Thomas Bonura

The Influence of Oxygen on the Yield of DNA Double-Strand Breaks in X-Irradiated Escherichia coli K-12

Using a procedure which includes lysis on neutral sucrose gradients containing pronase and detergent with sedimentation at low speeds, we have measured the rate of DNA double-strand breakage by x rays in E. coli K-12 in the presence and absence of oxygen. The number of DNA double-strand breaks increased linearly with dose in both air and nitrogen. The energy required to produce a double-strand break under aerobic irradiation conditions was 532 +- 28 eV and under anoxic conditions 1290 +- 126 eV giving an oxygen enhancement ratio for DNA double-strand breaks of 2.42 +- 0.39. The D

QUANTITATIVE EVIDENCE FOR ENZYMATICALLY‐INDUCED DNA DOUBLE‐STRAND BREAKS AS LETHAL LESIONS IN UV IRRADIATED pol+ AND polAl STRAINS OF E. COLI K‐12

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R.B.E. of 50 kVp X-rays and 660 keV γ-rays (137Cs) with Respect to the Production of DNA Damage, Repair and Cell-killing in Escherichia Coli K-12

values based upon cell killing kinetics were determined to be 2.29 krad for irradiations under aerobic conditions and 6.38 krad for irradiations under anoxic conditions. The resulting oxygen enhancement ratio was 2.79. From these data we have calculated that 1.3 to 1.4 double-strand breaks per genome were produced per lethal event, suggesting that DNA double-strand breaks may play a primary role in the killing of wild-type E. coli K-12 by x irradiation. (auth)

Recent Developments in the Enzymology of Excision Repair of DNA

Abstract— We have recently reported that DNA double‐strand breaks arise enzymatically during the course of excision repair in uvr+ strains of Escherichia coli K‐12. Survival curves for ultraviolet (UV) irradiated E. coli K‐12 pol+ (JG139) and polA1 (JG138) strains have a pronounced shoulder region. The regions of the survival curves at which killing approaches exponential correspond to the fiuences at which DNA double‐strand breaks (assumed to be lethal events) accumulate linearly. Reducing the number of UV photoproducts either by photoreactivation or fluence fractionation results in an increase in survival and a decrease in the yield of DNA double‐strand breaks in both strains. These data support the hypothesis that enzymatically‐induced DNA double‐strand breaks may be the lesion ultimately responsible for UV‐induced cell killing in the pol+ strain of E. coli K‐12. and perhaps also in the polA1 strain.

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

We have compared the efficiency of cell-killing, DNA single-strand breakage and double-strand breakage in an Escherichia coli K-12 wild-type strain after irradiation with soft X-rays (50kVp) and hard gamma-rays (600 keV) under aerobic conditions. Irradiation with 50 kVP x-rays resulted in 1.47 times more cell killing than was observed with 137Cs gamma-rays based on a comparison of D0 values evaluated from the survival curves. DNA sedimentation studies showed that, although 50 kVp X-rays were 1-93 times more effective than 137Cs gamma-rays in producing DNA double-strand breaks, there was no sigificant difference between the two qualities of radiation with respect to the initial number of single-strand breaks produced. When the cells were irradiated and allowed to repair maximally in minimal medium, 1-57 times more unrepaired DNA single-strand breaks remained per krad after irradiation with 50 kVp X-rays than with 137Cs gamma-rays. The increased yield of DNA double-strand breaks resulting from 50 kVp X-radiation may account for most of these additional unrepaired single-strand breaks, since single- and double-strand breaks are indistinguishable on alkaline sucrose gradients. These results suggest that the greater r.b.e. of 50 kVp X-rays may be related to an increased effectiveness for producing DNA double-strand breaks compared with the higher energy 137Cs gamma-rays.

Sensitization of Escherichia coli C to gamma-radiation by 5-bromouracil incorporation.

Publisher Summary This chapter describes the details of the enzymic mechanisms of excision repair of damaged DNA. It is the incision of UV irradiated DNA by an enzyme mechanism, involving a specific DNA glycosylase that is not universal. This argument notwithstanding, even in the two biological systems just mentioned, evidence indicates that pyrimidine dimers are excised from DNA as a part of a larger oligonucleotide, whereas in all known examples of base-excision repair, the products of excision are free bases. The chapter also describes an activity in extracts of M . luteus that recognizes 8-(2-hydroxyisopropy1) purines in DNA. Preliminary evidence indicates that this activity is a true endonuclease without associated DNA glycosylase activity. Thus, it is distinctly possible that the excision repair of cell damage by hydrolysis of N-glycosyl bonds involves a number of specific forms of base damage, in which the degree of structural distortion of DNA secondary structure is limited. Very bulky DNA adducts such as those produced by the interaction of DNA with polycyclic aromatic hydrocarbons, acetamidofluorene, etc., may generate relatively nonspecific but significant distortion of the DNA helix that is recognized by a limited number of “general” endonucleases.

14 Enzymes That Incise Damaged DNA

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 ([H5] 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 degrees 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 approximately 7 nucleotides. In contrast, the calculated average patch size in uninfected E. coli is approximately 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.

Inhibitor of uracil-DNA glycosylase induced by bacteriophage PBS2. Purification and preliminary characterization.

Escherichia coli C cells, unifilarly substituted with 5-bromouracil (BrUra) were 2-25 times as sensitive as unsubstituted cells to killing by gamma-irradiation under aerobic conditions. The yield of DNA double-strand breaks in BrUra-substituted cells was increased by a factor only 1-55, suggesting that other lesions also contribute to cell-killing. Alkaline sucrose density gradient analysis of the 3H-thymine labelled DNA strand showed there was less repair of gamma-ray-induced single-strand breaks when BrUra was in the complementary strand. Since there are more of these unrepaired breaks than can be accounted for by BrUra-induced DNA double-strand breakage, some fraction of the lethal events in BrUra-substituted E. coli cells may be unrepaired DNA single-strand breaks.

The Involvement of Indirect Effects in Cell-killing and DNA Double-strand Breakage in γ-irradiated Escherichia Coli K-12

Publisher Summary This chapter focuses on DNA-incising activities from Escherichia coli . E.coli has been the subject of extensive genetic and biochemical investigations on the excision repair of DNA. The excision of damaged or inappropriate nucleotides from DNA can occur by a number of different biochemical pathways, depending on both the nature of the specific base damage in question and on the particular organism under investigation. Most pathways of excision repair of DNA include the enzyme-catalyzed hydrolysis of phosphodiester bonds by specific enzymes. Such enzymes can be divided into two major classes (1) those that attack phosphodiester bonds in DNA subsequent to the hydrolysis of the associated glycosylic bond that links a nitrogenous base to the deoxyribose-phosphate backbone and (2) those that directly attack phosphodiester bonds in damaged DNA. The former class of enzymes is designated as apurinic/apyrimidinic (AP) endonucleases because their endonuclease activity is confined to sites of base loss in DNA. Such substrate sites arise by the spontaneous hydrolysis of N-glycosylic bonds in DNA, or by enzyme-catalyzed hydrolysis of these bonds by DNA glycosylases.