Reversible protein phosphorylation modulates nucleotide excision repair of damaged DNA by human cell extracts (original) (raw)

Abstract

Nucleotide excision repair of DNA in mammalian cells uses more than 20 polypeptides to remove DNA lesions caused by UV light and other mutagens. To investigate whether reversible protein phosphorylation can significantly modulate this repair mechanism we studied the effect of specific inhibitors of Ser/Thr protein phosphatases. The ability of HeLa cell extracts to carry out nucleotide excision repair in vitro was highly sensitive to three toxins (okadaic acid, microcystin-LR and tautomycin), which block PP1- and PP2A-type phosphatases. Repair was more sensitive to okadaic acid than to tautomycin, suggesting the involvement of a PP2A-type enzyme, and was insensitive to inhibitor-2, which exclusively inhibits PP1-type enzymes. In a repair synthesis assay the toxins gave 70% inhibition of activity. Full activity could be restored to toxin-inhibited extracts by addition of purified PP2A, but not PP1. The p34 subunit of replication protein A was hyperphosphorylated in cell extracts in the presence of phosphatase inhibitors, but we found no evidence that this affected repair. In a coupled incision/synthesis repair assay okadaic acid decreased the production of incision intermediates in the repair reaction. The formation of 25-30mer oligonucleotides by dual incision during repair was also inhibited by okadaic acid and inhibition could be reversed with PP2A. Thus Ser/Thr- specific protein phosphorylation plays an important role in the modulation of nucleotide excision repair in vitro.

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Selected References

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  1. Aboussekhra A., Biggerstaff M., Shivji M. K., Vilpo J. A., Moncollin V., Podust V. N., Protić M., Hübscher U., Egly J. M., Wood R. D. Mammalian DNA nucleotide excision repair reconstituted with purified protein components. Cell. 1995 Mar 24;80(6):859–868. doi: 10.1016/0092-8674(95)90289-9. [DOI] [PubMed] [Google Scholar]
  2. Aboussekhra A., Wood R. D. Repair of UV-damaged DNA by mammalian cells and Saccharomyces cerevisiae. Curr Opin Genet Dev. 1994 Apr;4(2):212–220. doi: 10.1016/s0959-437x(05)80047-4. [DOI] [PubMed] [Google Scholar]
  3. Alessi D. R., Street A. J., Cohen P., Cohen P. T. Inhibitor-2 functions like a chaperone to fold three expressed isoforms of mammalian protein phosphatase-1 into a conformation with the specificity and regulatory properties of the native enzyme. Eur J Biochem. 1993 May 1;213(3):1055–1066. doi: 10.1111/j.1432-1033.1993.tb17853.x. [DOI] [PubMed] [Google Scholar]
  4. Barker H. M., Craig S. P., Spurr N. K., Cohen P. T. Sequence of human protein serine/threonine phosphatase 1 gamma and localization of the gene (PPP1CC) encoding it to chromosome bands 12q24.1-q24.2. Biochim Biophys Acta. 1993 Aug 18;1178(2):228–233. doi: 10.1016/0167-4889(93)90014-g. [DOI] [PubMed] [Google Scholar]
  5. Bialojan C., Takai A. Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics. Biochem J. 1988 Nov 15;256(1):283–290. doi: 10.1042/bj2560283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bootsma D., Weeda G., Vermeulen W., van Vuuren H., Troelstra C., van der Spek P., Hoeijmakers J. Nucleotide excision repair syndromes: molecular basis and clinical symptoms. Philos Trans R Soc Lond B Biol Sci. 1995 Jan 30;347(1319):75–81. doi: 10.1098/rstb.1995.0012. [DOI] [PubMed] [Google Scholar]
  7. Brush G. S., Anderson C. W., Kelly T. J. The DNA-activated protein kinase is required for the phosphorylation of replication protein A during simian virus 40 DNA replication. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12520–12524. doi: 10.1073/pnas.91.26.12520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bøe R., Gjertsen B. T., Vintermyr O. K., Houge G., Lanotte M., Døskeland S. O. The protein phosphatase inhibitor okadaic acid induces morphological changes typical of apoptosis in mammalian cells. Exp Cell Res. 1991 Jul;195(1):237–246. doi: 10.1016/0014-4827(91)90523-w. [DOI] [PubMed] [Google Scholar]
  9. Calsou P., Salles B. Measurement of damage-specific DNA incision by nucleotide excision repair in vitro. Biochem Biophys Res Commun. 1994 Jul 29;202(2):788–795. doi: 10.1006/bbrc.1994.1999. [DOI] [PubMed] [Google Scholar]
  10. Carty M. P., Zernik-Kobak M., McGrath S., Dixon K. UV light-induced DNA synthesis arrest in HeLa cells is associated with changes in phosphorylation of human single-stranded DNA-binding protein. EMBO J. 1994 May 1;13(9):2114–2123. doi: 10.1002/j.1460-2075.1994.tb06487.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cohen P., Alemany S., Hemmings B. A., Resink T. J., Strålfors P., Tung H. Y. Protein phosphatase-1 and protein phosphatase-2A from rabbit skeletal muscle. Methods Enzymol. 1988;159:390–408. doi: 10.1016/0076-6879(88)59039-0. [DOI] [PubMed] [Google Scholar]
  12. Cohen P. Classification of protein-serine/threonine phosphatases: identification and quantitation in cell extracts. Methods Enzymol. 1991;201:389–398. doi: 10.1016/0076-6879(91)01035-z. [DOI] [PubMed] [Google Scholar]
  13. Cohen P., Klumpp S., Schelling D. L. An improved procedure for identifying and quantitating protein phosphatases in mammalian tissues. FEBS Lett. 1989 Jul 3;250(2):596–600. doi: 10.1016/0014-5793(89)80803-8. [DOI] [PubMed] [Google Scholar]
  14. Cohen P. The discovery of protein phosphatases: from chaos and confusion to an understanding of their role in cell regulation and human disease. Bioessays. 1994 Aug;16(8):583–588. doi: 10.1002/bies.950160812. [DOI] [PubMed] [Google Scholar]
  15. Cohen P. The structure and regulation of protein phosphatases. Annu Rev Biochem. 1989;58:453–508. doi: 10.1146/annurev.bi.58.070189.002321. [DOI] [PubMed] [Google Scholar]
  16. Coverley D., Kenny M. K., Lane D. P., Wood R. D. A role for the human single-stranded DNA binding protein HSSB/RPA in an early stage of nucleotide excision repair. Nucleic Acids Res. 1992 Aug 11;20(15):3873–3880. doi: 10.1093/nar/20.15.3873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Din S., Brill S. J., Fairman M. P., Stillman B. Cell-cycle-regulated phosphorylation of DNA replication factor A from human and yeast cells. Genes Dev. 1990 Jun;4(6):968–977. doi: 10.1101/gad.4.6.968. [DOI] [PubMed] [Google Scholar]
  18. Dutta A., Stillman B. cdc2 family kinases phosphorylate a human cell DNA replication factor, RPA, and activate DNA replication. EMBO J. 1992 Jun;11(6):2189–2199. doi: 10.1002/j.1460-2075.1992.tb05278.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fotedar R., Roberts J. M. Cell cycle regulated phosphorylation of RPA-32 occurs within the replication initiation complex. EMBO J. 1992 Jun;11(6):2177–2187. doi: 10.1002/j.1460-2075.1992.tb05277.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Garfin D. E. One-dimensional gel electrophoresis. Methods Enzymol. 1990;182:425–441. doi: 10.1016/0076-6879(90)82035-z. [DOI] [PubMed] [Google Scholar]
  21. Haystead T. A., Sim A. T., Carling D., Honnor R. C., Tsukitani Y., Cohen P., Hardie D. G. Effects of the tumour promoter okadaic acid on intracellular protein phosphorylation and metabolism. Nature. 1989 Jan 5;337(6202):78–81. doi: 10.1038/337078a0. [DOI] [PubMed] [Google Scholar]
  22. Hubbard M. J., Cohen P. On target with a new mechanism for the regulation of protein phosphorylation. Trends Biochem Sci. 1993 May;18(5):172–177. doi: 10.1016/0968-0004(93)90109-z. [DOI] [PubMed] [Google Scholar]
  23. Hubbard M. J., Cohen P. Targeting subunits for protein phosphatases. Methods Enzymol. 1991;201:414–427. doi: 10.1016/0076-6879(91)01038-4. [DOI] [PubMed] [Google Scholar]
  24. Hunter T. Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell. 1995 Jan 27;80(2):225–236. doi: 10.1016/0092-8674(95)90405-0. [DOI] [PubMed] [Google Scholar]
  25. Hunter T., Sefton B. M. Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1311–1315. doi: 10.1073/pnas.77.3.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kenny M. K., Schlegel U., Furneaux H., Hurwitz J. The role of human single-stranded DNA binding protein and its individual subunits in simian virus 40 DNA replication. J Biol Chem. 1990 May 5;265(13):7693–7700. [PubMed] [Google Scholar]
  27. MacKintosh C., Beattie K. A., Klumpp S., Cohen P., Codd G. A. Cyanobacterial microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants. FEBS Lett. 1990 May 21;264(2):187–192. doi: 10.1016/0014-5793(90)80245-e. [DOI] [PubMed] [Google Scholar]
  28. MacKintosh C., Klumpp S. Tautomycin from the bacterium Streptomyces verticillatus. Another potent and specific inhibitor of protein phosphatases 1 and 2A. FEBS Lett. 1990 Dec 17;277(1-2):137–140. doi: 10.1016/0014-5793(90)80828-7. [DOI] [PubMed] [Google Scholar]
  29. Mermoud J. E., Cohen P. T., Lamond A. I. Regulation of mammalian spliceosome assembly by a protein phosphorylation mechanism. EMBO J. 1994 Dec 1;13(23):5679–5688. doi: 10.1002/j.1460-2075.1994.tb06906.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mermoud J. E., Cohen P., Lamond A. I. Ser/Thr-specific protein phosphatases are required for both catalytic steps of pre-mRNA splicing. Nucleic Acids Res. 1992 Oct 25;20(20):5263–5269. doi: 10.1093/nar/20.20.5263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mu D., Park C. H., Matsunaga T., Hsu D. S., Reardon J. T., Sancar A. Reconstitution of human DNA repair excision nuclease in a highly defined system. J Biol Chem. 1995 Feb 10;270(6):2415–2418. doi: 10.1074/jbc.270.6.2415. [DOI] [PubMed] [Google Scholar]
  32. O'Donovan A., Davies A. A., Moggs J. G., West S. C., Wood R. D. XPG endonuclease makes the 3' incision in human DNA nucleotide excision repair. Nature. 1994 Sep 29;371(6496):432–435. doi: 10.1038/371432a0. [DOI] [PubMed] [Google Scholar]
  33. Pan Z. Q., Amin A. A., Gibbs E., Niu H., Hurwitz J. Phosphorylation of the p34 subunit of human single-stranded-DNA-binding protein in cyclin A-activated G1 extracts is catalyzed by cdk-cyclin A complex and DNA-dependent protein kinase. Proc Natl Acad Sci U S A. 1994 Aug 30;91(18):8343–8347. doi: 10.1073/pnas.91.18.8343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pan Z. Q., Park C. H., Amin A. A., Hurwitz J., Sancar A. Phosphorylated and unphosphorylated forms of human single-stranded DNA-binding protein are equally active in simian virus 40 DNA replication and in nucleotide excision repair. Proc Natl Acad Sci U S A. 1995 May 9;92(10):4636–4640. doi: 10.1073/pnas.92.10.4636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sancar A., Tang M. S. Nucleotide excision repair. Photochem Photobiol. 1993 May;57(5):905–921. doi: 10.1111/j.1751-1097.1993.tb09233.x. [DOI] [PubMed] [Google Scholar]
  36. Schönthal A., Feramisco J. R. Inhibition of histone H1 kinase expression, retinoblastoma protein phosphorylation, and cell proliferation by the phosphatase inhibitor okadaic acid. Oncogene. 1993 Feb;8(2):433–441. [PubMed] [Google Scholar]
  37. Shivji K. K., Kenny M. K., Wood R. D. Proliferating cell nuclear antigen is required for DNA excision repair. Cell. 1992 Apr 17;69(2):367–374. doi: 10.1016/0092-8674(92)90416-a. [DOI] [PubMed] [Google Scholar]
  38. Shivji M. K., Grey S. J., Strausfeld U. P., Wood R. D., Blow J. J. Cip1 inhibits DNA replication but not PCNA-dependent nucleotide excision-repair. Curr Biol. 1994 Dec 1;4(12):1062–1068. doi: 10.1016/s0960-9822(00)00244-x. [DOI] [PubMed] [Google Scholar]
  39. Wood R. D., Aboussekhra A., Biggerstaff M., Jones C. J., O'Donovan A., Shivji M. K., Szymkowski D. E. Nucleotide excision repair of DNA by mammalian cell extracts and purified proteins. Cold Spring Harb Symp Quant Biol. 1993;58:625–632. doi: 10.1101/sqb.1993.058.01.069. [DOI] [PubMed] [Google Scholar]
  40. Wood R. D. Proteins that participate in nucleotide excision repair of DNA in mammalian cells. Philos Trans R Soc Lond B Biol Sci. 1995 Jan 30;347(1319):69–74. doi: 10.1098/rstb.1995.0011. [DOI] [PubMed] [Google Scholar]
  41. Wood R. D., Robins P., Lindahl T. Complementation of the xeroderma pigmentosum DNA repair defect in cell-free extracts. Cell. 1988 Apr 8;53(1):97–106. doi: 10.1016/0092-8674(88)90491-6. [DOI] [PubMed] [Google Scholar]