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In Vitro Analysis of the Role of Replication Protein A (RPA) and RPA Phosphorylation in ATR-mediated Checkpoint Signaling*
- Publication Year :
- 2012
- Publisher :
- American Society for Biochemistry and Molecular Biology, 2012.
-
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.
- Subjects :
- inorganic chemicals
Cell cycle checkpoint
DNA Repair
DNA repair
DNA, Single-Stranded
Cell Cycle Proteins
Ataxia Telangiectasia Mutated Proteins
Biology
DNA and Chromosomes
Protein Serine-Threonine Kinases
Biochemistry
complex mixtures
Cell Line
Replication Protein A
Humans
Protein phosphorylation
CHEK1
Phosphorylation
Molecular Biology
Replication protein A
Cell-Free System
Cell Biology
Cell Cycle Checkpoints
G2-M DNA damage checkpoint
Molecular biology
enzymes and coenzymes (carbohydrates)
Checkpoint Kinase 1
Mutation
bacteria
biological phenomena, cell phenomena, and immunity
Tumor Suppressor Protein p53
Ataxia telangiectasia and Rad3 related
Protein Kinases
Nucleotide excision repair
Signal Transduction
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Accession number :
- edsair.doi.dedup.....cb39765f989a7f4dc32b1716db5955f8