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. 1978 Jun;75(6):2598–2602. doi: 10.1073/pnas.75.6.2598

Phage T4 endonuclease V stimulates DNA repair replication in isolated nuclei from ultraviolet-irradiated human cells, including xeroderma pigmentosum fibroblasts

Charles Allen Smith 1, Philip C Hanawalt 1,*
PMCID: PMC392609  PMID: 275829

Abstract

The repair mode of DNA replication has been demonstrated in isolated nuclei from UV-irradiated human cells. Nuclei are incubated in a mixture containing [3H]thymidine triphosphate and bromodeoxyuridine triphosphate in a 1:5 ratio. The 3H at the density of parental DNA in alkaline CsCl density gradients is then a measure of repair. In nuclei prepared from WI38 cells 30 min after irradiation, repair replication is UV dependent and proceeds at approximately the in vivo rate for 5 min. Repair replication is reduced in irradiated nuclei or in nuclei prepared immediately after irradiation. It is Mg2+-dependent and stimulated by added ATP and deoxyribonucleoside triphosphates. No repair replication is observed in nuclei from xeroderma pigmentosum (complementation group A) cells. However, upon addition of coliphage T4 endonuclease V, which specifically nicks DNA containing pyrimidine dimers, repair replication is observed in nuclei from irradiated xeroderma pigmentosum cells and is stimulated in WI38 nuclei. The reaction then persists for an hour and is dependent upon added ATP and deoxyribonucleoside triphosphates. The repair label is in stretches of roughly 35 nucleotides, as it is in intact cells. Added pancreatic DNase does not promote UV-dependent repair synthesis. Our results support the view that xeroderma pigmentosum (group A) cells are defective in the incision step of the DNA excision repair pathway, and demonstrate the utility of this system for probing DNA repair mechanisms.

Keywords: excision repair, pyrimidine dimer

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Selected References

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

  1. Cleaver J. E. Defective repair replication of DNA in xeroderma pigmentosum. Nature. 1968 May 18;218(5142):652–656. doi: 10.1038/218652a0. [DOI] [PubMed] [Google Scholar]
  2. Cleaver J. E. Xeroderma pigmentosum: a human disease in which an initial stage of DNA repair is defective. Proc Natl Acad Sci U S A. 1969 Jun;63(2):428–435. doi: 10.1073/pnas.63.2.428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Edenberg H., Hanawalt P. Size of repair patches in the DNA of ultraviolet-irradiated HeLa cells. Biochim Biophys Acta. 1972 Jul 20;272(3):361–372. doi: 10.1016/0005-2787(72)90389-9. [DOI] [PubMed] [Google Scholar]
  4. Friedberg E. C., King J. J. Dark repair of ultraviolet-irradiated deoxyribonucleic acid by bacteriophage T4: purification and characterization of a dimer-specific phage-induced endonuclease. J Bacteriol. 1971 May;106(2):500–507. doi: 10.1128/jb.106.2.500-507.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hudson B., Upholt W. B., Devinny J., Vinograd J. The use of an ethidium analogue in the dye-buoyant density procedure for the isolation of closed circular DNA: the variation of the superhelix density of mitochondrial DNA. Proc Natl Acad Sci U S A. 1969 Mar;62(3):813–820. doi: 10.1073/pnas.62.3.813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ikenaga M., Ishii Y., Tada M., Kakunaga T., Takebe H. Excision-repair of 4-nitroquinolin-1-oxide damage responsible for killing, mutation, and cancer. Basic Life Sci. 1975;5B:763–771. doi: 10.1007/978-1-4684-2898-8_54. [DOI] [PubMed] [Google Scholar]
  7. Masker W. E., Hanawalt P. C. Ultraviolet-stimulated DNA synthesis in toluenzied Escherichia coli deficient in DNA polymerase I. Proc Natl Acad Sci U S A. 1973 Jan;70(1):129–133. doi: 10.1073/pnas.70.1.129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Minton K., Durphy M., Taylor R., Friedberg E. C. The ultraviolet endonuclease of bacteriophage T4. Further characterization. J Biol Chem. 1975 Apr 25;250(8):2823–2829. [PubMed] [Google Scholar]
  9. Mortelmans K., Friedberg E. C., Slor H., Thomas G., Cleaver J. E. Defective thymine dimer excision by cell-free extracts of xeroderma pigmentosum cells. Proc Natl Acad Sci U S A. 1976 Aug;73(8):2757–2761. doi: 10.1073/pnas.73.8.2757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Robbins J. H., Kraemer K. H., Lutzner M. A., Festoff B. W., Coon H. G. Xeroderma pigmentosum. An inherited diseases with sun sensitivity, multiple cutaneous neoplasms, and abnormal DNA repair. Ann Intern Med. 1974 Feb;80(2):221–248. doi: 10.7326/0003-4819-80-2-221. [DOI] [PubMed] [Google Scholar]
  11. Rupp W. D., Howard-Flanders P. Discontinuities in the DNA synthesized in an excision-defective strain of Escherichia coli following ultraviolet irradiation. J Mol Biol. 1968 Jan 28;31(2):291–304. doi: 10.1016/0022-2836(68)90445-2. [DOI] [PubMed] [Google Scholar]
  12. Seki S., Lemahieu M., Mueller G. C. A permeable cell system for studying DNA replication in synchronized HeLa cells. Biochim Biophys Acta. 1975 Feb 10;378(3):333–343. doi: 10.1016/0005-2787(75)90178-1. [DOI] [PubMed] [Google Scholar]
  13. Setlow R. B., Regan J. D., German J., Carrier W. L. Evidence that xeroderma pigmentosum cells do not perform the first step in the repair of ultraviolet damage to their DNA. Proc Natl Acad Sci U S A. 1969 Nov;64(3):1035–1041. doi: 10.1073/pnas.64.3.1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Simon T. J., Smith C. A., Friedberg E. C. Action of bacteriophage T4 ultraviolet endonuclease on duplex DNA containing one ultraviolet-irradiated strand. J Biol Chem. 1975 Nov 25;250(22):8748–8752. [PubMed] [Google Scholar]
  15. Smith C. A., Hanawalt P. C. Repair replication in cultured normal and transformed human fibroblasts. Biochim Biophys Acta. 1976 Oct 4;447(2):121–132. doi: 10.1016/0005-2787(76)90335-x. [DOI] [PubMed] [Google Scholar]
  16. Smith C. A., Hanawalt P. C. Repair replication in human cells. Simplified determination utilizing hydroxyurea. Biochim Biophys Acta. 1976 May 19;432(3):336–347. doi: 10.1016/0005-2787(76)90143-x. [DOI] [PubMed] [Google Scholar]
  17. Taketo A., Yasuda S., Sekiguchi M. Initial step of excision repair in Escherichia coli: replacement of defective function of uvr mutants by T4 endonuclease V. J Mol Biol. 1972 Sep 14;70(1):1–14. doi: 10.1016/0022-2836(72)90160-x. [DOI] [PubMed] [Google Scholar]
  18. Tanaka K., Hayakawa H., Sekiguchi M., Okada Y. Specific action of T4 endonuclease V on damaged DNA in xeroderma pigmentosum cells in vivo. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2958–2962. doi: 10.1073/pnas.74.7.2958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Tanaka K., Sekiguchi M., Okada Y. Restoration of ultraviolet-induced unscheduled DNA synthesis of xeroderma pigmentosum cells by the concomitant treatment with bacteriophage T4 endonuclease V and HVJ (Sendai virus). Proc Natl Acad Sci U S A. 1975 Oct;72(10):4071–4075. doi: 10.1073/pnas.72.10.4071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Weissbach A. Eukaryotic DNA polymerases. Annu Rev Biochem. 1977;46:25–47. doi: 10.1146/annurev.bi.46.070177.000325. [DOI] [PubMed] [Google Scholar]

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