Alteration of Uracil-Dna Glycosylase Activity by Uracil Dimers in Dna

Duker, Nahum J.; Davies, Wayne A.; Hart, Donna M.

doi:10.1111/j.1751-1097.1981.tb09346.x

Cited by 18 publications

(7 citation statements)

References 35 publications

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“…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.…”

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confidence: 99%

“…Photoalkylated DNA was assayed for uracil-containing dimers as described (7). Other pyrimidine damage was investigated by enzymic digestion of DNA to deoxynucleosides (18) and precipitation of the enzymes with a% trichloroacetic acid.…”

mentioning

confidence: 99%

“…The inhibitions observed here might be due to blockage ofprocessive movement, perhaps as a consequence of structural distortion of DNA or of a high affinity of the glycosylase for these forms of DNA damage. This would result in tight binding of the enzyme to forms of DNA damage other than those involving uracil (7).…”

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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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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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“…Fifty microliter aliquots of either DNA were irradiated by monochromatic light in a 1 mm quartz cell under air at room temperature. The light source consisted of a 200 W Hg Osram lamp and a Bausch & Lomb high intensity monochromator (Duker et al, 1981). Irradiances were measured using an International Light ILSOOA research radiometer with a calibrated photodetector.…”

Section: Methodsmentioning

confidence: 99%

Wavelength Dependence of Dna Incision by a Human Ultraviolet Endonuclease

Gallagher

Weiss

Brent

et al. 1989

Photochem & Photobiology

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The wavelength dependence of an ultraviolet irradiation of the DNA substrate for a human endonuclease was determined. Sites of DNA incision for all UVB and UVC wavelengths examined were at cytosines which were neither cyclobutane pyrimidine dimers nor 6-4'-(pyrimidin-2-one)pyrimidines. The optimal wavelengths for formation of these cytosine photoproducts were between 270 and 295 nm. This human endonuclease therefore has a similar ultraviolet substrate specificity to endonuclease III.

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“…The DNA was labeled to a specific activity of 10000-15000 dpm/^g by addition of 20 /¿Ci/rnL [methyl-3H] thymidine (6.7 Ci/mmol, New England Nuclear, Boston, MA) and purified as previously described (Frenkel et al, 1981). The DNA was reacted with AAAF (obtained from the National Cancer Institute Carcinogen Repository, Bethesda, MD) under the conditions of Santella et al (1981) and the carcinogen extracted according to Duker J/M2) in the presence of the photosensitizer AgN03 according to Rahn & Landry (1973) with the apparatus previously described (Duker et al, 1981). The irradiated DNA was separated from silver ions by sequential dialysis into 10 mM Tris-HCl/10 mM EDTA, pH 7.8, containing 1.0, 0.5,0.2, and 0.1 M NaCl, respectively.…”

Section: Methodsmentioning

confidence: 99%

Inhibition of enzymic incision of thymine dimers by covalently bound 2-[N-[(deoxyguanosin-8-yl)acetyl]amino]fluorene in deoxyribonucleic acid

Duker¹,

Merkel²

1985

Biochemistry

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The effects of DNA adducts of the carcinogen 2-[N-(acetoxyacetyl)amino]fluorene on enzymic incision of thymine dimers was investigated. Escherichia coli DNA labeled with [3H]thymidine was reacted with the carcinogen. Thymine dimers were then introduced into the modified DNA by irradiation with monochromatic 254-nm light in the presence of the photosensitizer silver nitrate. This DNA containing both types of damages, mainly 2-[N-[(deoxyguanosin-8-yl)acetyl]fluorene and thymine dimers, was then used as substrate for pyrimidine dimer-DNA glycosylase, purified from E. coli infected by bacteriophage T4. Activity was assayed by measuring release of free labeled thymine after photoreversal of the enzyme-reacted DNA by 254-nm light. The Vmax of the enzyme was decreased when it was reacted with the extensively arylamidated substrate. This inhibition of incision of pyrimidine dimers was increased with the number of carcinogen-DNA adducts, although no enzymic activity against modified guanines was present. Therefore, carcinogen-modified purine moieties can interfere with initiation of excision repair of ultraviolet-induced pyrimidine dimers. This suggests an indirect pathway by which modified DNA bases can be mutagenic.

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Alteration of Uracil-Dna Glycosylase Activity by Uracil Dimers in Dna

Cited by 18 publications

References 35 publications

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

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

Wavelength Dependence of Dna Incision by a Human Ultraviolet Endonuclease

Inhibition of enzymic incision of thymine dimers by covalently bound 2-[N-[(deoxyguanosin-8-yl)acetyl]amino]fluorene in deoxyribonucleic acid

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