Pyrimidine dimer-DNA glycosylases: Studies on bacteriophage T4-infected and on uninfected Escherichia coli

T. Bonura, E. H. Radany, S. McMillan, J. D. Love, R. A. Schultz, H. J. Edenberg, E. C. Friedberg

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

Pyrimidine dimer (PD)-DNA glycosylase activity has been reported in both the M. luteus and phage T4 UV endonucleases. In the present studies the T4 PD-DNA glycosylase has been purified close to physical homogeneity using an assay that measures the release of free thymine from UV-irradiated poly ([H3] dT):poly (dA), after the photo-reversal of thymine-thymine dimers. The activity has also been demonstrated in vivo following infection of UV-irradiated E. coli uvr- cells with phage T4. Under these conditions the T4 PD-DNA glycosylase accounts quantitatively for all thymine-containing PD excised from [3H] labeled E. coli DNA. In vitro the T4 PD-DNA glycosylase has an associated AP endonuclease activity that incises UV-irradiated DNA 3 to the apyrimidinic sites created by the glycosylase. However, the glycosylase/AP endonuclease reaction mechanism in vitro does not appear to be a concerted one. In addition, a T4 phage with a temperature-sensitive mutation in the denV gene shows wild-type levels of survival at the permissive temperature, despite the fact that in vitro, extracts of E. coli infected with this mutant show no detectable phage-coded AP endonuclease at 28°C. Thus the exact role of the T4 AP endonuclease in the incision of UV-irradiated DNA dimer in vivo is not clear. The ratio of excised non-containing nucleotides to dimer-containing nucleotides following infection of UV-irradiated E. coli with phage T4 denV+ yields a calculated average repair patch size of ∼ 7 nucleotides. In contrast, the calculated average patch size in uninfected E. coli is ∼ 70 nucleotides. Thus the extent of excision/resynthesis of UV-irradiated DNA may be determined by the specific mode of incision of the DNA at PD. When uninfected E. coli (uvr+) is exposed to UV radiation, a fraction of the excised thymine-containing PD contain photolabile thymine, suggesting the presence of PD-DNA glycosylase in E. coli. The role of this putative activity in the metabolism of UV-irradiated DNA is under investigation.

Original languageEnglish (US)
Pages (from-to)643-654
Number of pages12
JournalBiochimie
Volume64
Issue number8-9
DOIs
StatePublished - Sep 29 1982

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Keywords

  • DNA repair
  • Escherichia coli
  • apurinic-apyrimidinic endonucleases
  • bacteriophage T4
  • pyrimidine dimer-DNA glycosylases

ASJC Scopus subject areas

  • Biochemistry

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