Human uracil DNA N-glycosidase: studies in normal and repair defective cultured fibroblasts

Kühnlein, U.; Lee, Betty; Linn, Stuart

doi:10.1093/nar/5.1.117

Cited by 42 publications

(10 citation statements)

References 23 publications

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“…The results with the double-stranded [U-3H]DNAs indicated that the uracil/thymine ratio in the repaired strand influenced both K,,, and V (Table4). The K,,, values for substrates 1 and 2 were respectively 0.36 pM and 0.13 pM dUMP residues in DNA, in good agreement with similar results for mammalian enzymes already published [6,9,23] ; too many uracil residues thus decrease the apparent affinity of the enzyme for the substrate. On the other hand, V was six-times higher for substrate 1 (33 fmol .…”

Section: Effect Of Strandedness and Uracillthymine Ratio : Inhibitionsupporting

confidence: 89%

Intracellular Localization of Rat‐Liver Uracil‐DNA Glycosylase

Colson

Verly

1983

European Journal of Biochemistry

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Most of the uracil-DNA glycosylase of the rat liver cell is located in chromatin; there is, however, some activity in the nuclear sap and in the cytoplasm. The chromatin uracil-DNA glycosylase has been purified; the preparation is devoid of endonuclease and exonuclease activities ; the enzyme does not need divalent cations, has a broad optimum pH around 8, is strongly inhibitcd by increasing ionic strength and free uracil.The apparent K, is independent of the strandedness of the DNA substrate containing uracil, but V is slightly higher with the single-stranded substrate. The frequency of uracil substitution in the double-stranded DNA influences the kinetic parameters: a higher frequency increases both K, and V.The inhibitory effects of NaCl and free uracil are greater when the substrate is double-stranded rather than single-stranded. It is speculated that, acting either on the D N A or on the enzyme, both oppose the opening of the double helix necessary for the formation of the enzyme-substrate complex.The increased reaction rate with a higher frequency of uracil residues in double-stranded D N A is interpreted as a tendency for the repair enzyme to work in a processive way. It is supposed that pmcessivity also occurs with single-stranded D N A and that it is opposed by both NaCl and free uracil, explaining a greater inhibition when the single-stranded substrate has a higher uracil content.

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Section: Effect Of Strandedness and Uracillthymine Ratio : Inhibitionsupporting

confidence: 89%

Intracellular Localization of Rat‐Liver Uracil‐DNA Glycosylase

Colson

Verly

1983

European Journal of Biochemistry

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“…The biological significance of in vivo deamination of uracil to cytosine has been demonstrated in E. coli mutants lacking dUra(DNA) glycosylase (4). The extent of such deamination in human DNA has not been 'determined because no similar human mutant cell lines are available (39,40). Therefore, the efficient repair of uracil renders impossible the detection of deamination of cytosine.…”

Section: Discussionmentioning

confidence: 99%

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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“…i) are initiated by a family of enzymes called N-glycosidases or, better still, glycosylases. These remove damaged bases by cleaving the bond between base and deoxyribose without breaking the DNA strand (Kirtikar & Goldthwait, 1974;Wist, Unhjem & Krokan, 1978;Kuhnlein, Lee & Linn, 1978). Subsequently, however, the base-free site is attacked by apurinic/apyrimidinic endonucleases which incise the DNA and allow degradation and resynthesis to take place (Laval, 1977;Friedberg et al, 1977).…”

Section: Repair Of Single Strand Damagementioning

confidence: 99%

DNA repair in human diseases

Giannelli

1980

Clin Exp Dermatol

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Human uracil DNA N-glycosidase: studies in normal and repair defective cultured fibroblasts

Cited by 42 publications

References 23 publications

Intracellular Localization of Rat‐Liver Uracil‐DNA Glycosylase

Intracellular Localization of Rat‐Liver Uracil‐DNA Glycosylase

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

DNA repair in human diseases

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