Photoalkylation of Purines in Dna

Ben-Ishai, R.; Green, M.; Graff, E.; Elad, D.; Steinmaus, H.; Salomon, J.

doi:10.1111/j.1751-1097.1973.tb06345.x

Cited by 40 publications

(14 citation statements)

References 37 publications

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“…Therefore, the major products ofphotoalkylation ofPBS-2 DNA were photoalkylated purines. As with native thymine-containing DNAs, no photoalkylated pyrimidines were detected (9,14). This is consistent with the suppression of photoalkylation of pyrimidines by equimolar purines, reported for both uracil and thymine bases (28) and for PM2 DNA (9).…”

supporting

confidence: 88%

“…The degree of purine modification in DNA was assessed by quantitating labeled purines liberated by acid hydrolysis (14) utilizing paper chromatography (14) mGua yields in Me2SO4-treated DNA, the elution buffer was 0.5 M and the guanine, mGua, and adenine peaks were detected at 23, 32, and 38 min after sample application, respectively. In the analysis for HPG, the buffer was 0.8 M and HPG, guanine and adenine peaks were detected at 14, 21, and 36 min, respectively.…”

mentioning

confidence: 99%

“…HPG marker was made by photoalkylation of guanine (16). Identities of both were confirmed by paper chromatography (9,14). mGua was obtained from Vega Biochemicals (Tucson, AZ).…”

mentioning

confidence: 99%

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Perturbations of enzymic uracil excision due to purine damage in DNA.

Duker¹,

Jensen²,

Hart³

et al. 1982

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

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Phage PBS-2 DNA, which contains uracil in place of thymine, was selectively damaged and then used as substrate for purified Bacillu subtilis uracil-DNA glycosylase. This enzyme releases uracil from DNA in a limited processive manner. Irradiation by ultraviolet light (>305 nm) in the presence of isopropanol and a free radical photoinitiator introduced covalently bound 8-(2-hydroxy-2-propyl)purines into DNA. Methylation by dimethylsulfate yielded 7-methylguanine. Apurinic sites were produced by gentle heating of methylated DNA. Rates of enzymic release of uracil from DNA varied among these three substrates.The Vm. was markedly decreased for DNA containing 8-(2-hydroxy-2-propyl)purines and apurinic sites but was unaffected by the presence of larger quantities of 7-methylguanine. This suggests that certain types of damaged DNA moieties may decrease the capacity for uracil excision. Therefore, interference with enzymic excision of this potentially mutagenic base may constitute a common mechanism of action of the reaction products of several unrelated DNA damaging agents.Uracil-DNA glycosylase [dUra(DNA) glycosylase] specifically removes uracil from DNA. Such uracil may result either from incorporation in place of thymine during DNA synthesis or deamination of DNA cytosine (1), which may occur at physiological conditions (2). Left unrepaired, deaminated cytosines would result in transition mutations (3). Escherichia coli mutants deficient in dUra(DNA) glycosylase (ung-) are mutators, and in vivo deamination of cytosines is the source of these mutations (4,5). This implies that dUra(DNA) glycosylase activity is necessary to preserve the integrity of DNA in the face ofcontinuous potentially mutagenic damage. The finding that this enzyme is apparently ubiquitous supports the suggestions that this is its prime function (1, 4,6).The presence of UV photoproducts results in alteration of dUra(DNA) glycosylase activity toward phage PBS-2 DNA, which contains uracil in place of thymine. The rate of enzymic release ofuracil from such UV-irradiated DNA decreases as the number of photodimers increases (7). This indicates that the presence of pyrimidine dimers in DNA may affect excision of uracil.We investigated whether this alteration occurs with other types of DNA modifications. Three different types of purine damage were introduced into PBS-2 DNA, which was then used as substrate for dUra(DNA) glycosylase. Photoalkylation produced the modified purines 8-(2-hydroxy-2-propyl)guanine (HPG) and 8-(2-hydroxy-2-propyl)adenine (HPA) in DNA. (9), except that the samples were not flushed with nitrogen before irradiation. Actinometry (10) showed the incident dose to be 6.6 x 10-5 einstein cm-2 min-1. Photoalkylated DNA was extensively dialyzed into 10 mM Tris HCl/1 mM EDTA, pH 8.0. DNA was methylated with 10 mM Me2SO4 for 1 hr (11) and purified by passage through a Sephadex G-50 column in 10 mM Tris HCI/ 1 mM EDTA, pH 8.0 (12). In some experiments, methylated PBS-2 DNA was partially depurinated by heat (13) followed by dialysis ...

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confidence: 88%

mentioning

confidence: 99%

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Perturbations of enzymic uracil excision due to purine damage in DNA.

Duker¹,

Jensen²,

Hart³

et al. 1982

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

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“…It should be recalled that the addition of this type of free radical to DNA constituents, under our conditions, produces only a thymine adduct in addition to 8-(2-hydroxy-2-propyl) purines (27). Because the thymine addition product was not present in our DNA substrate (unpublished data), we can conclude that the endonuclease is directed towards 8-(2-hydroxy-2-propyl) purines in DNA.…”

Section: Methodsmentioning

confidence: 65%

“…This effect of protection of pyrimidines from reacting with free radicals was attributed to the degree of the involvement of pyrimidine moieties in base-stacking interactions (24)(25)(26). The application of these reactions to DNA (27)(28)(29) provided the basis for the preparation of damaged DNA substrates containing 8-substituted purines as the only lesion. These substrates are used in this work as a probe for the detection and characterization of specific repair endonucleases and have potential for the eventual study of damage-enzyme interactions.…”

mentioning

confidence: 99%

Endonucleolytic activity directed towards 8-(2-hydroxy-2-propyl) purines in double-stranded DNA.

Livneh¹,

Elad²,

Sperling³

1979

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

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Photoalkylation of circular covalently closed DNA from phage PM2 with isopropyl alcohol by using a free radical photoinitiator and UV light of X > 305 nm led to the specific 8-substitution of purine moieties in the DNA, yielding 842-hydroxv-2-propyl)adenine and 8(2-hydroxy-2-propyl)guanine as the only detectable damage in the DNA. Using this specifically photoalkylated DNA as a substrate, we discovered in extracts of Micrococcus luteus an endonucleolytic activity that is directed towards 8(2-hydroxy-2-propyl) purines in DNA. The activity is not a combination of a DNA-glycosylase and an apurinic site endonuclease. It is not inhibited by single-stranded DNA, by UV-or y-irradiated single-stranded DNA, or by normal or depurinated double-stranded DNA; however, 'y-or UV-(254 nm) irradiated double-stranded DNAs do inhibit the activity, hinting at the possibility of a common type of lesion in these damaged DNAs. Divalent cations are not required for the incising activity, and it is fully active in 1 mM EDTA, whereas caffeine and ATP cause inhibition. Extracts of mutant M. luteus lacking pyrimidine-dimer-directed endonucleases were found to contain the endonucleolytic activity in levels comparable to those present in the wild type. After the incision, we could demonstrate the specific excision of the 8-alkylated purines from the damaged DNA. The special conformational consequences of the 8-a kylation of purines, at the nucleotide level, namely their nonregular syn conformation, suggest that it is the distortion in the DNA that is recognized by the endonuclease. Nucleotide excision repair is one of the main repair mechanisms that acts to restore the structural integrity of damaged DNA. It is initiated by an endonuclease that incises the damaged DNA strand in the neighborhood of the damage, followved by excision of the damaged sequences by an exonuclease, repolymerization by a DNA polymerase, and finally covalent sealing by DNA ligase to restore the DNA molecule (1-3).The nucleotide excision repair is initiated by specific endonucleases that recognize chemically modified regions, or consequent conformational changes in the DNA, and incise the DNA at the vicinity of such lesions. So far only two endonucleases that are directed towards known chemically defined lesions have been described. These are the pyrimidine-dimer endonuclease (4-6) and the apurinic/apyrimidinic site endonuclease (7-13). Other endonuicleases have been found to incise UV-, 'y-ray-, or chemically damaged DNA, but their specificities are not yet known (14)(15)(16)(17)(18)(19)(20)(21)(22). This is mainly due to the fact that the damaged DNA substrates usually contain a multiplicity of products, not all of them chemically characterized, thus preventing an unambiguous assignment of a specific enzyme to a defined lesion. This difficulty interferes with the characterization of repair endontucleases and with the elucidation of the molecular basis of the damage to enzyme interactions, which play a key role in the initiation of the repair process.We have a...

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Photochemistry of the Nucleic Acids

Kittler¹,

Löber²

1977

Photochemical and Photobiological Reviews

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Photoalkylation of Purines in Dna

Cited by 40 publications

References 37 publications

Perturbations of enzymic uracil excision due to purine damage in DNA.

Perturbations of enzymic uracil excision due to purine damage in DNA.

Endonucleolytic activity directed towards 8-(2-hydroxy-2-propyl) purines in double-stranded DNA.

Photochemistry of the Nucleic Acids

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