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. 1994 May 1;13(9):2114–2123. doi: 10.1002/j.1460-2075.1994.tb06487.x

UV light-induced DNA synthesis arrest in HeLa cells is associated with changes in phosphorylation of human single-stranded DNA-binding protein.

M P Carty 1, M Zernik-Kobak 1, S McGrath 1, K Dixon 1
PMCID: PMC395063  PMID: 8187764

Abstract

We show that DNA replication activity in extracts of human HeLa cells decreases following UV irradiation. Alterations in replication activity in vitro parallel the UV-induced block in cell cycle progression of these cells in culture. UV irradiation also induces specific changes in the pattern of phosphorylation of the 34 kDa subunit of a DNA replication protein, human single-stranded DNA-binding protein (hSSB). The appearance of a hyperphosphorylated form of hSSB correlates with reduced in vitro DNA replication activity in extracts of UV-irradiated cells. Replication activity can be restored to these extracts in vitro by addition of purified hSSB. These results suggest that UV-induced DNA synthesis arrest may be mediated in part through phosphorylation-related alterations in the activity of hSSB, an essential component of the DNA replication apparatus.

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Selected References

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  1. Bishop J. M. Molecular themes in oncogenesis. Cell. 1991 Jan 25;64(2):235–248. doi: 10.1016/0092-8674(91)90636-d. [DOI] [PubMed] [Google Scholar]
  2. Boyle W. J., van der Geer P., Hunter T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 1991;201:110–149. doi: 10.1016/0076-6879(91)01013-r. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Brash D. E., Rudolph J. A., Simon J. A., Lin A., McKenna G. J., Baden H. P., Halperin A. J., Pontén J. A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10124–10128. doi: 10.1073/pnas.88.22.10124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brill S. J., Stillman B. Replication factor-A from Saccharomyces cerevisiae is encoded by three essential genes coordinately expressed at S phase. Genes Dev. 1991 Sep;5(9):1589–1600. doi: 10.1101/gad.5.9.1589. [DOI] [PubMed] [Google Scholar]
  6. Brill S. J., Stillman B. Yeast replication factor-A functions in the unwinding of the SV40 origin of DNA replication. Nature. 1989 Nov 2;342(6245):92–95. doi: 10.1038/342092a0. [DOI] [PubMed] [Google Scholar]
  7. Carty M. P., Hauser J., Levine A. S., Dixon K. Replication and mutagenesis of UV-damaged DNA templates in human and monkey cell extracts. Mol Cell Biol. 1993 Jan;13(1):533–542. doi: 10.1128/mcb.13.1.533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Coverley D., Kenny M. K., Munn M., Rupp W. D., Lane D. P., Wood R. D. Requirement for the replication protein SSB in human DNA excision repair. Nature. 1991 Feb 7;349(6309):538–541. doi: 10.1038/349538a0. [DOI] [PubMed] [Google Scholar]
  9. D'Urso G., Marraccino R. L., Marshak D. R., Roberts J. M. Cell cycle control of DNA replication by a homologue from human cells of the p34cdc2 protein kinase. Science. 1990 Nov 9;250(4982):786–791. doi: 10.1126/science.2173140. [DOI] [PubMed] [Google Scholar]
  10. Denekamp J. Cell kinetics and radiation biology. Int J Radiat Biol Relat Stud Phys Chem Med. 1986 Feb;49(2):357–380. doi: 10.1080/09553008514552591. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Dutta A., Din S., Brill S. J., Stillman B. Phosphorylation of replication protein A: a role for cdc2 kinase in G1/S regulation. Cold Spring Harb Symp Quant Biol. 1991;56:315–324. doi: 10.1101/sqb.1991.056.01.038. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Fairman M. P., Stillman B. Cellular factors required for multiple stages of SV40 DNA replication in vitro. EMBO J. 1988 Apr;7(4):1211–1218. doi: 10.1002/j.1460-2075.1988.tb02933.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Gatti R. A., Boder E., Vinters H. V., Sparkes R. S., Norman A., Lange K. Ataxia-telangiectasia: an interdisciplinary approach to pathogenesis. Medicine (Baltimore) 1991 Mar;70(2):99–117. [PubMed] [Google Scholar]
  17. Hartwell L. H., Weinert T. A. Checkpoints: controls that ensure the order of cell cycle events. Science. 1989 Nov 3;246(4930):629–634. doi: 10.1126/science.2683079. [DOI] [PubMed] [Google Scholar]
  18. Hauser J., Levine A. S., Dixon K. Fidelity of DNA synthesis in a mammalian in vitro replication system. Mol Cell Biol. 1988 Aug;8(8):3267–3271. doi: 10.1128/mcb.8.8.3267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. He Z., Brinton B. T., Greenblatt J., Hassell J. A., Ingles C. J. The transactivator proteins VP16 and GAL4 bind replication factor A. Cell. 1993 Jun 18;73(6):1223–1232. doi: 10.1016/0092-8674(93)90650-f. [DOI] [PubMed] [Google Scholar]
  20. Herrlich P., Ponta H., Rahmsdorf H. J. DNA damage-induced gene expression: signal transduction and relation to growth factor signaling. Rev Physiol Biochem Pharmacol. 1992;119:187–223. doi: 10.1007/3540551921_7. [DOI] [PubMed] [Google Scholar]
  21. Kastan M. B., Onyekwere O., Sidransky D., Vogelstein B., Craig R. W. Participation of p53 protein in the cellular response to DNA damage. Cancer Res. 1991 Dec 1;51(23 Pt 1):6304–6311. [PubMed] [Google Scholar]
  22. Kastan M. B., Zhan Q., el-Deiry W. S., Carrier F., Jacks T., Walsh W. V., Plunkett B. S., Vogelstein B., Fornace A. J., Jr A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell. 1992 Nov 13;71(4):587–597. doi: 10.1016/0092-8674(92)90593-2. [DOI] [PubMed] [Google Scholar]
  23. Kaufmann W. K., Cleaver J. E., Painter R. B. Ultraviolet radiation inhibits replicon initiation in S phase human cells. Biochim Biophys Acta. 1980 Jun 27;608(1):191–195. doi: 10.1016/0005-2787(80)90147-1. [DOI] [PubMed] [Google Scholar]
  24. Kenny M. K., Lee S. H., Hurwitz J. Multiple functions of human single-stranded-DNA binding protein in simian virus 40 DNA replication: single-strand stabilization and stimulation of DNA polymerases alpha and delta. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9757–9761. doi: 10.1073/pnas.86.24.9757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Kuerbitz S. J., Plunkett B. S., Walsh W. V., Kastan M. B. Wild-type p53 is a cell cycle checkpoint determinant following irradiation. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7491–7495. doi: 10.1073/pnas.89.16.7491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  28. Lane D. P. Cancer. p53, guardian of the genome. Nature. 1992 Jul 2;358(6381):15–16. doi: 10.1038/358015a0. [DOI] [PubMed] [Google Scholar]
  29. Li J. J., Kelly T. J. Simian virus 40 DNA replication in vitro. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6973–6977. doi: 10.1073/pnas.81.22.6973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Li R., Botchan M. R. The acidic transcriptional activation domains of VP16 and p53 bind the cellular replication protein A and stimulate in vitro BPV-1 DNA replication. Cell. 1993 Jun 18;73(6):1207–1221. doi: 10.1016/0092-8674(93)90649-b. [DOI] [PubMed] [Google Scholar]
  31. Murray A. W. Creative blocks: cell-cycle checkpoints and feedback controls. Nature. 1992 Oct 15;359(6396):599–604. doi: 10.1038/359599a0. [DOI] [PubMed] [Google Scholar]
  32. Painter R. B., Young B. R. Radiosensitivity in ataxia-telangiectasia: a new explanation. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7315–7317. doi: 10.1073/pnas.77.12.7315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Painter R. B., Young B. R. X-ray-induced inhibition of DNA synthesis in Chinese hamster ovary, human HeLa, and Mouse L cells. Radiat Res. 1975 Dec;64(3):648–656. [PubMed] [Google Scholar]
  34. Radler-Pohl A., Sachsenmaier C., Gebel S., Auer H. P., Bruder J. T., Rapp U., Angel P., Rahmsdorf H. J., Herrlich P. UV-induced activation of AP-1 involves obligatory extranuclear steps including Raf-1 kinase. EMBO J. 1993 Mar;12(3):1005–1012. doi: 10.1002/j.1460-2075.1993.tb05741.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Roberts J. M., D'Urso G. An origin unwinding activity regulates initiation of DNA replication during mammalian cell cycle. Science. 1988 Sep 16;241(4872):1486–1489. doi: 10.1126/science.2843984. [DOI] [PubMed] [Google Scholar]
  36. Rossman T. G., Klein C. B. From DNA damage to mutation in mammalian cells: a review. Environ Mol Mutagen. 1988;11(1):119–133. doi: 10.1002/em.2850110111. [DOI] [PubMed] [Google Scholar]
  37. Seidman M. M., Dixon K., Razzaque A., Zagursky R. J., Berman M. L. A shuttle vector plasmid for studying carcinogen-induced point mutations in mammalian cells. Gene. 1985;38(1-3):233–237. doi: 10.1016/0378-1119(85)90222-7. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Stillman B. W., Gluzman Y. Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells. Mol Cell Biol. 1985 Aug;5(8):2051–2060. doi: 10.1128/mcb.5.8.2051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tsurimoto T., Stillman B. Functions of replication factor C and proliferating-cell nuclear antigen: functional similarity of DNA polymerase accessory proteins from human cells and bacteriophage T4. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1023–1027. doi: 10.1073/pnas.87.3.1023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Weinberg D. H., Collins K. L., Simancek P., Russo A., Wold M. S., Virshup D. M., Kelly T. J. Reconstitution of simian virus 40 DNA replication with purified proteins. Proc Natl Acad Sci U S A. 1990 Nov;87(22):8692–8696. doi: 10.1073/pnas.87.22.8692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Weinert T. A., Hartwell L. H. The RAD9 gene controls the cell cycle response to DNA damage in Saccharomyces cerevisiae. Science. 1988 Jul 15;241(4863):317–322. doi: 10.1126/science.3291120. [DOI] [PubMed] [Google Scholar]
  43. Wobbe C. R., Dean F., Weissbach L., Hurwitz J. In vitro replication of duplex circular DNA containing the simian virus 40 DNA origin site. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5710–5714. doi: 10.1073/pnas.82.17.5710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wobbe C. R., Weissbach L., Borowiec J. A., Dean F. B., Murakami Y., Bullock P., Hurwitz J. Replication of simian virus 40 origin-containing DNA in vitro with purified proteins. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1834–1838. doi: 10.1073/pnas.84.7.1834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wold M. S., Li J. J., Kelly T. J. Initiation of simian virus 40 DNA replication in vitro: large-tumor-antigen- and origin-dependent unwinding of the template. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3643–3647. doi: 10.1073/pnas.84.11.3643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wold M. S., Weinberg D. H., Virshup D. M., Li J. J., Kelly T. J. Identification of cellular proteins required for simian virus 40 DNA replication. J Biol Chem. 1989 Feb 15;264(5):2801–2809. [PubMed] [Google Scholar]

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