Near‐uv‐induced Breaks in Phage Dna: Sensitization by Hydrogen Peroxide (A Tryptophan Photoproduct)

Ananthaswamy, Honnavara N.; Eisenstark, A.

doi:10.1111/j.1751-1097.1976.tb06851.x

Cited by 79 publications

(25 citation statements)

References 15 publications

(10 reference statements)

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“…As noted previously (6,9), the contribution of SS DNA breaks to death in T7 is insignificant. However, DS breakage could account for the striking enhancement of NUV lethality by H202, because x-ray inactivation of DNA-containing phages, including T7, is known to be due mainly to DS DNA breaks (23), which can impede injection (15,24).…”

supporting

confidence: 51%

“…H202 also enhances NUV lethality in phage T7 (6,9), thus providing a simple model system for the study of these synergistic interactions. While action spectrum analysis indicates synergism is maximal at 340 nm, the frequency of SS DNA break induction could not be correlated with peak of cell killing (9); i.e., there is no apparent direct relationship between SS breaks and cell death.…”

mentioning

confidence: 99%

“…6 (9,25). Second, the shoulder in the inactivation curve is far less for synergistic H202 plus NUV than for NUV alone (6). The shoulder reflects phage-encoded repair capacity (26); its reduction implicates a defect in either adsorption or injection without opportunity for DNA repair.…”

mentioning

confidence: 99%

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Inactivation of phage T7 by near-ultraviolet radiation plus hydrogen peroxide: DNA-protein crosslinks prevent DNA injection.

Hartman¹,

Eisenstark²,

Pauw³

1979

Proc. Natl. Acad. Sci. U.S.A.

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A nonlethal concentration of H202 (0.05%) greatly enhances near-ultraviolet (NUV) inactivation of phage T7. Simultaneous treatment with H202 and NUV reduces the amount of DNA injected into the bacterial host, but not-the number of phage adsorbed. Not only were recombination and gene expression of late markers reduced upon treatment of phage '11 with NUV plus H202, but also a gradient of recombination resulted, with markers injected first reduced to a lesser extent than those injected last. Labeling Techniques. Phage used for adsorption experiments were labeled as reported (9). The method of Hawkins (11) was used in double labeling. Both procedures yielded from 10-6 to 10-7 cpm per phage.Adsorption and Injection Studies. Phage were separated by the polyethylene glycol/dextran sulfate method (12) and further purified by differential centrifugation (13) prior to gradient centrifugal studies. Titers of ca. 5 X 1012 plaqueforming units per ml were routinely obtained. For phage adsorption and injection assays (14) E. coli B was infected with treated phage; 8 min after infection of cells, chloramphenicol was added, a sample was removed to assay radioactivity, and infected bacteria were removed by centrifugation. The pellet was resuspended and an aliquot was withdrawn to determine percentage of phage adsorbed. To determine the fraction of DNA injected into the host, cells were lysed, treated with DNase I, and centrifuged to remove bacterial debris as well as whole phage particles, and the supernatant was then assayed for radioactivity. This procedure is based on the assumption that the solubilized radioactivity represents the amount of DNA that is injected into the bacterial host, because only it is available for digestion by deoxyribonuclease.Genetic Recombination. For genetic crosses between amber mutants (15, 16) E. colh 011' (Su+) was coinfected with either T7 amli (gene 8) or am28 (gene 5), and one of a series of different amber mutants (helper phage) located throughout the T7 geqome. Lysates were assayed on 011' (Su+) and B (Su-) for total progeny and wild-type recombinants, respectively. Results are expressed as percentage of normal recombination (16). Average frequencies of wild-type recombinants for the control crosses were compatible with those in previous reports (15,16).Gel Electrophoresis Studies. E. coli B was grown at 37°C to 4 X 108 cells per ml in M9 medium plus 2 mg of casamino acids per ml, irradiated with 72 J-M2 of far-UV radiation to suppress host cell protein synthesis, and incubated for 30 min at 30°C in the dark. Aliquots (2 ml) were then infected with appropriately treated T7 at a multiplicity of infection of 10 and incubated with 10 ,tCi (1 Ci = 3.7 X 1010 becquerels) of [-5S]-methionine (New England Nuclear) per ml for 24 min at 30°C. Samples were washed and concentrated 10-fold by centrifugation and applied to polyacrylamide gels. Sodium dodecyl sulfate/polyacrylamide gradient slab gels were prepared 1.5 mm thick with a linear gradient of 8-13% acrylamide and a 5% acrylamide stackin...

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supporting

confidence: 51%

mentioning

confidence: 99%

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Inactivation of phage T7 by near-ultraviolet radiation plus hydrogen peroxide: DNA-protein crosslinks prevent DNA injection.

Hartman¹,

Eisenstark²,

Pauw³

1979

Proc. Natl. Acad. Sci. U.S.A.

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“…Oxidative stress via near-ultraviolet radiation (NUV) damages cells, at least in part, by single-strand breaks in DNA (1). In Escherichia coli these single-strand breaks result from direct damage by hydrogen peroxide (H202) (2).…”

mentioning

confidence: 99%

Exonuclease III and the catalase hydroperoxidase II in Escherichia coli are both regulated by the katF gene product.

Sak

Eisenstark²,

Touati³

1989

Proc. Natl. Acad. Sci. U.S.A.

155

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The levels of both exonuclease HI (exo m, product of xthA) and hydroperoxidase H (HP-II, product of katE) activity in Escherichia coli were influenced by a functional katF gene. The katF gene product is also necessary for synthesis of HP-Il. Mutations in either katF or xthA, but not katE, result in sensitivity to H202 and near-UV (300-400 nm) radiation. Exo HI, encoded by the xthA locus, recognizes and removes nucleoside 5'-monophosphates near apurinic and apyrimidinic sites in damaged DNA. Extracts of katF mutant strains had little detectable exo HI activity. When a katF+ plasmid was introduced into the katF mutant, exo m activity exceeded wild-type levels. We propose that the katF gene is a trans-acting positive regulator of exo m and HP-II enzymes, both of which are involved in cellular recovery from oxidative damage.

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“…Cellular death and DNA damage by near-UV occur mainly by indirect photosensitization pathways that produce reactive intermediates from intracellular chromophores such as porphyrin, flavins, and reduced nicotinamide coenzymes; however, near-UV may also damage DNA (4), key enzymes (5), and thiolated tRNA (1) directly. Near-UV generation of reactive oxygen species (e.g., hydroxyl radicals, hydrogen peroxide, superoxide anion, and singlet oxygen) kills cells; near-UV radiation in the absence of oxygen significantly reduces cell death (6).…”

mentioning

confidence: 99%

Near-Ultraviolet Mutagenesis in Superoxide Dismutase-deficient Strains of Escherichia coli.

Knowles

Eisenstark

1994

Environ Health Perspect

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We compared mutagenic spectra induced by polychromatic near-ultraviolet radiation (near-UV; 300-400 nm) with superoxide anion (O2-) -dependent mutagenesis using a set of Escherichia coli tester strains. Near-UV radiation produced increased frequencies of G:C to A:T transitions, G:C to T:A and A:T to T:A transversions, and small increases in frameshift mutations in wild-type cells. Tester strains lacking superoxide dismutase (SOD) activity (sodAsodB double mutants) demonstrated high spontaneous mutation frequencies and increased near-UV sensitivity. The double mutants also showed increased mutations induced by near-UV compared to either isogenic wild type, sodA or sodB single mutants. Futhermore, these mutants had an unusual spontaneous mutation spectrum, with a predominance of A:T to T:A transversions, followed by G:C to T:A transversions and frameshifts generated in runs of adenines in both the +1 and -1 direction. Other frameshifts were detected to a lesser degree. The oxygen dependency and the type of mutations spontaneously induced in SOD-deficient cells indicated that this mutagenic spectrum was caused by oxidative DNA damage. However, no apparent synergistic action between near-UV radiation and an increased flux of O2- could be detected. From the frequency and types of mutations induced by the two agents, we speculate that near-UV-induced mutagenesis and O2--dependent mutagenesis involve, in part, different lesion(s) and/or mechanism(s). The nature and possible mutagenic pathways of each are discussed. Images Figure 1. Figure 2. Figure 3. Figure 4. Figure 5.

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Near‐uv‐induced Breaks in Phage Dna: Sensitization by Hydrogen Peroxide (A Tryptophan Photoproduct)

Cited by 79 publications

References 15 publications

Inactivation of phage T7 by near-ultraviolet radiation plus hydrogen peroxide: DNA-protein crosslinks prevent DNA injection.

Inactivation of phage T7 by near-ultraviolet radiation plus hydrogen peroxide: DNA-protein crosslinks prevent DNA injection.

Exonuclease III and the catalase hydroperoxidase II in Escherichia coli are both regulated by the katF gene product.

Near-Ultraviolet Mutagenesis in Superoxide Dismutase-deficient Strains of Escherichia coli.

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