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A PP4 phosphatase complex dephosphorylates RPA2 to facilitate DNA repair via homologous recombination.

ABSTRACT: Double-stranded DNA breaks (DSBs) induce a phosphorylation-mediated signaling cascade, but the role of phosphatases in this pathway remains unclear. Here we show that human protein phosphatase 4 (PP4) dephosphorylates replication protein A (RPA) subunit RPA2, regulating its role in the DSB response. PP4R2, a regulatory subunit of PP4, mediates the DNA damage-dependent association between RPA2 and the PP4C catalytic subunit. PP4 efficiently dephosphorylates phospho-RPA2 in vitro, and silencing PP4R2 in cells alters the kinetics and pattern of RPA2 phosphorylation. Depletion of PP4R2 impedes homologous recombination (HR) via inefficient loading of the essential HR factor RAD51, causing an extended G2-M checkpoint and hypersensitivity to DNA damage. Cells expressing phosphomimetic RPA2 mutants have a comparable phenotype, suggesting that PP4-mediated dephosphorylation of RPA2 is necessary for an efficient DNA-damage response. These observations provide new insight into the role and regulation of RPA phosphorylation in HR-mediated repair.

SUBMITTER: Lee DH

PROVIDER: S-EPMC3057140 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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A PP4 phosphatase complex dephosphorylates RPA2 to facilitate DNA repair via homologous recombination.

Lee Dong-Hyun DH Pan Yunfeng Y Kanner Shlomo S Sung Patrick P Borowiec James A JA Chowdhury Dipanjan D

Nature structural & molecular biology 20100214 3

Double-stranded DNA breaks (DSBs) induce a phosphorylation-mediated signaling cascade, but the role of phosphatases in this pathway remains unclear. Here we show that human protein phosphatase 4 (PP4) dephosphorylates replication protein A (RPA) subunit RPA2, regulating its role in the DSB response. PP4R2, a regulatory subunit of PP4, mediates the DNA damage-dependent association between RPA2 and the PP4C catalytic subunit. PP4 efficiently dephosphorylates phospho-RPA2 in vitro, and silencing PP ...[more]

PMID: 20154705

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Project description:BackgroundRAD51-dependent homologous recombination (HR) is one of the most important pathways for repairing DNA double-strand breaks (DSBs), and its regulation is crucial to maintain genome integrity. Elp1 gene encodes IKAP/ELP1, a core subunit of the Elongator complex, which has been implicated in translational regulation. However, how ELP1 contributes to genome maintenance is unclear.MethodsTo investigate the function of Elp1, Elp1-deficient mouse embryonic fibroblasts (MEFs) were generated. Metaphase chromosome spreading, immunofluorescence, and comet assays were used to access chromosome abnormalities and DSB formation. Functional roles of Elp1 in MEFs were evaluated by cell viability, colony forming capacity, and apoptosis assays. HR-dependent DNA repair was assessed by reporter assay, immunofluorescence, and western blot. Polysome profiling was used to evaluate translational efficiency. Differentially expressed proteins and signaling pathways were identified using a label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics approach.ResultsHere, we report that Elp1 depletion enhanced genomic instability, manifested as chromosome breakage and genotoxic stress-induced genomic DNA fragmentation upon ionizing radiation (IR) exposure. Elp1-deficient cells were hypersensitive to DNA damage and exhibited impaired cell proliferation and defective HR repair. Moreover, Elp1 depletion reduced the formation of IR-induced RAD51 foci and decreased RAD51 protein levels. Polysome profiling analysis revealed that ELP1 regulated RAD51 expression by promoting its translation in response to DNA damage. Notably, the requirement for ELP1 in DSB repair could be partially rescued in Elp1-deficient cells by reintroducing RAD51, suggesting that Elp1-mediated HR-directed repair of DSBs is RAD51-dependent. Finally, using proteome analyses, we identified several proteins involved in cancer pathways and DNA damage responses as being differentially expressed upon Elp1 depletion.ConclusionsOur study uncovered a molecular mechanism underlying Elp1-mediated regulation of HR activity and provides a novel link between translational regulation and genome stability.

Dataset Information

A PP4 phosphatase complex dephosphorylates RPA2 to facilitate DNA repair via homologous recombination.

Publications

A PP4 phosphatase complex dephosphorylates RPA2 to facilitate DNA repair via homologous recombination.

Similar Datasets

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