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, Howard Edenberg, E. C. Friedberg

Research output: Contribution to journalArticle

8 Citations (Scopus)

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

Fingerprint

Bacteriophage T4
Bacteriophages
Escherichia coli
Thymine
DNA-(Apurinic or Apyrimidinic Site) Lyase
Pyrimidine Dimers
DNA
Nucleotides
Dimers
Poly dA-dT
Temperature
Infection
deoxyribopyrimidine endonucleosidase
Metabolism
Ultraviolet radiation
Assays
Repair
Genes
Radiation
Mutation

Keywords

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

ASJC Scopus subject areas

  • Biochemistry

Cite this

Bonura, T., Radany, E. H., McMillan, S., Love, J. D., Schultz, R. A., Edenberg, H., & Friedberg, E. C. (1982). Pyrimidine dimer-DNA glycosylases: Studies on bacteriophage T4-infected and on uninfected Escherichia coli. Biochimie, 64(8-9), 643-654. https://doi.org/10.1016/S0300-9084(82)80104-1

Pyrimidine dimer-DNA glycosylases : Studies on bacteriophage T4-infected and on uninfected Escherichia coli. / Bonura, T.; Radany, E. H.; McMillan, S.; Love, J. D.; Schultz, R. A.; Edenberg, Howard; Friedberg, E. C.

In: Biochimie, Vol. 64, No. 8-9, 29.09.1982, p. 643-654.

Research output: Contribution to journalArticle

Bonura, T, Radany, EH, McMillan, S, Love, JD, Schultz, RA, Edenberg, H & Friedberg, EC 1982, 'Pyrimidine dimer-DNA glycosylases: Studies on bacteriophage T4-infected and on uninfected Escherichia coli', Biochimie, vol. 64, no. 8-9, pp. 643-654. https://doi.org/10.1016/S0300-9084(82)80104-1
Bonura, T. ; Radany, E. H. ; McMillan, S. ; Love, J. D. ; Schultz, R. A. ; Edenberg, Howard ; Friedberg, E. C. / Pyrimidine dimer-DNA glycosylases : Studies on bacteriophage T4-infected and on uninfected Escherichia coli. In: Biochimie. 1982 ; Vol. 64, No. 8-9. pp. 643-654.
@article{179b9e81544f4af692a0143839e6e9b5,
title = "Pyrimidine dimer-DNA glycosylases: Studies on bacteriophage T4-infected and on uninfected Escherichia coli",
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.",
keywords = "apurinic-apyrimidinic endonucleases, bacteriophage T4, DNA repair, Escherichia coli, pyrimidine dimer-DNA glycosylases",
author = "T. Bonura and Radany, {E. H.} and S. McMillan and Love, {J. D.} and Schultz, {R. A.} and Howard Edenberg and Friedberg, {E. C.}",
year = "1982",
month = "9",
day = "29",
doi = "10.1016/S0300-9084(82)80104-1",
language = "English",
volume = "64",
pages = "643--654",
journal = "Biochimie",
issn = "0300-9084",
publisher = "Elsevier",
number = "8-9",

}

TY - JOUR

T1 - Pyrimidine dimer-DNA glycosylases

T2 - Studies on bacteriophage T4-infected and on uninfected Escherichia coli

AU - Bonura, T.

AU - Radany, E. H.

AU - McMillan, S.

AU - Love, J. D.

AU - Schultz, R. A.

AU - Edenberg, Howard

AU - Friedberg, E. C.

PY - 1982/9/29

Y1 - 1982/9/29

N2 - 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.

AB - 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.

KW - apurinic-apyrimidinic endonucleases

KW - bacteriophage T4

KW - DNA repair

KW - Escherichia coli

KW - pyrimidine dimer-DNA glycosylases

UR - http://www.scopus.com/inward/record.url?scp=0020168793&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0020168793&partnerID=8YFLogxK

U2 - 10.1016/S0300-9084(82)80104-1

DO - 10.1016/S0300-9084(82)80104-1

M3 - Article

C2 - 6753948

AN - SCOPUS:0020168793

VL - 64

SP - 643

EP - 654

JO - Biochimie

JF - Biochimie

SN - 0300-9084

IS - 8-9

ER -