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. 1992 Aug 11;20(15):3873–3880. doi: 10.1093/nar/20.15.3873

A role for the human single-stranded DNA binding protein HSSB/RPA in an early stage of nucleotide excision repair.

D Coverley 1, M K Kenny 1, D P Lane 1, R D Wood 1
PMCID: PMC334061  PMID: 1508673

Abstract

The human single-stranded DNA binding protein (HSSB/RPA) is involved in several processes that maintain the integrity of the genome including DNA replication, homologous recombination, and nucleotide excision repair of damaged DNA. We report studies that analyze the role of HSSB in DNA repair. Specific protein-protein interactions appear to be involved in the repair function of HSSB, since it cannot be replaced by heterologous single-stranded DNA binding proteins. Anti-HSSB antibodies that inhibit the ability of HSSB to stimulate DNA polymerase alpha also inhibit repair synthesis mediated by human cell-free extracts. However, antibodies that neutralize DNA polymerase alpha do not inhibit repair synthesis. Repair is sensitive to aphidicolin, suggesting that DNA polymerase epsilon or delta participates in nucleotide excision repair by cell extracts. HSSB has a role other than generally stimulating synthesis by DNA polymerases, as it does not enhance the residual damage-dependent background synthesis displayed by repair-deficient extracts from xeroderma pigmentosum cells. Significantly, when damaged DNA is incised by the Escherichia coli UvrABC repair enzyme, human cell extracts can carry out repair synthesis even when HSSB has been neutralized with antibodies. This suggests that HSSB functions in an early stage of repair, rather than exclusively in repair synthesis. A model for the role of HSSB in repair is presented.

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Selected References

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

  1. Biggerstaff M., Robins P., Coverley D., Wood R. D. Effect of exogenous DNA fragments on human cell extract-mediated DNA repair synthesis. Mutat Res. 1991 May;254(3):217–224. doi: 10.1016/0921-8777(91)90059-x. [DOI] [PubMed] [Google Scholar]
  2. Biggerstaff M., Wood R. D. Requirement for ERCC-1 and ERCC-3 gene products in DNA excision repair in vitro. Complementation using rodent and human cell extracts. J Biol Chem. 1992 Apr 5;267(10):6879–6885. [PubMed] [Google Scholar]
  3. Bravo R., Frank R., Blundell P. A., Macdonald-Bravo H. Cyclin/PCNA is the auxiliary protein of DNA polymerase-delta. Nature. 1987 Apr 2;326(6112):515–517. doi: 10.1038/326515a0. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Burgers P. M. Saccharomyces cerevisiae replication factor C. II. Formation and activity of complexes with the proliferating cell nuclear antigen and with DNA polymerases delta and epsilon. J Biol Chem. 1991 Nov 25;266(33):22698–22706. [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Dornreiter I., Erdile L. F., Gilbert I. U., von Winkler D., Kelly T. J., Fanning E. Interaction of DNA polymerase alpha-primase with cellular replication protein A and SV40 T antigen. EMBO J. 1992 Feb;11(2):769–776. doi: 10.1002/j.1460-2075.1992.tb05110.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dresler S. L. Comparative enzymology of ultraviolet-induced DNA repair synthesis and semiconservative DNA replication in permeable diploid human fibroblasts. J Biol Chem. 1984 Nov 25;259(22):13947–13952. [PubMed] [Google Scholar]
  10. Dresler S. L., Frattini M. G. DNA replication and UV-induced DNA repair synthesis in human fibroblasts are much less sensitive than DNA polymerase alpha to inhibition by butylphenyl-deoxyguanosine triphosphate. Nucleic Acids Res. 1986 Sep 11;14(17):7093–7102. doi: 10.1093/nar/14.17.7093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Erdile L. F., Wold M. S., Kelly T. J. The primary structure of the 32-kDa subunit of human replication protein A. J Biol Chem. 1990 Feb 25;265(6):3177–3182. [PubMed] [Google Scholar]
  12. 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]
  13. Grossman L., Yeung A. T. The UvrABC endonuclease of Escherichia coli. Photochem Photobiol. 1990 Jun;51(6):749–755. [PubMed] [Google Scholar]
  14. Hansson J., Grossman L., Lindahl T., Wood R. D. Complementation of the xeroderma pigmentosum DNA repair synthesis defect with Escherichia coli UvrABC proteins in a cell-free system. Nucleic Acids Res. 1990 Jan 11;18(1):35–40. doi: 10.1093/nar/18.1.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hansson J., Munn M., Rupp W. D., Kahn R., Wood R. D. Localization of DNA repair synthesis by human cell extracts to a short region at the site of a lesion. J Biol Chem. 1989 Dec 25;264(36):21788–21792. [PubMed] [Google Scholar]
  16. Heyer W. D., Rao M. R., Erdile L. F., Kelly T. J., Kolodner R. D. An essential Saccharomyces cerevisiae single-stranded DNA binding protein is homologous to the large subunit of human RP-A. EMBO J. 1990 Jul;9(7):2321–2329. doi: 10.1002/j.1460-2075.1990.tb07404.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Huang J. C., Svoboda D. L., Reardon J. T., Sancar A. Human nucleotide excision nuclease removes thymine dimers from DNA by incising the 22nd phosphodiester bond 5' and the 6th phosphodiester bond 3' to the photodimer. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3664–3668. doi: 10.1073/pnas.89.8.3664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hunting D. J., Gowans B. J., Dresler S. L. DNA polymerase delta mediates excision repair in growing cells damaged with ultraviolet radiation. Biochem Cell Biol. 1991 Apr;69(4):303–308. doi: 10.1139/o91-046. [DOI] [PubMed] [Google Scholar]
  19. Ikeda J. E., Enomoto T., Hurwitz J. Replication of adenovirus DNA-protein complex with purified proteins. Proc Natl Acad Sci U S A. 1981 Feb;78(2):884–888. doi: 10.1073/pnas.78.2.884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ishimi Y., Claude A., Bullock P., Hurwitz J. Complete enzymatic synthesis of DNA containing the SV40 origin of replication. J Biol Chem. 1988 Dec 25;263(36):19723–19733. [PubMed] [Google Scholar]
  21. Jones C. J., Edwards S. M., Waters R. The repair of identified large DNA adducts induced by 4-nitroquinoline-1-oxide in normal or xeroderma pigmentosum group A human fibroblasts, and the role of DNA polymerases alpha or delta. Carcinogenesis. 1989 Jul;10(7):1197–1201. doi: 10.1093/carcin/10.7.1197. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Keyse S. M., Tyrrell R. M. Excision repair in permeable arrested human skin fibroblasts damaged by UV (254 nm) radiation: evidence that alpha- and beta-polymerases act sequentially at the repolymerisation step. Mutat Res. 1985 Jul;146(1):109–119. doi: 10.1016/0167-8817(85)90061-6. [DOI] [PubMed] [Google Scholar]
  25. Kim C., Snyder R. O., Wold M. S. Binding properties of replication protein A from human and yeast cells. Mol Cell Biol. 1992 Jul;12(7):3050–3059. doi: 10.1128/mcb.12.7.3050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kong X. P., Onrust R., O'Donnell M., Kuriyan J. Three-dimensional structure of the beta subunit of E. coli DNA polymerase III holoenzyme: a sliding DNA clamp. Cell. 1992 May 1;69(3):425–437. doi: 10.1016/0092-8674(92)90445-i. [DOI] [PubMed] [Google Scholar]
  27. Kumar A., Widen S. G., Williams K. R., Kedar P., Karpel R. L., Wilson S. H. Studies of the domain structure of mammalian DNA polymerase beta. Identification of a discrete template binding domain. J Biol Chem. 1990 Feb 5;265(4):2124–2131. [PubMed] [Google Scholar]
  28. Lee S. H., Kwong A. D., Pan Z. Q., Hurwitz J. Studies on the activator 1 protein complex, an accessory factor for proliferating cell nuclear antigen-dependent DNA polymerase delta. J Biol Chem. 1991 Jan 5;266(1):594–602. [PubMed] [Google Scholar]
  29. Lee S. H., Pan Z. Q., Kwong A. D., Burgers P. M., Hurwitz J. Synthesis of DNA by DNA polymerase epsilon in vitro. J Biol Chem. 1991 Nov 25;266(33):22707–22717. [PubMed] [Google Scholar]
  30. Manley J. L., Fire A., Samuels M., Sharp P. A. In vitro transcription: whole-cell extract. Methods Enzymol. 1983;101:568–582. doi: 10.1016/0076-6879(83)01038-1. [DOI] [PubMed] [Google Scholar]
  31. Moore S. P., Erdile L., Kelly T., Fishel R. The human homologous pairing protein HPP-1 is specifically stimulated by the cognate single-stranded binding protein hRP-A. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9067–9071. doi: 10.1073/pnas.88.20.9067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mosbaugh D. W., Linn S. Gap-filling DNA synthesis by HeLa DNA polymerase alpha in an in vitro base excision DNA repair scheme. J Biol Chem. 1984 Aug 25;259(16):10247–10251. [PubMed] [Google Scholar]
  33. Mosig G. Bacteriophage T4 gene 32 participates in excision repair as well as recombinational repair of UV damages. Genetics. 1985 Jun;110(2):159–171. doi: 10.1093/genetics/110.2.159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nichols A. F., Sancar A. Purification of PCNA as a nucleotide excision repair protein. Nucleic Acids Res. 1992 Jul 11;20(13):2441–2446. doi: 10.1093/nar/20.10.2441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Nishida C., Reinhard P., Linn S. DNA repair synthesis in human fibroblasts requires DNA polymerase delta. J Biol Chem. 1988 Jan 5;263(1):501–510. [PubMed] [Google Scholar]
  36. Orren D. K., Selby C. P., Hearst J. E., Sancar A. Post-incision steps of nucleotide excision repair in Escherichia coli. Disassembly of the UvrBC-DNA complex by helicase II and DNA polymerase I. J Biol Chem. 1992 Jan 15;267(2):780–788. [PubMed] [Google Scholar]
  37. Popanda O., Thielmann H. W. The function of DNA polymerases in DNA repair synthesis of ultraviolet-irradiated human fibroblasts. Biochim Biophys Acta. 1992 Jan 6;1129(2):155–160. doi: 10.1016/0167-4781(92)90480-n. [DOI] [PubMed] [Google Scholar]
  38. Prelich G., Tan C. K., Kostura M., Mathews M. B., So A. G., Downey K. M., Stillman B. Functional identity of proliferating cell nuclear antigen and a DNA polymerase-delta auxiliary protein. Nature. 1987 Apr 2;326(6112):517–520. doi: 10.1038/326517a0. [DOI] [PubMed] [Google Scholar]
  39. Robins P., Jones C. J., Biggerstaff M., Lindahl T., Wood R. D. Complementation of DNA repair in xeroderma pigmentosum group A cell extracts by a protein with affinity for damaged DNA. EMBO J. 1991 Dec;10(12):3913–3921. doi: 10.1002/j.1460-2075.1991.tb04961.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Satokata I., Tanaka K., Miura N., Miyamoto I., Satoh Y., Kondo S., Okada Y. Characterization of a splicing mutation in group A xeroderma pigmentosum. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9908–9912. doi: 10.1073/pnas.87.24.9908. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Selby C. P., Sancar A. Structure and function of the (A)BC excinuclease of Escherichia coli. Mutat Res. 1990 Sep-Nov;236(2-3):203–211. doi: 10.1016/0921-8777(90)90005-p. [DOI] [PubMed] [Google Scholar]
  42. Seo Y. S., Lee S. H., Hurwitz J. Isolation of a DNA helicase from HeLa cells requiring the multisubunit human single-stranded DNA-binding protein for activity. J Biol Chem. 1991 Jul 15;266(20):13161–13170. [PubMed] [Google Scholar]
  43. Sheaff R., Ilsley D., Kuchta R. Mechanism of DNA polymerase alpha inhibition by aphidicolin. Biochemistry. 1991 Sep 3;30(35):8590–8597. doi: 10.1021/bi00099a014. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. Sibghatullah, Husain I., Carlton W., Sancar A. Human nucleotide excision repair in vitro: repair of pyrimidine dimers, psoralen and cisplatin adducts by HeLa cell-free extract. Nucleic Acids Res. 1989 Jun 26;17(12):4471–4484. doi: 10.1093/nar/17.12.4471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Syväoja J., Suomensaari S., Nishida C., Goldsmith J. S., Chui G. S., Jain S., Linn S. DNA polymerases alpha, delta, and epsilon: three distinct enzymes from HeLa cells. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6664–6668. doi: 10.1073/pnas.87.17.6664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tanaka S., Hu S. Z., Wang T. S., Korn D. Preparation and preliminary characterization of monoclonal antibodies against human DNA polymerase alpha. J Biol Chem. 1982 Jul 25;257(14):8386–8390. [PubMed] [Google Scholar]
  48. Toulmé J. J., Behmoaras T., Guigues M., Hélène C. Recognition of chemically damaged DNA by the gene 32 protein from bacteriophage T4. EMBO J. 1983;2(4):505–510. doi: 10.1002/j.1460-2075.1983.tb01454.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tsurimoto T., Stillman B. Multiple replication factors augment DNA synthesis by the two eukaryotic DNA polymerases, alpha and delta. EMBO J. 1989 Dec 1;8(12):3883–3889. doi: 10.1002/j.1460-2075.1989.tb08567.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Tsurimoto T., Stillman B. Replication factors required for SV40 DNA replication in vitro. II. Switching of DNA polymerase alpha and delta during initiation of leading and lagging strand synthesis. J Biol Chem. 1991 Jan 25;266(3):1961–1968. [PubMed] [Google Scholar]
  51. Van Houten B. Nucleotide excision repair in Escherichia coli. Microbiol Rev. 1990 Mar;54(1):18–51. doi: 10.1128/mr.54.1.18-51.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wang T. S. Eukaryotic DNA polymerases. Annu Rev Biochem. 1991;60:513–552. doi: 10.1146/annurev.bi.60.070191.002501. [DOI] [PubMed] [Google Scholar]
  53. Weeda G., van Ham R. C., Vermeulen W., Bootsma D., van der Eb A. J., Hoeijmakers J. H. A presumed DNA helicase encoded by ERCC-3 is involved in the human repair disorders xeroderma pigmentosum and Cockayne's syndrome. Cell. 1990 Aug 24;62(4):777–791. doi: 10.1016/0092-8674(90)90122-u. [DOI] [PubMed] [Google Scholar]
  54. Wiebauer K., Jiricny J. Mismatch-specific thymine DNA glycosylase and DNA polymerase beta mediate the correction of G.T mispairs in nuclear extracts from human cells. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5842–5845. doi: 10.1073/pnas.87.15.5842. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. 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]
  56. Wold M. S., Kelly T. Purification and characterization of replication protein A, a cellular protein required for in vitro replication of simian virus 40 DNA. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2523–2527. doi: 10.1073/pnas.85.8.2523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. 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]
  58. 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]
  59. 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]
  60. Yeung A. T., Mattes W. B., Oh E. Y., Yoakum G. H., Grossman L. The purification of the Escherichia coli UvrABC incision system. Nucleic Acids Res. 1986 Nov 11;14(21):8535–8556. doi: 10.1093/nar/14.21.8535. [DOI] [PMC free article] [PubMed] [Google Scholar]

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