In Vitro Analysis of the Role of Replication Protein A (RPA) and RPA Phosphorylation in ATR-mediated Checkpoint Signaling

Laura A Lindsey-Boltz; Joyce T Reardon; Marc S Wold; Aziz Sancar

doi:10.1074/jbc.M112.407825

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In Vitro Analysis of the Role of Replication Protein A (RPA) and RPA Phosphorylation in ATR-mediated Checkpoint Signaling

Journal article

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In Vitro Analysis of the Role of Replication Protein A (RPA) and RPA Phosphorylation in ATR-mediated Checkpoint Signaling

Laura A Lindsey-Boltz, Joyce T Reardon, Marc S Wold and Aziz Sancar

The Journal of biological chemistry, Vol.287(43), pp.36123-36131

10/19/2012

DOI: 10.1074/jbc.M112.407825

PMCID: PMC3476280

PMID: 22948311

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Abstract

Background: The function of RPA and RPA phosphorylation in the activation of ATR is unknown. Results: RPA with phosphomimetic mutations cannot support ATR kinase function yet maintains functions in nucleotide excision repair. Conclusion: These results reveal a RPA separation of function for checkpoint activation and excision repair. Significance: RPA phosphorylation may modulate ATR checkpoint signaling while maintaining other cellular functions of RPA. 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 ( a taxia t elangiectasia mutated and R ad3-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.

DNA Nucleotide Excision Repair

DNA and Chromosomes

Checkpoint Control

Phosphorylation

Protein Kinases

DNA-Protein Interaction

TopBP1

DNA Binding Protein

Rad17-RFC

Protein phosphorylation

p53

Details

Title: Subtitle: In Vitro Analysis of the Role of Replication Protein A (RPA) and RPA Phosphorylation in ATR-mediated Checkpoint Signaling
Creators: Laura A Lindsey-Boltz - From the
Joyce T Reardon - From the
Marc S Wold - the
Aziz Sancar - From the
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.287(43), pp.36123-36131
DOI: 10.1074/jbc.M112.407825
PMID: 22948311
PMCID: PMC3476280
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: American Society for Biochemistry and Molecular Biology; 9650 Rockville Pike, Bethesda, MD 20814, U.S.A
Grant note: GM32833; GM44721 / National Institutes of Health
Alternative title: RPA and RPA Phosphorylation in ATR-mediated Checkpoint Signaling
Language: English
Date published: 10/19/2012
Academic Unit: Radiation Oncology; Biochemistry and Molecular Biology
Record Identifier: 9984025251002771

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