proteins by the ATR kinase requires enlargement of the excision gap generated by the excision repair system by the 5' to 3' exonuclease activity of EXO1. We conclude that, in addition to damaged DNA, RPA, XPA, XPC, TFIIH, XPG, XPF-ERCC1, ATR-ATRIP, TopBP1, and EXO1 constitute the minimum essential set of factors for ATR-mediated DNA damage checkpoint response.
authors.affiliation: From the Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7260 and.
authors.forename: Laura A
authors.initials: LA
authors.lastname: Lindsey-Boltz
authors.forename: Michael G
authors.initials: MG
authors.lastname: Kemp
authors.forename: Joyce T
authors.initials: JT
authors.lastname: Reardon
authors.forename: Vanessa
authors.initials: V
authors.lastname: DeRocco
authors.forename: Ravi R
authors.initials: RR
authors.lastname: Iyer
authors.forename: Paul
authors.initials: P
authors.lastname: Modrich
authors.forename: Aziz
authors.initials: A
authors.lastname: Sancar
mesh_terms: D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D005090:Exodeoxyribonucleases / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D008954:Models, Biological; D010766:Phosphorylation; D051716:Replication Protein A / Q000378:metabolism; D015398:Signal Transduction; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: D051716:Replication Protein A; D016159:Tumor Suppressor Protein p53; D004247:DNA; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I
keywords: Checkpoint Control; DNA Damage Response; DNA Nucleotide Excision Repair; Protein Kinases; p53