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

Abstract

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 ...

A role for DNA mismatch repair in sensing and responding to fluoropyrimidine damage

“…The probability that the second scenario may occur in mammalian cells is consistent with observations of Duker et al (1982) showing that AP sites antagonize their own repair if closely spaced in DNA. Thus, under conditions leading to high dUMP and/or FdUMP incorporation densities, mispaired loops involving AP sites could be more common than anticipated at SSBs, providing lesion sites that are recognized by MMR and leading to a cascade of events resulting in progressively larger repair patches in DNA until completion of normal repair processes becomes impossible.…”

Endonucleolytic Incision of Uvb‐irradiated Dna

“…DNA was irradiated with a Bausch & Lomb unit equipped with a 200-W Osram Hg bulb. Broadspectrum UV irradiation was conducted through a Schott WG 320 filter at an incident dose of 6.6 x einstein, cm*/min as previously described (Duker et al, 1982(Duker et al, , 1985Weiss and Duker, 1986). Monochromatic UV irradiation of PM2 phage DNA was performed at the wavelengths and doses indicated through a Bausch & Lomb highintensity monochromator as previously described (Duke1 et al, 1981).…”

Ionizing radiation-induced mutagenesis

“…Indeed the glycolase activity for radiation ring damaged thymine is also an endonuclease (Breimer & Lindahl, 1984 -see above). It has been reported that simultaneous presence of AP sites and base lesions in the same phage DNA interferes with the efficiency of their respective repair (Duker et al, 1982). Tindall et al (1987) reported a preferential substitution of A.T for any original base pair in irradiated lambda phage assayed in SOS induced hosts, and from Kunkel's work (1984), suggested that AP sites were the mutagenic intermediate.…”