Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1990 Oct;64(10):4957–4967. doi: 10.1128/jvi.64.10.4957-4967.1990

Herpes simplex virus DNA synthesis at a preformed replication fork in vitro.

S D Rabkin 1, B Hanlon 1
PMCID: PMC247987  PMID: 2168979

Abstract

Proteins from herpes simplex virus (HSV)-infected cells were used to reconstitute DNA synthesis in vitro on a preformed replication fork. The preformed replication fork consisted of a nicked, double-stranded, circular DNA molecule with a 5' single-strand tail that was noncomplementary to the template. The products of DNA synthesis on this substrate were rolling-circle molecules, as demonstrated by electron microscopy and alkaline agarose gel electrophoresis. The tails contained double-stranded regions, indicating that both leading- and lagging-strand DNA syntheses occurred. Rolling-circle DNA replication was dependent upon HSV DNA polymerase and ATP and was stimulated by a crude fraction containing ICP8 (HSV DNA-binding protein). Similar protein fractions from mock-infected cells were unable to support rolling-circle DNA replication. This in vitro DNA replication system should prove useful in the identification and characterization of the enzymatic activities required at the HSV replication fork.

Full text

PDF
4957

Images in this article

4959Image
on p.4959
4960Image
on p.4960
4961Image
on p.4961
4961Image
on p.4961
4962Image
on p.4962
4962Image
on p.4962
4963Image
on p.4963
4964Image
on p.4964

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alberts B. M. The DNA enzymology of protein machines. Cold Spring Harb Symp Quant Biol. 1984;49:1–12. doi: 10.1101/sqb.1984.049.01.003. [DOI] [PubMed] [Google Scholar]
  2. Bayliss G. J., Marsden H. S., Hay J. Herpes simplex virus proteins: DNA-binding proteins in infected cells and in the virus structure. Virology. 1975 Nov;68(1):124–134. doi: 10.1016/0042-6822(75)90154-3. [DOI] [PubMed] [Google Scholar]
  3. Becker Y., Chejanovsky N., Fridlender B. DNA-binding proteins in the nuclei of herpes simplex virus-infected, arginine-deprived, BSC-1 cells. J Gen Virol. 1980 Oct;50(2):259–267. doi: 10.1099/0022-1317-50-2-259. [DOI] [PubMed] [Google Scholar]
  4. Bedinger P., Munn M., Alberts B. M. Sequence-specific pausing during in vitro DNA replication on double-stranded DNA templates. J Biol Chem. 1989 Oct 5;264(28):16880–16886. [PubMed] [Google Scholar]
  5. Ben-Porat T., Blankenship M. L., DeMarchi J. M., Kaplan A. S. Replication of herpesvirus DNA. III. Rate of DNA elongation. J Virol. 1977 Jun;22(3):734–741. doi: 10.1128/jvi.22.3.734-741.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Caradonna S. J., Cheng Y. C. Induction of uracil-DNA glycosylase and dUTP nucleotidohydrolase activity in herpes simplex virus-infected human cells. J Biol Chem. 1981 Oct 10;256(19):9834–9837. [PubMed] [Google Scholar]
  7. Cha T. A., Alberts B. M. The bacteriophage T4 DNA replication fork. Only DNA helicase is required for leading strand DNA synthesis by the DNA polymerase holoenzyme. J Biol Chem. 1989 Jul 25;264(21):12220–12225. [PubMed] [Google Scholar]
  8. Challberg M. D. A method for identifying the viral genes required for herpesvirus DNA replication. Proc Natl Acad Sci U S A. 1986 Dec;83(23):9094–9098. doi: 10.1073/pnas.83.23.9094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Challberg M. D., Englund P. T. The effect of template secondary structure on vaccinia DNA polymerase. J Biol Chem. 1979 Aug 25;254(16):7820–7826. [PubMed] [Google Scholar]
  10. Cohen G. H. Ribonucleotide reductase activity of synchronized KB cells infected with herpes simplex virus. J Virol. 1972 Mar;9(3):408–418. doi: 10.1128/jvi.9.3.408-418.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Crute J. J., Tsurumi T., Zhu L. A., Weller S. K., Olivo P. D., Challberg M. D., Mocarski E. S., Lehman I. R. Herpes simplex virus 1 helicase-primase: a complex of three herpes-encoded gene products. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2186–2189. doi: 10.1073/pnas.86.7.2186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Elias P., O'Donnell M. E., Mocarski E. S., Lehman I. R. A DNA binding protein specific for an origin of replication of herpes simplex virus type 1. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6322–6326. doi: 10.1073/pnas.83.17.6322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gallo M. L., Dorsky D. I., Crumpacker C. S., Parris D. S. The essential 65-kilodalton DNA-binding protein of herpes simplex virus stimulates the virus-encoded DNA polymerase. J Virol. 1989 Dec;63(12):5023–5029. doi: 10.1128/jvi.63.12.5023-5029.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Heilbronn R., zur Hausen H. A subset of herpes simplex virus replication genes induces DNA amplification within the host cell genome. J Virol. 1989 Sep;63(9):3683–3692. doi: 10.1128/jvi.63.9.3683-3692.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jacob R. J., Morse L. S., Roizman B. Anatomy of herpes simplex virus DNA. XII. Accumulation of head-to-tail concatemers in nuclei of infected cells and their role in the generation of the four isomeric arrangements of viral DNA. J Virol. 1979 Feb;29(2):448–457. doi: 10.1128/jvi.29.2.448-457.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. KIT S., DUBBS D. R. Acquisition of thymidine kinase activity by herpes simplex-infected mouse fibroblast cells. Biochem Biophys Res Commun. 1963 Apr 2;11:55–59. doi: 10.1016/0006-291x(63)90027-5. [DOI] [PubMed] [Google Scholar]
  17. Kaguni L. S., Clayton D. A. Template-directed pausing in in vitro DNA synthesis by DNA polymerase a from Drosophila melanogaster embryos. Proc Natl Acad Sci U S A. 1982 Feb;79(4):983–987. doi: 10.1073/pnas.79.4.983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kornberg A., Scott J. F., Bertsch L. L. ATP utilization by rep protein in the catalytic separation of DNA strands at a replicating fork. J Biol Chem. 1978 May 10;253(9):3298–3304. [PubMed] [Google Scholar]
  19. LaDuca R. J., Fay P. J., Chuang C., McHenry C. S., Bambara R. A. Site-specific pausing of deoxyribonucleic acid synthesis catalyzed by four forms of Escherichia coli DNA polymerase III. Biochemistry. 1983 Oct 25;22(22):5177–5188. doi: 10.1021/bi00291a018. [DOI] [PubMed] [Google Scholar]
  20. Lechner R. L., Richardson C. C. A preformed, topologically stable replication fork. Characterization of leading strand DNA synthesis catalyzed by T7 DNA polymerase and T7 gene 4 protein. J Biol Chem. 1983 Sep 25;258(18):11185–11196. [PubMed] [Google Scholar]
  21. Lee M. S., Marians K. J. The Escherichia coli primosome can translocate actively in either direction along a DNA strand. J Biol Chem. 1989 Aug 25;264(24):14531–14542. [PubMed] [Google Scholar]
  22. Lehman I. R., Kaguni L. S. DNA polymerase alpha. J Biol Chem. 1989 Mar 15;264(8):4265–4268. [PubMed] [Google Scholar]
  23. Matthews J. T., Carroll R. D., Stevens J. T., Haffey M. L. In vitro mutagenesis of the herpes simplex virus type 1 DNA polymerase gene results in altered drug sensitivity of the enzyme. J Virol. 1989 Nov;63(11):4913–4918. doi: 10.1128/jvi.63.11.4913-4918.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Matz B. Herpes simplex virus infection generates large tandemly reiterated simian virus 40 DNA molecules in a transformed hamster cell line. J Virol. 1987 May;61(5):1427–1434. doi: 10.1128/jvi.61.5.1427-1434.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Matz B., Subak-Sharpe J. H., Preston V. G. Physical mapping of temperature-sensitive mutations of herpes simplex virus type 1 using cloned restriction endonuclease fragments. J Gen Virol. 1983 Oct;64(Pt 10):2261–2270. doi: 10.1099/0022-1317-64-10-2261. [DOI] [PubMed] [Google Scholar]
  26. McGeoch D. J., Dalrymple M. A., Davison A. J., Dolan A., Frame M. C., McNab D., Perry L. J., Scott J. E., Taylor P. The complete DNA sequence of the long unique region in the genome of herpes simplex virus type 1. J Gen Virol. 1988 Jul;69(Pt 7):1531–1574. doi: 10.1099/0022-1317-69-7-1531. [DOI] [PubMed] [Google Scholar]
  27. Mok M., Marians K. J. The Escherichia coli preprimosome and DNA B helicase can form replication forks that move at the same rate. J Biol Chem. 1987 Dec 5;262(34):16644–16654. [PubMed] [Google Scholar]
  28. Morrison J. M., Keir H. M. A new DNA-exonuclease in cells infected with herpes virus: partial purification and properties of the enzyme. J Gen Virol. 1968 Dec;3(3):337–347. doi: 10.1099/0022-1317-3-3-337. [DOI] [PubMed] [Google Scholar]
  29. Nakai H., Richardson C. C. Leading and lagging strand synthesis at the replication fork of bacteriophage T7. Distinct properties of T7 gene 4 protein as a helicase and primase. J Biol Chem. 1988 Jul 15;263(20):9818–9830. [PubMed] [Google Scholar]
  30. Nakai H., Richardson C. C. The effect of the T7 and Escherichia coli DNA-binding proteins at the replication fork of bacteriophage T7. J Biol Chem. 1988 Jul 15;263(20):9831–9839. [PubMed] [Google Scholar]
  31. O'Donnell M. E., Elias P., Lehman I. R. Processive replication of single-stranded DNA templates by the herpes simplex virus-induced DNA polymerase. J Biol Chem. 1987 Mar 25;262(9):4252–4259. [PubMed] [Google Scholar]
  32. Olivo P. D., Nelson N. J., Challberg M. D. Herpes simplex virus DNA replication: the UL9 gene encodes an origin-binding protein. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5414–5418. doi: 10.1073/pnas.85.15.5414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Parris D. S., Cross A., Haarr L., Orr A., Frame M. C., Murphy M., McGeoch D. J., Marsden H. S. Identification of the gene encoding the 65-kilodalton DNA-binding protein of herpes simplex virus type 1. J Virol. 1988 Mar;62(3):818–825. doi: 10.1128/jvi.62.3.818-825.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Powell K. L., Littler E., Purifoy D. J. Nonstructural proteins of herpes simplex virus. II. Major virus-specific DNa-binding protein. J Virol. 1981 Sep;39(3):894–902. doi: 10.1128/jvi.39.3.894-902.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Powell K. L., Purifoy D. J. Nonstructural proteins of herpes simplex virus. I. Purification of the induced DNA polymerase. J Virol. 1977 Nov;24(2):618–626. doi: 10.1128/jvi.24.2.618-626.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Purifoy D. J., Lewis R. B., Powell K. L. Identification of the herpes simplex virus DNA polymerase gene. Nature. 1977 Oct 13;269(5629):621–623. doi: 10.1038/269621a0. [DOI] [PubMed] [Google Scholar]
  37. Rabkin S. D., Richardson C. C. Initiation of DNA replication at cloned origins of bacteriophage T7. J Mol Biol. 1988 Dec 20;204(4):903–916. doi: 10.1016/0022-2836(88)90050-2. [DOI] [PubMed] [Google Scholar]
  38. Stow N. D. Localization of an origin of DNA replication within the TRS/IRS repeated region of the herpes simplex virus type 1 genome. EMBO J. 1982;1(7):863–867. doi: 10.1002/j.1460-2075.1982.tb01261.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tabor S., Huber H. E., Richardson C. C. Escherichia coli thioredoxin confers processivity on the DNA polymerase activity of the gene 5 protein of bacteriophage T7. J Biol Chem. 1987 Nov 25;262(33):16212–16223. [PubMed] [Google Scholar]
  40. Teraoka H., Sumikawa T., Tsukada K. Purification of DNA ligase II from calf thymus and preparation of rabbit antibody against calf thymus DNA ligase II. J Biol Chem. 1986 May 25;261(15):6888–6892. [PubMed] [Google Scholar]
  41. Vaughan P. J., Purifoy D. J., Powell K. L. DNA-binding protein associated with herpes simplex virus DNA polymerase. J Virol. 1985 Feb;53(2):501–508. doi: 10.1128/jvi.53.2.501-508.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Weissbach A., Hong S. C., Aucker J., Muller R. Characterization of herpes simplex virus-induced deoxyribonucleic acid polymerase. J Biol Chem. 1973 Sep 25;248(18):6270–6277. [PubMed] [Google Scholar]
  43. Weller S. K., Carmichael E. P., Aschman D. P., Goldstein D. J., Schaffer P. A. Genetic and phenotypic characterization of mutants in four essential genes that map to the left half of HSV-1 UL DNA. Virology. 1987 Nov;161(1):198–210. doi: 10.1016/0042-6822(87)90186-3. [DOI] [PubMed] [Google Scholar]
  44. Wohlrab F., Francke B. Deoxyribopyrimidine triphosphatase activity specific for cells infected with herpes simplex virus type 1. Proc Natl Acad Sci U S A. 1980 Apr;77(4):1872–1876. doi: 10.1073/pnas.77.4.1872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wu C. A., Nelson N. J., McGeoch D. J., Challberg M. D. Identification of herpes simplex virus type 1 genes required for origin-dependent DNA synthesis. J Virol. 1988 Feb;62(2):435–443. doi: 10.1128/jvi.62.2.435-443.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES