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. 1990 Dec 25;18(24):7425–7432. doi: 10.1093/nar/18.24.7425

Excision repair of UV-damaged plasmid DNA in Xenopus oocytes is mediated by DNA polymerase alpha (and/or delta).

J K Saxena 1, J B Hays 1, E J Ackerman 1
PMCID: PMC332881  PMID: 2175436

Abstract

We studied DNA repair by injecting plasmids containing random pyrimidine dimers into Xenopus oocytes. We demonstrated excision repair by recovering plasmids and analyzing them with T4 UV endonuclease treatment and alkaline agarose gel electrophoresis. The mechanism for excision repair of these plasmids appears to be processive, rather than distributive, since repair occurs in 'all or none' fashion. At less than 4-5 dimers/plasmid, nearly all repair occurs within 4-6 hours (approximately 10(10) dimers repaired per oocyte); the oocyte, therefore, has abundant repair activity. Specific antibodies and inhibitors were used to determine enzymes involved in repair. We conclude that DNA polymerase alpha (and/or delta) is required because repair is inhibited by antibodies to human DNA polymerase alpha, as well as by aphidicolin, an inhibitor of polymerases alpha (and/or delta). Repair was not inhibited by hydroxyurea, cytosine beta-D-arabinofuranoside, or inhibitors of topoisomerase II (novobiocin). Oocyte repair does not activate semi-conservative DNA replication, nor is protein synthesis required. Photoreactivation cannot account for repair because dimer removal is independent of exogenous light.

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Selected References

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

  1. Ackerman E. J., Saxena S. K., Ulbrich N. Alpha-sarcin causes a specific cut in 28 S rRNA when microinjected into Xenopus oocytes. J Biol Chem. 1988 Nov 15;263(32):17076–17083. [PubMed] [Google Scholar]
  2. Bohr V. A., Smith C. A., Okumoto D. S., Hanawalt P. C. DNA repair in an active gene: removal of pyrimidine dimers from the DHFR gene of CHO cells is much more efficient than in the genome overall. Cell. 1985 Feb;40(2):359–369. doi: 10.1016/0092-8674(85)90150-3. [DOI] [PubMed] [Google Scholar]
  3. Chao C. C., Rosenstein B. S. Use of metabolic inhibitors to investigate the excision repair of pyrimidine dimers and non-dimer DNA damages induced in human and ICR 2A frog cells by solar ultraviolet radiation. Photochem Photobiol. 1986 Feb;43(2):165–170. doi: 10.1111/j.1751-1097.1986.tb09509.x. [DOI] [PubMed] [Google Scholar]
  4. Cortese R., Harland R., Melton D. Transcription of tRNA genes in vivo: single-stranded compared to double-stranded templates. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4147–4151. doi: 10.1073/pnas.77.7.4147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ford C. C., Woodland H. R. DNA synthesis in ocytes and eggs of Xenopus laevis injected with DNA. Dev Biol. 1975 Mar;43(1):189–199. doi: 10.1016/0012-1606(75)90140-2. [DOI] [PubMed] [Google Scholar]
  6. Gurdon J. B. Methods for nuclear transplantation in amphibia. Methods Cell Biol. 1977;16:125–139. doi: 10.1016/s0091-679x(08)60096-5. [DOI] [PubMed] [Google Scholar]
  7. Gurdon J. B., Wickens M. P. The use of Xenopus oocytes for the expression of cloned genes. Methods Enzymol. 1983;101:370–386. doi: 10.1016/0076-6879(83)01028-9. [DOI] [PubMed] [Google Scholar]
  8. Harland R. M., Laskey R. A. Regulated replication of DNA microinjected into eggs of Xenopus laevis. Cell. 1980 Oct;21(3):761–771. doi: 10.1016/0092-8674(80)90439-0. [DOI] [PubMed] [Google Scholar]
  9. Hays J. B., Ackerman E. J., Pang Q. S. Rapid and apparently error-prone excision repair of nonreplicating UV-irradiated plasmids in Xenopus laevis oocytes. Mol Cell Biol. 1990 Jul;10(7):3505–3511. doi: 10.1128/mcb.10.7.3505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ikegami S., Taguchi T., Ohashi M., Oguro M., Nagano H., Mano Y. Aphidicolin prevents mitotic cell division by interfering with the activity of DNA polymerase-alpha. Nature. 1978 Oct 5;275(5679):458–460. doi: 10.1038/275458a0. [DOI] [PubMed] [Google Scholar]
  11. Legerski R. J., Penkala J. E., Peterson C. A., Wright D. A. Repair of UV-induced lesions in Xenopus laevis oocytes. Mol Cell Biol. 1987 Dec;7(12):4317–4323. doi: 10.1128/mcb.7.12.4317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Mellon I., Bohr V. A., Smith C. A., Hanawalt P. C. Preferential DNA repair of an active gene in human cells. Proc Natl Acad Sci U S A. 1986 Dec;83(23):8878–8882. doi: 10.1073/pnas.83.23.8878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Saxena S. K., O'Brien A. D., Ackerman E. J. Shiga toxin, Shiga-like toxin II variant, and ricin are all single-site RNA N-glycosidases of 28 S RNA when microinjected into Xenopus oocytes. J Biol Chem. 1989 Jan 5;264(1):596–601. [PubMed] [Google Scholar]
  15. SenGupta D. N., Kumar P., Zmudzka B. Z., Coughlin S., Vishwanatha J. K., Robey F. A., Parrott C., Wilson S. H. Mammalian alpha-polymerase: cloning of partial complementary DNA and immunobinding of catalytic subunit in crude homogenate protein blots. Biochemistry. 1987 Feb 10;26(3):956–963. doi: 10.1021/bi00377a041. [DOI] [PubMed] [Google Scholar]
  16. Valerie K., Henderson E. E., de Riel J. K. Expression of a cloned denV gene of bacteriophage T4 in Escherichia coli. Proc Natl Acad Sci U S A. 1985 Jul;82(14):4763–4767. doi: 10.1073/pnas.82.14.4763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. van Zeeland A. A., Natarajan A. T., Verdegaal-Immerzeel E. A., Filon A. R. Photoreactivation of UV induced cell killing, chromosome aberrations, sister chromatid exchanges, mutations and pyrimidine dimers in Xenopus laevis fibroblasts. Mol Gen Genet. 1980;180(3):495–500. doi: 10.1007/BF00268052. [DOI] [PubMed] [Google Scholar]

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