Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1990 May;64(5):2082–2089. doi: 10.1128/jvi.64.5.2082-2089.1990

Characterization of a major DNA-binding domain in the herpes simplex virus type 1 DNA-binding protein (ICP8).

Y S Wang 1, J D Hall 1
PMCID: PMC249364  PMID: 2157871

Abstract

We have studied the major DNA-binding protein (ICP8) from herpes simplex virus type 1 to identify its DNA-binding site. Since we obtained our protein from a cell line carrying multiple chromosomally located copies of the ICP8 gene, we first analyzed this protein to assess its similarity to the corresponding viral protein. Our protein resembled the viral protein by molecular weight, response to antibody, preference for binding single-stranded DNA, and ability to lower the melting temperature of poly(dA-dT). To define the DNA-binding domain, we subjected the protein to limited trypsin digestion and separated the peptide products on a sodium dodecyl sulfate-polyacrylamide gel. These fragments were then transferred to a nitrocellulose membrane, renatured in situ, and tested for their ability to bind DNA. From this assay, we identified four fragments which both bound DNA and exhibited the expected binding preference for single-stranded DNA. The sequence of the smallest of these fragments was determined and corresponds to a polypeptide spanning residues 300 to 849 in the intact protein. This peptide contains several regions which may be important for DNA binding based on sequence similarities in single-stranded DNA-binding proteins from other herpesviruses and, in one case, on a conserved sequence found in more distant procaryotic and eucaryotic proteins.

Full text

PDF
2082

Images in this article

2084Image
on p.2084
2085Image
on p.2085

Selected References

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

  1. Aebersold R. H., Teplow D. B., Hood L. E., Kent S. B. Electroblotting onto activated glass. High efficiency preparation of proteins from analytical sodium dodecyl sulfate-polyacrylamide gels for direct sequence analysis. J Biol Chem. 1986 Mar 25;261(9):4229–4238. [PubMed] [Google Scholar]
  2. Alberts B. M., Amodio F. J., Jenkins M., Gutmann E. D., Ferris F. L. Studies with DNA-cellulose chromatography. I. DNA-binding proteins from Escherichia coli. Cold Spring Harb Symp Quant Biol. 1968;33:289–305. doi: 10.1101/sqb.1968.033.01.033. [DOI] [PubMed] [Google Scholar]
  3. Alberts B. M., Frey L. T4 bacteriophage gene 32: a structural protein in the replication and recombination of DNA. Nature. 1970 Sep 26;227(5265):1313–1318. doi: 10.1038/2271313a0. [DOI] [PubMed] [Google Scholar]
  4. Ariga H., Klein H., Levine A. J., Horwitz M. S. A cleavage product of the adenovirus DNA binding protein is active in DNA replication in vitro. Virology. 1980 Feb;101(1):307–310. doi: 10.1016/0042-6822(80)90510-3. [DOI] [PubMed] [Google Scholar]
  5. Arsenakis M., Roizman B. A post-alpha gene function turns off the capacity of a host protein to bind DNA in cells infected with herpes simplex virus 1. J Virol. 1984 Mar;49(3):813–818. doi: 10.1128/jvi.49.3.813-818.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Baer R., Bankier A. T., Biggin M. D., Deininger P. L., Farrell P. J., Gibson T. J., Hatfull G., Hudson G. S., Satchwell S. C., Séguin C. DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature. 1984 Jul 19;310(5974):207–211. doi: 10.1038/310207a0. [DOI] [PubMed] [Google Scholar]
  7. Bowen B., Steinberg J., Laemmli U. K., Weintraub H. The detection of DNA-binding proteins by protein blotting. Nucleic Acids Res. 1980 Jan 11;8(1):1–20. doi: 10.1093/nar/8.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brayer G. D., McPherson A. Mechanism of DNA binding to the gene 5 protein of bacteriophage fd. Biochemistry. 1984 Jan 17;23(2):340–349. doi: 10.1021/bi00297a025. [DOI] [PubMed] [Google Scholar]
  9. Campbell J. L. Eukaryotic DNA replication. Annu Rev Biochem. 1986;55:733–771. doi: 10.1146/annurev.bi.55.070186.003505. [DOI] [PubMed] [Google Scholar]
  10. Casas-Finet J. R., Khamis M. I., Maki A. H., Chase J. W. Tryptophan 54 and phenylalanine 60 are involved synergistically in the binding of E. coli SSB protein to single-stranded polynucleotides. FEBS Lett. 1987 Aug 17;220(2):347–352. doi: 10.1016/0014-5793(87)80844-x. [DOI] [PubMed] [Google Scholar]
  11. Chase J. W., Williams K. R. Single-stranded DNA binding proteins required for DNA replication. Annu Rev Biochem. 1986;55:103–136. doi: 10.1146/annurev.bi.55.070186.000535. [DOI] [PubMed] [Google Scholar]
  12. Conley A. J., Knipe D. M., Jones P. C., Roizman B. Molecular genetics of herpes simplex virus. VII. Characterization of a temperature-sensitive mutant produced by in vitro mutagenesis and defective in DNA synthesis and accumulation of gamma polypeptides. J Virol. 1981 Jan;37(1):191–206. doi: 10.1128/jvi.37.1.191-206.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cuypers T., van der Ouderaa F. J., de Jong W. W. The amino acid sequence of gene 5 protein of bacteriophage M 13. Biochem Biophys Res Commun. 1974 Jul 24;59(2):557–563. doi: 10.1016/s0006-291x(74)80016-1. [DOI] [PubMed] [Google Scholar]
  14. Davison A. J., Scott J. E. The complete DNA sequence of varicella-zoster virus. J Gen Virol. 1986 Sep;67(Pt 9):1759–1816. doi: 10.1099/0022-1317-67-9-1759. [DOI] [PubMed] [Google Scholar]
  15. Delius H., Mantell N. J., Alberts B. Characterization by electron microscopy of the complex formed between T4 bacteriophage gene 32-protein and DNA. J Mol Biol. 1972 Jun 28;67(3):341–350. doi: 10.1016/0022-2836(72)90454-8. [DOI] [PubMed] [Google Scholar]
  16. Gao M., Bouchey J., Curtin K., Knipe D. M. Genetic identification of a portion of the herpes simplex virus ICP8 protein required for DNA-binding. Virology. 1988 Apr;163(2):319–329. doi: 10.1016/0042-6822(88)90272-3. [DOI] [PubMed] [Google Scholar]
  17. Gao M., Knipe D. M. Genetic evidence for multiple nuclear functions of the herpes simplex virus ICP8 DNA-binding protein. J Virol. 1989 Dec;63(12):5258–5267. doi: 10.1128/jvi.63.12.5258-5267.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gauss P., Krassa K. B., McPheeters D. S., Nelson M. A., Gold L. Zinc (II) and the single-stranded DNA binding protein of bacteriophage T4. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8515–8519. doi: 10.1073/pnas.84.23.8515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Giedroc D. P., Keating K. M., Williams K. R., Konigsberg W. H., Coleman J. E. Gene 32 protein, the single-stranded DNA binding protein from bacteriophage T4, is a zinc metalloprotein. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8452–8456. doi: 10.1073/pnas.83.22.8452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Godowski P. J., Knipe D. M. Identification of a herpes simplex virus function that represses late gene expression from parental viral genomes. J Virol. 1985 Aug;55(2):357–365. doi: 10.1128/jvi.55.2.357-365.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Godowski P. J., Knipe D. M. Mutations in the major DNA-binding protein gene of herpes simplex virus type 1 result in increased levels of viral gene expression. J Virol. 1983 Sep;47(3):478–486. doi: 10.1128/jvi.47.3.478-486.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hall J. D., Coen D. M., Fisher B. L., Weisslitz M., Randall S., Almy R. E., Gelep P. T., Schaffer P. A. Generation of genetic diversity in herpes simplex virus: an antimutator phenotype maps to the DNA polymerase locus. Virology. 1984 Jan 15;132(1):26–37. doi: 10.1016/0042-6822(84)90088-6. [DOI] [PubMed] [Google Scholar]
  23. Hosoda J., Moise H. Purification and physicochemical properties of limited proteolysis products of T4 helix destabilizing protein (gene 32 protein). J Biol Chem. 1978 Oct 25;253(20):7547–7558. [PubMed] [Google Scholar]
  24. Khamis M. I., Casas-Finet J. R., Maki A. H., Murphy J. B., Chase J. W. Investigation of the role of individual tryptophan residues in the binding of Escherichia coli single-stranded DNA binding protein to single-stranded polynucleotides. A study by optical detection of magnetic resonance and site-selected mutagenesis. J Biol Chem. 1987 Aug 15;262(23):10938–10945. [PubMed] [Google Scholar]
  25. Khamis M. I., Casas-Finet J. R., Maki A. H., Murphy J. B., Chase J. W. Role of tryptophan 54 in the binding of E. coli single-stranded DNA-binding protein to single-stranded polynucleotides. FEBS Lett. 1987 Jan 26;211(2):155–159. doi: 10.1016/0014-5793(87)81427-8. [DOI] [PubMed] [Google Scholar]
  26. Klein H., Maltzman W., Levine A. J. Structure-function relationships of the adenovirus DNA-binding protein. J Biol Chem. 1979 Nov 10;254(21):11051–11060. [PubMed] [Google Scholar]
  27. Klessig D. F., Grodzicker T. Mutations that allow human Ad2 and Ad5 to express late genes in monkey cells map in the viral gene encoding the 72K DNA binding protein. Cell. 1979 Aug;17(4):957–966. doi: 10.1016/0092-8674(79)90335-0. [DOI] [PubMed] [Google Scholar]
  28. Kowalczykowski S. C., Lonberg N., Newport J. W., von Hippel P. H. Interactions of bacteriophage T4-coded gene 32 protein with nucleic acids. I. Characterization of the binding interactions. J Mol Biol. 1981 Jan 5;145(1):75–104. doi: 10.1016/0022-2836(81)90335-1. [DOI] [PubMed] [Google Scholar]
  29. Krevolin M. D., Horwitz M. S. Functional interactions of the domains of the adenovirus DNA binding protein. Virology. 1987 Jan;156(1):167–170. doi: 10.1016/0042-6822(87)90448-x. [DOI] [PubMed] [Google Scholar]
  30. Kruijer W., van Schaik F. M., Sussenbach J. S. Structure and organization of the gene coding for the DNA binding protein of adenovirus type 5. Nucleic Acids Res. 1981 Sep 25;9(18):4439–4457. doi: 10.1093/nar/9.18.4439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Lee C. K., Knipe D. M. An immunoassay for the study of DNA-binding activities of herpes simplex virus protein ICP8. J Virol. 1985 Jun;54(3):731–738. doi: 10.1128/jvi.54.3.731-738.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Leinbach S. S., Heath L. S. Characterization of the single-stranded DNA-binding domain of the herpes simplex virus protein ICP8. Biochim Biophys Acta. 1989 Aug 14;1008(3):281–286. doi: 10.1016/0167-4781(89)90017-1. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  36. Neale G. A., Kitchingman G. R. Biochemical analysis of adenovirus type 5 DNA-binding protein mutants. J Biol Chem. 1989 Feb 25;264(6):3153–3159. [PubMed] [Google Scholar]
  37. Nevins J. R., Winkler J. J. Regulation of early adenovirus transcription: a protein product of early region 2 specifically represses region 4 transcription. Proc Natl Acad Sci U S A. 1980 Apr;77(4):1893–1897. doi: 10.1073/pnas.77.4.1893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. O'Donnell M. E., Elias P., Funnell B. E., Lehman I. R. Interaction between the DNA polymerase and single-stranded DNA-binding protein (infected cell protein 8) of herpes simplex virus 1. J Biol Chem. 1987 Mar 25;262(9):4260–4266. [PubMed] [Google Scholar]
  39. Orberg P. K., Schaffer P. A. Expression of herpes simplex virus type 1 major DNA-binding protein, ICP8, in transformed cell lines: complementation of deletion mutants and inhibition of wild-type virus. J Virol. 1987 Apr;61(4):1136–1146. doi: 10.1128/jvi.61.4.1136-1146.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Peeters B. P., Konings R. N., Schoenmakers J. G. Characterization of the DNA binding protein encoded by the N-specific filamentous Escherichia coli phage IKe. Binding properties of the protein and nucleotide sequence of the gene. J Mol Biol. 1983 Sep 5;169(1):197–215. doi: 10.1016/s0022-2836(83)80180-6. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Prasad B. V., Chiu W. Sequence comparison of single-stranded DNA binding proteins and its structural implications. J Mol Biol. 1987 Feb 5;193(3):579–584. doi: 10.1016/0022-2836(87)90268-3. [DOI] [PubMed] [Google Scholar]
  43. Prigodich R. V., Shamoo Y., Williams K. R., Chase J. W., Konigsberg W. H., Coleman J. E. 1H NMR (500 MHz) identification of aromatic residues of gene 32 protein involved in DNA binding by use of protein containing perdeuterated aromatic residues and by site-directed mutagenesis. Biochemistry. 1986 Jun 17;25(12):3666–3672. doi: 10.1021/bi00360a029. [DOI] [PubMed] [Google Scholar]
  44. Quinn C. O., Kitchingman G. R. Functional analysis of the adenovirus type 5 DNA-binding protein: site-directed mutants which are defective for adeno-associated virus helper activity. J Virol. 1986 Nov;60(2):653–661. doi: 10.1128/jvi.60.2.653-661.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Quinn J. P., McGeoch D. J. DNA sequence of the region in the genome of herpes simplex virus type 1 containing the genes for DNA polymerase and the major DNA binding protein. Nucleic Acids Res. 1985 Nov 25;13(22):8143–8163. doi: 10.1093/nar/13.22.8143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Roberts C. R., Weir A. C., Hay J., Straus S. E., Ruyechan W. T. DNA-binding proteins present in varicella-zoster virus-infected cells. J Virol. 1985 Jul;55(1):45–53. doi: 10.1128/jvi.55.1.45-53.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Ruyechan W. T. The major herpes simplex virus DNA-binding protein holds single-stranded DNA in an extended configuration. J Virol. 1983 May;46(2):661–666. doi: 10.1128/jvi.46.2.661-666.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Ruyechan W. T., Weir A. C. Interaction with nucleic acids and stimulation of the viral DNA polymerase by the herpes simplex virus type 1 major DNA-binding protein. J Virol. 1984 Dec;52(3):727–733. doi: 10.1128/jvi.52.3.727-733.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sancar A., Williams K. R., Chase J. W., Rupp W. D. Sequences of the ssb gene and protein. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4274–4278. doi: 10.1073/pnas.78.7.4274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Schaffer P. A., Bone D. R., Courtney R. J. DNA-negative temperature-sensitive mutants of herpes simplex virus type 1: patterns of viral DNA synthesis after temperature shift-up. J Virol. 1976 Mar;17(3):1043–1048. doi: 10.1128/jvi.17.3.1043-1048.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Shamoo Y., Ghosaini L. R., Keating K. M., Williams K. R., Sturtevant J. M., Konigsberg W. H. Site-specific mutagenesis of T4 gene 32: the role of tyrosine residues in protein-nucleic acid interactions. Biochemistry. 1989 Sep 5;28(18):7409–7417. doi: 10.1021/bi00444a039. [DOI] [PubMed] [Google Scholar]
  52. Sigal N., Delius H., Kornberg T., Gefter M. L., Alberts B. A DNA-unwinding protein isolated from Escherichia coli: its interaction with DNA and with DNA polymerases. Proc Natl Acad Sci U S A. 1972 Dec;69(12):3537–3541. doi: 10.1073/pnas.69.12.3537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Struhl K. Helix-turn-helix, zinc-finger, and leucine-zipper motifs for eukaryotic transcriptional regulatory proteins. Trends Biochem Sci. 1989 Apr;14(4):137–140. doi: 10.1016/0968-0004(89)90145-X. [DOI] [PubMed] [Google Scholar]
  54. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Vos H. L., van der Lee F. M., Reemst A. M., van Loon A. E., Sussenbach J. S. The genes encoding the DNA binding protein and the 23K protease of adenovirus types 40 and 41. Virology. 1988 Mar;163(1):1–10. doi: 10.1016/0042-6822(88)90227-9. [DOI] [PubMed] [Google Scholar]
  56. Weller S. K., Lee K. J., Sabourin D. J., Schaffer P. A. Genetic analysis of temperature-sensitive mutants which define the gene for the major herpes simplex virus type 1 DNA-binding protein. J Virol. 1983 Jan;45(1):354–366. doi: 10.1128/jvi.45.1.354-366.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Wilbur W. J., Lipman D. J. Rapid similarity searches of nucleic acid and protein data banks. Proc Natl Acad Sci U S A. 1983 Feb;80(3):726–730. doi: 10.1073/pnas.80.3.726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Williams K. R., LoPresti M. B., Setoguchi M. Primary structure of the bacteriophage T4 DNA helix-destabilizing protein. J Biol Chem. 1981 Feb 25;256(4):1754–1762. [PubMed] [Google Scholar]
  59. 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