title: In vitro analysis of the role of replication protein A (RPA) and RPA phosphorylation in ATR-mediated checkpoint signaling.
abstract: Replication protein A (RPA) plays essential roles in DNA metabolism, including replication, checkpoint, and repair. Recently, we described an in vitro system in which the phosphorylation of human Chk1 kinase by ATR (ataxia telangiectasia mutated and Rad3-related) is dependent on RPA bound to single-stranded DNA. Here, we report that phosphorylation of other ATR targets, p53 and Rad17, has the same requirements and that RPA is also phosphorylated in this system. At high p53 or Rad17 concentrations, RPA phosphorylation is inhibited and, in this system, RPA with phosphomimetic mutations cannot support ATR kinase function, whereas a non-phosphorylatable RPA mutant exhibits full activity. Phosphorylation of these ATR substrates depends on the recruitment of ATR and the substrates by RPA to the RPA-ssDNA complex. Finally, mutant RPAs lacking checkpoint function exhibit essentially normal activity in nucleotide excision repair, revealing RPA separation of function for checkpoint and excision repair.
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7260, USA.
authors.forename: Laura A
authors.initials: LA
authors.lastname: Lindsey-Boltz
authors.forename: Joyce T
authors.initials: JT
authors.lastname: Reardon
authors.forename: Marc S
authors.initials: MS
authors.lastname: Wold
authors.forename: Aziz
authors.initials: A
authors.lastname: Sancar
mesh_terms: D064007:Ataxia Telangiectasia Mutated Proteins; D059447:Cell Cycle Checkpoints; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism; D000071877:Checkpoint Kinase 1; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009154:Mutation; D010766:Phosphorylation / Q000502:physiology; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051716:Replication Protein A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D004277:DNA, Single-Stranded; C113575:Rad17 protein, human; D051716:Replication Protein A; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D011494:Protein Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases