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

Nature Structural & Molecular Biology volume 17, pages 365–372 (2010)Cite this article

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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.

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Figure 1: A PP4 complex interacts with RPA2 in the context of DNA damage, and silencing the PP4 complex enhances RPA2 phosphorylation.
Figure 2: PP4 dephosphorylates RPA2 in vitro and influences the kinetics and pattern of RPA2 phosphorylation in cells.
Figure 3: Hyperphosphorylation of RPA2 affects HR-mediated repair of DSBs and the DNA-damage response.
Figure 4: Premature formation of hyperphosphorylated RPA2 impedes recruitment of RPA and RAD51 to chromatinized DNA damage–induced foci.

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References

  1. Matsuoka, S. et al. ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. Science 316, 1160–1166 (2007).

    Article  CAS  Google Scholar 

  2. Chowdhury, D. et al. γ-H2AX dephosphorylation by protein phosphatase 2A facilitates DNA double-strand break repair. Mol. Cell 20, 801–809 (2005).

    Article  CAS  Google Scholar 

  3. Chowdhury, D. et al. A PP4-phosphatase complex dephosphorylates γ-H2AX generated during DNA replication. Mol. Cell 31, 33–46 (2008).

    Article  CAS  Google Scholar 

  4. Keogh, M.C. et al. A phosphatase complex that dephosphorylates γ-H2AX regulates DNA damage checkpoint recovery. Nature 439, 497–501 (2006).

    Article  CAS  Google Scholar 

  5. Honkanen, R.E. & Golden, T. Regulators of serine/threonine protein phosphatases at the dawn of a clinical era? Curr. Med. Chem. 9, 2055–2075 (2002).

    Article  CAS  Google Scholar 

  6. Virshup, D.M. & Shenolikar, S. From promiscuity to precision: protein phosphatases get a makeover. Mol. Cell 33, 537–545 (2009).

    Article  CAS  Google Scholar 

  7. Janssens, V., Goris, J. & Van Hoof, C. PP2A: the expected tumor suppressor. Curr. Opin. Genet. Dev. 15, 34–41 (2005).

    Article  CAS  Google Scholar 

  8. Janssens, V., Longin, S. & Goris, J. PP2A holoenzyme assembly: in cauda venenum (the sting is in the tail). Trends Biochem. Sci. 33, 113–121 (2008).

    Article  CAS  Google Scholar 

  9. Hastie, C.J., Carnegie, G.K., Morrice, N. & Cohen, P.T. A novel 50 kDa protein forms complexes with protein phosphatase 4 and is located at centrosomal microtubule organizing centres. Biochem. J. 347, 845–855 (2000).

    Article  CAS  Google Scholar 

  10. Nakada, S., Chen, G.I., Gingras, A.C. & Durocher, D. PP4 is a γ-H2AX phosphatase required for recovery from the DNA damage checkpoint. EMBO Rep. 9, 1019–1026 (2008).

    Article  CAS  Google Scholar 

  11. Anantha, R.W. & Borowiec, J.A. Mitotic crisis: the unmasking of a novel role for RPA. Cell Cycle 8, 357–361 (2009).

    Article  CAS  Google Scholar 

  12. Fanning, E., Klimovich, V. & Nager, A.R. A dynamic model for replication protein A (RPA) function in DNA processing pathways. Nucleic Acids Res. 34, 4126–4137 (2006).

    Article  CAS  Google Scholar 

  13. Zou, Y., Liu, Y., Wu, X. & Shell, S.M. Functions of human replication protein A (RPA): from DNA replication to DNA damage and stress responses. J. Cell. Physiol. 208, 267–273 (2006).

    Article  CAS  Google Scholar 

  14. Binz, S.K., Sheehan, A.M. & Wold, M.S. Replication protein A phosphorylation and the cellular response to DNA damage. DNA Repair (Amst.) 3, 1015–1024 (2004).

    Article  CAS  Google Scholar 

  15. Iftode, C., Daniely, Y. & Borowiec, J.A. Replication protein A (RPA): the eukaryotic SSB. Crit. Rev. Biochem. Mol. Biol. 34, 141–180 (1999).

    Article  CAS  Google Scholar 

  16. Anantha, R.W., Vassin, V.M. & Borowiec, J.A. Sequential and synergistic modification of human RPA stimulates chromosomal DNA repair. J. Biol. Chem. 282, 35910–35923 (2007).

    Article  CAS  Google Scholar 

  17. Wang, H. et al. Replication protein A2 phosphorylation after DNA damage by the coordinated action of ataxia telangiectasia-mutated and DNA-dependent protein kinase. Cancer Res. 61, 8554–8563 (2001).

    CAS  PubMed  Google Scholar 

  18. Lu, X., Nannenga, B. & Donehower, L.A. PPM1D dephosphorylates Chk1 and p53 and abrogates cell cycle checkpoints. Genes Dev. 19, 1162–1174 (2005).

    Article  CAS  Google Scholar 

  19. Xia, Y., Ongusaha, P., Lee, S.W. & Liou, Y.C. Loss of Wip1 sensitizes cells to stress- and DNA damage-induced apoptosis. J. Biol. Chem. 284, 17428–17437 (2009).

    Article  CAS  Google Scholar 

  20. Gingras, A.C. et al. A novel, evolutionarily conserved protein phosphatase complex involved in cisplatin sensitivity. Mol. Cell. Proteomics 4, 1725–1740 (2005).

    Article  CAS  Google Scholar 

  21. Xu, Y., Chen, Y., Zhang, P., Jeffrey, P.D. & Shi, Y. Structure of a protein phosphatase 2A holoenzyme: insights into B55-mediated τ dephosphorylation. Mol. Cell 31, 873–885 (2008).

    Article  CAS  Google Scholar 

  22. Sakasai, R. et al. Differential involvement of phosphatidylinositol 3-kinase-related protein kinases in hyperphosphorylation of replication protein A2 in response to replication-mediated DNA double-strand breaks. Genes Cells 11, 237–246 (2006).

    Article  CAS  Google Scholar 

  23. Cruet-Hennequart, S., Glynn, M.T., Murillo, L.S., Coyne, S. & Carty, M.P. Enhanced DNA-PK-mediated RPA2 hyperphosphorylation in DNA polymerase eta-deficient human cells treated with cisplatin and oxaliplatin. DNA Repair (Amst.) 7, 582–596 (2008).

    Article  CAS  Google Scholar 

  24. Vassin, V.M., Wold, M.S. & Borowiec, J.A. Replication protein A (RPA) phosphorylation prevents RPA association with replication centers. Mol. Cell. Biol. 24, 1930–1943 (2004).

    Article  CAS  Google Scholar 

  25. Olson, E., Nievera, C.J., Klimovich, V., Fanning, E. & Wu, X. RPA2 is a direct downstream target for ATR to regulate the S-phase checkpoint. J. Biol. Chem. 281, 39517–39533 (2006).

    Article  CAS  Google Scholar 

  26. Oakley, G.G. et al. RPA phosphorylation in mitosis alters DNA binding and protein-protein interactions. Biochemistry 42, 3255–3264 (2003).

    Article  CAS  Google Scholar 

  27. Patrick, S.M., Oakley, G.G., Dixon, K. & Turchi, J.J. DNA damage induced hyperphosphorylation of replication protein A. 2. Characterization of DNA binding activity, protein interactions, and activity in DNA replication and repair. Biochemistry 44, 8438–8448 (2005).

    Article  CAS  Google Scholar 

  28. Zernik-Kobak, M., Vasunia, K., Connelly, M., Anderson, C.W. & Dixon, K. Sites of UV-induced phosphorylation of the p34 subunit of replication protein A from HeLa cells. J. Biol. Chem. 272, 23896–23904 (1997).

    Article  CAS  Google Scholar 

  29. Shao, R.G. et al. Replication-mediated DNA damage by camptothecin induces phosphorylation of RPA by DNA-dependent protein kinase and dissociates RPA:DNA-PK complexes. EMBO J. 18, 1397–1406 (1999).

    Article  CAS  Google Scholar 

  30. Anantha, R.W., Sokolova, E. & Borowiec, J.A. RPA phosphorylation facilitates mitotic exit in response to mitotic DNA damage. Proc. Natl. Acad. Sci. USA 105, 12903–12908 (2008).

    Article  CAS  Google Scholar 

  31. Kim, S.H., Holway, A.H., Wolff, S., Dillin, A. & Michael, W.M. SMK-1/PPH-4.1-mediated silencing of the CHK-1 response to DNA damage in early C. elegans embryos. J. Cell Biol. 179, 41–52 (2007).

    Article  CAS  Google Scholar 

  32. Cimprich, K.A. & Cortez, D. ATR: an essential regulator of genome integrity. Nat. Rev. Mol. Cell Biol. 9, 616–627 (2008).

    Article  CAS  Google Scholar 

  33. San Filippo, J., Sung, P. & Klein, H. Mechanism of eukaryotic homologous recombination. Annu. Rev. Biochem. 77, 229–257 (2008).

    Article  CAS  Google Scholar 

  34. Weinstock, D.M., Nakanishi, K., Helgadottir, H.R. & Jasin, M. Assaying double-strand break repair pathway choice in mammalian cells using a targeted endonuclease or the RAG recombinase. Methods Enzymol. 409, 524–540 (2006).

    Article  CAS  Google Scholar 

  35. Sleeth, K.M. et al. RPA mediates recombination repair during replication stress and is displaced from DNA by checkpoint signalling in human cells. J. Mol. Biol. 373, 38–47 (2007).

    Article  CAS  Google Scholar 

  36. Wang, X. & Haber, J.E. Role of Saccharomyces single-stranded DNA-binding protein RPA in the strand invasion step of double-strand break repair. PLoS Biol. 2, E21 (2004).

    Article  Google Scholar 

  37. Sugiyama, T., Zaitseva, E.M. & Kowalczykowski, S.C. A single-stranded DNA-binding protein is needed for efficient presynaptic complex formation by the Saccharomyces cerevisiae Rad51 protein. J. Biol. Chem. 272, 7940–7945 (1997).

    Article  CAS  Google Scholar 

  38. Wu, X., Yang, Z., Liu, Y. & Zou, Y. Preferential localization of hyperphosphorylated replication protein A to double-strand break repair and checkpoint complexes upon DNA damage. Biochem. J. 391, 473–480 (2005).

    Article  CAS  Google Scholar 

  39. Douglas, P., Moorhead, G.B., Ye, R. & Lees-Miller, S.P. Protein phosphatases regulate DNA-dependent protein kinase activity. J. Biol. Chem. 276, 18992–18998 (2001).

    Article  CAS  Google Scholar 

  40. Feng, J. et al. PP2A-dependent dephosphorylation of replication protein A (RPA) is required for the repair of DNA breaks induced by replication stress. Mol. Cell. Biol. 29, 5696–5709 (2009).

    Article  CAS  Google Scholar 

  41. Speit, G. & Schutz, P. The effect of inhibited replication on DNA migration in the comet assay in relation to cytotoxicity and clastogenicity. Mutat. Res. 655, 22–27 (2008).

    Article  CAS  Google Scholar 

  42. Petersen, P. et al. Protein phosphatase 2A antagonizes ATM and ATR in a Cdk2- and Cdc7-independent DNA damage checkpoint. Mol. Cell. Biol. 26, 1997–2011 (2006).

    Article  CAS  Google Scholar 

  43. Chen, G.I. et al. PP4R4/KIAA1622 forms a novel stable cytosolic complex with phosphoprotein phosphatase 4. J. Biol. Chem. 283, 29273–29284 (2008).

    Article  CAS  Google Scholar 

  44. Pandey, A.V., Mellon, S.H. & Miller, W.L. Protein phosphatase 2A and phosphoprotein SET regulate androgen production by P450c17. J. Biol. Chem. 278, 2837–2844 (2003).

    Article  CAS  Google Scholar 

  45. Xu, X. & Stern, D.F. NFBD1/KIAA0170 is a chromatin-associated protein involved in DNA damage signaling pathways. J. Biol. Chem. 278, 8795–8803 (2003).

    Article  CAS  Google Scholar 

  46. Lal, A. et al. miR-24-mediated downregulation of H2AX suppresses DNA repair in terminally differentiated blood cells. Nat. Struct. Mol. Biol. 16, 492–498 (2009).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank M. Michael and members of the Chowdhury and Borowiec laboratories for useful discussions. This work was supported by the Joint Center for Radiation Therapy and a Barr Award (D.C.), US National Institutes of Health grant GM083185 and an Exceptional Project Award Grant from the Breast Cancer Alliance (J.A.B.).

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Authors and Affiliations

  1. Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA

    Dong-Hyun Lee, Yunfeng Pan & Dipanjan Chowdhury

  2. Department of Radiation Oncology, Harvard Medical School, Boston, Massachusetts, USA

    Dong-Hyun Lee, Yunfeng Pan & Dipanjan Chowdhury

  3. Department of Biochemistry, New York University School of Medicine, New York, New York, USA

    Shlomo Kanner & James A Borowiec

  4. New York University Cancer Institute, New York University School of Medicine, New York, New York, USA

    Shlomo Kanner & James A Borowiec

  5. Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut, USA

    Patrick Sung

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Contributions

Most of the experiments were performed by D.-H.L. with assistance from Y.P.; RAD51 foci staining was done by S.K.; J.A.B. and D.C. wrote the paper and conceived all the experiments with assistance from P.S.

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Correspondence to Dipanjan Chowdhury.

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Lee, DH., Pan, Y., Kanner, S. et al. A PP4 phosphatase complex dephosphorylates RPA2 to facilitate DNA repair via homologous recombination. Nat Struct Mol Biol 17, 365–372 (2010). https://doi.org/10.1038/nsmb.1769

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