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. 1996 Aug 15;24(16):3149–3157. doi: 10.1093/nar/24.16.3149

Inhibition of initiation of simian virus 40 DNA replication during acute response of cells irradiated by ultraviolet light.

Y C Wang 1, M T Hsu 1
PMCID: PMC146069  PMID: 8774894

Abstract

To study the mechanism by which ultraviolet (UV) light inhibits DNA replication, we examined the effects of UV 254 nm irradiation on the replication of simian virus 40 (SV40) DNA and SV40-based plasmid in monkey cells. The study was designed to determine the relative contributions made by inhibition of replication initiation and chain elongation to the immediate inhibition of DNA replication following UV irradiation. We used two-dimensional neutral-alkaline electrophoresis to examine the behaviour of replication intermediates unambiguously. Kinetic analysis using this technique showed that initiation of replication started to decline at 15 min post-irradiation. When the pulse label incorporated in SV40 replication intermediates before irradiation was chased for 1 h, most of the label was found in mature Form I and II molecules. This indicated that replication elongation took place on damaged template. We also used a transfection technique to show that heavily irradiated plasmids replicated efficiently in unirradiated transfected cells. By the transfection technique, we observed that UV irradiation of host cells dose-dependently inhibited replication of transfected non-irradiated plasmids, suggesting that the inhibition of DNA replication is due to a global change in cellular physiology induced by UV. This change was also apparent from poor staining of the chromatin by fluorescent-DNA-binding dyes immediately after UV irradiation of intact cells. We conclude that a significant fraction of chain elongation proceeds on damaged templates and DNA replication during the acute response of cells irradiated with UV is mainly controlled by the inhibition of replication initiation.

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Selected References

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  1. Bredberg A., Kraemer K. H., Seidman M. M. Restricted ultraviolet mutational spectrum in a shuttle vector propagated in xeroderma pigmentosum cells. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8273–8277. doi: 10.1073/pnas.83.21.8273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brown T. C., Cerutti P. A. Ultraviolet radiation inactivates SV40 by disrupting at least four genetic functions. EMBO J. 1986 Jan;5(1):197–203. doi: 10.1002/j.1460-2075.1986.tb04196.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brown T. C., Cerutti P. Gene expression rather than the initiation of DNA replication is the principal target of lethal u.v.-induced damage in a regulatory region of SV40 DNA. Carcinogenesis. 1987 Aug;8(8):1133–1136. doi: 10.1093/carcin/8.8.1133. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Clark J. M., Hanawalt P. C. Replicative intermediates in UV-irradiated simian virus 40. Mutat Res. 1984 Jul-Aug;132(1-2):1–14. doi: 10.1016/0167-8817(84)90061-0. [DOI] [PubMed] [Google Scholar]
  7. Cleaver J. E. Replication of nuclear and mitochondrial DNA in X-ray-damaged cells: evidence for a nuclear-specific mechanism that down-regulates replication. Radiat Res. 1992 Sep;131(3):338–344. [PubMed] [Google Scholar]
  8. Devary Y., Gottlieb R. A., Smeal T., Karin M. The mammalian ultraviolet response is triggered by activation of Src tyrosine kinases. Cell. 1992 Dec 24;71(7):1081–1091. doi: 10.1016/s0092-8674(05)80058-3. [DOI] [PubMed] [Google Scholar]
  9. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  10. Hoy C. A., Carswell C., Schimke R. T. Bromodeoxyuridine/DNA analysis of replication in CHO cells after exposure to UV light. Mutat Res. 1993 Dec;290(2):217–230. doi: 10.1016/0027-5107(93)90162-9. [DOI] [PubMed] [Google Scholar]
  11. Hurley D. L., Skantar A. M., Deering R. A., Skantarz A. M. Nuclear DNA synthesis is blocked by UV irradiation in Dictyostelium discoideum. Mutat Res. 1989 Jan;217(1):25–32. doi: 10.1016/0921-8777(89)90032-3. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Kaufmann W. K., Cleaver J. E. Mechanisms of inhibition of DNA replication by ultraviolet light in normal human and xeroderma pigmentosum fibroblasts. J Mol Biol. 1981 Jun 25;149(2):171–187. doi: 10.1016/0022-2836(81)90297-7. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Lamb J. R., Petit-Frère C., Broughton B. C., Lehmann A. R., Green M. H. Inhibition of DNA replication by ionizing radiation is mediated by a trans-acting factor. Int J Radiat Biol. 1989 Aug;56(2):125–130. doi: 10.1080/09553008914551271. [DOI] [PubMed] [Google Scholar]
  16. Larner J. M., Lee H., Hamlin J. L. Radiation effects on DNA synthesis in a defined chromosomal replicon. Mol Cell Biol. 1994 Mar;14(3):1901–1908. doi: 10.1128/mcb.14.3.1901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Livneh Z. Mechanism of replication of ultraviolet-irradiated single-stranded DNA by DNA polymerase III holoenzyme of Escherichia coli. Implications for SOS mutagenesis. J Biol Chem. 1986 Jul 15;261(20):9526–9533. [PubMed] [Google Scholar]
  18. Livneh Z. Replication of UV-irradiated single-stranded DNA by DNA polymerase III holoenzyme of Escherichia coli: evidence for bypass of pyrimidine photodimers. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4599–4603. doi: 10.1073/pnas.83.13.4599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Nawotka K. A., Huberman J. A. Two-dimensional gel electrophoretic method for mapping DNA replicons. Mol Cell Biol. 1988 Apr;8(4):1408–1413. doi: 10.1128/mcb.8.4.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. O'Day C. L., Burgers P. M., Taylor J. S. PCNA-induced DNA synthesis past cis-syn and trans-syn-I thymine dimers by calf thymus DNA polymerase delta in vitro. Nucleic Acids Res. 1992 Oct 25;20(20):5403–5406. doi: 10.1093/nar/20.20.5403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Painter R. B. Inhibition and recovery of DNA synthesis in human cells after exposure to ultraviolet light. Mutat Res. 1985 Jan-Mar;145(1-2):63–69. doi: 10.1016/0167-8817(85)90041-0. [DOI] [PubMed] [Google Scholar]
  22. Rabkin S. D., Moore P. D., Strauss B. S. In vitro bypass of UV-induced lesions by Escherichia coli DNA polymerase I: specificity of nucleotide incorporation. Proc Natl Acad Sci U S A. 1983 Mar;80(6):1541–1545. doi: 10.1073/pnas.80.6.1541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Sarasin A. R., Hanawalt P. C. Replication of ultraviolet-irradiated simian virus 40 in monkey kidney cells. J Mol Biol. 1980 Apr;138(2):299–319. doi: 10.1016/0022-2836(80)90288-0. [DOI] [PubMed] [Google Scholar]
  25. Scaria A., Edenberg H. J. Preirradiation of host cells does not alter blockage of simian virus 40 replication forks by pyrimidine dimers. Mutat Res. 1988 Jan;193(1):11–20. doi: 10.1016/0167-8817(88)90003-x. [DOI] [PubMed] [Google Scholar]
  26. Seetharam S., Protić-Sabljić M., Seidman M. M., Kraemer K. H. Abnormal ultraviolet mutagenic spectrum in plasmid DNA replicated in cultured fibroblasts from a patient with the skin cancer-prone disease, xeroderma pigmentosum. J Clin Invest. 1987 Dec;80(6):1613–1617. doi: 10.1172/JCI113248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sen A., Laipis P. J., Levine A. J. DNA replication in SV40-infected cells. XI. The properties of SV40 DNA and nucleoprotein complex synthesized in the presence of cycloheximide. Intervirology. 1975;5(3-4):122–136. doi: 10.1159/000149890. [DOI] [PubMed] [Google Scholar]
  28. Shavitt O., Livneh Z. The beta subunit modulates bypass and termination at UV lesions during in vitro replication with DNA polymerase III holoenzyme of Escherichia coli. J Biol Chem. 1989 Jul 5;264(19):11275–11281. [PubMed] [Google Scholar]
  29. Spivak G., Hanawalt P. C. Translesion DNA synthesis in the dihydrofolate reductase domain of UV-irradiated CHO cells. Biochemistry. 1992 Jul 28;31(29):6794–6800. doi: 10.1021/bi00144a021. [DOI] [PubMed] [Google Scholar]
  30. Sundin O., Varshavsky A. Terminal stages of SV40 DNA replication proceed via multiply intertwined catenated dimers. Cell. 1980 Aug;21(1):103–114. doi: 10.1016/0092-8674(80)90118-x. [DOI] [PubMed] [Google Scholar]
  31. Sussman D. J., Milman G. Short-term, high-efficiency expression of transfected DNA. Mol Cell Biol. 1984 Aug;4(8):1641–1643. doi: 10.1128/mcb.4.8.1641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Thomas D. C., Kunkel T. A. Replication of UV-irradiated DNA in human cell extracts: evidence for mutagenic bypass of pyrimidine dimers. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7744–7748. doi: 10.1073/pnas.90.16.7744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wang Y., Cheong N., Iliakis G. Persistent inhibition of DNA synthesis in irradiated rat embryo fibroblasts expressing the oncogenes H-ras plus v-myc derives from inhibition of replicon initiation and is mitigated by staurosporine. Cancer Res. 1993 Mar 15;53(6):1213–1217. [PubMed] [Google Scholar]
  34. Wang Y., Huq M. S., Cheng X., Iliakis G. Regulation of DNA replication in irradiated cells by trans-acting factors. Radiat Res. 1995 May;142(2):169–175. [PubMed] [Google Scholar]
  35. Williams J. I., Cleaver J. E. Perturbations in simian virus 40 DNA synthesis by ultraviolet light. Mutat Res. 1978 Dec;52(3):301–311. doi: 10.1016/0027-5107(78)90169-0. [DOI] [PubMed] [Google Scholar]
  36. Wong M. L., Hsu M. T. Linear adenovirus DNA is organized into supercoiled domains in virus particles. Nucleic Acids Res. 1989 May 11;17(9):3535–3550. doi: 10.1093/nar/17.9.3535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. al-Khodairy F., Carr A. M. DNA repair mutants defining G2 checkpoint pathways in Schizosaccharomyces pombe. EMBO J. 1992 Apr;11(4):1343–1350. doi: 10.1002/j.1460-2075.1992.tb05179.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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