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Perturbations of Enzymic Uracil Excision Due to Purine Damage in DNA* (Carcinogens/DNA Repair) NAHUM J

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Perturbations of Enzymic Uracil Excision Due to Purine Damage in DNA* (Carcinogens/DNA Repair) NAHUM J Proc. Nat. Acad. Sci. USA Vol. 79, pp. 4878-4882, August 1982 Biochemistry Perturbations of enzymic uracil excision due to purine damage in DNA* (carcinogens/DNA repair) NAHUM J. DUKERt, DAVID E. JENSENt, DONNA M. HARTt, AND DEBBIE E. FISHBEINt tDepartment of Pathology and Fels Research Institute, Temple University School of Medicine, Philadelphia, Pennsylvania 19140; and WFels Research Institute, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 Communicated by Sidney Weinhouse, May 6, 1982 ABSTRACT Phage PBS-2 DNA, which contains uracil in place DNA contained either apurinic sites or photoalkylated purines, of thymine, was selectively damaged and then used as substrate but it was unaffected by the introduction of mGua. Thus, the for purified Bacillu subtilis uracil-DNA glycosylase. This enzyme presence of specific DNA modifications may decrease the ca- releases uracil from DNA in a limited processive manner. Irra- pacity for uracil excision. This suggests the possibility that in- diation by ultraviolet light (>305 nm) in the presence of isopro- terference with the enzymic excision of this potentially muta- panol and a free radical photoinitiator introduced covalently genic base might constitute a common mechanism of action of bound 8-(2-hydroxy-2-propyl)purines into DNA. Methylation by several DNA-damaging agents. dimethylsulfate yielded 7-methylguanine. Apurinic sites were pro- duced by gentle heating of methylated DNA. Rates of enzymic release ofuracil from DNA varied among these three substrates. MATERIALS AND METHODS The Vm. was markedly decreased for DNA containing 8-(2-hy- DNA Preparations. Phage PBS-2 was grown in B. subtilis, droxy-2-propyl)purines and apurinic sites but was unaffected by and the DNA was purified and stored at 100 jig/ml according the presence of larger quantities of 7-methylguanine. This sug- to LindahletaL (8). PBS-2 DNAwas labeled in uracilby addition gests that certain types of damaged DNA moieties may decrease of [5-3H]uridine (New England Nuclear; final concentration, the capacity for uracil excision. Therefore, interference with en- 20 uCi/ml; 1 Ci = 3.7 X 1010 becquerels) or [2-14C]uridine zymic excision of this potentially mutagenic base may constitute (New England Nuclear; final concentration, 1.0 ,tCi/ml) 5 min a common mechanism ofaction ofthe reaction products ofseveral after infection. PBS-2 DNA was also prepared with the purines unrelated DNA damaging agents. radiolabeled by addition of [U-'4C]adenine (Amersham; final concentration, 1.0 uCi/ml) or [U-14C]guanosine (Amersham; Uracil-DNA glycosylase [dUra(DNA) glycosylase] specifically final concentration, 0.3 jCi/ml). Specific activities were: DNA removes uracil from DNA. Such uracil may result either from labeled with [3H]uridine, 250,000 dpm/;,g; DNA labeled with incorporation in place of thymine during DNA synthesis or [14C]uridine, 49,000 dpm/ttg; DNA labeled with [14C]adenine, deamination of DNA cytosine (1), which may occur at physio- 15,000 dpm/,ig with 98% of the label in adenine and 2% in logical conditions (2). Left unrepaired, deaminated cytosines guanine; DNA labeled with [14C]guanosine, 3,000 dpm/tug would result in transition mutations (3). Escherichia coli mu- with 50% of the label in sugar, 47% in guanine, and 3% in tants deficient in dUra(DNA) glycosylase (ung-) are mutators, adenine. and in vivo deamination ofcytosines is the source ofthese mu- Preparation ofDamaged DNA Substrates. PBS-2 DNA was tations (4, 5). This implies that dUra(DNA) glycosylase activity photoalkylated according to Livneh et aL (9), except that the is necessary to preserve the integrity of DNA in the face ofcon- samples were not flushed with nitrogen before irradiation. Ac- tinuous potentially mutagenic damage. The finding that this tinometry (10) showed the incident dose to be 6.6 x 10-5 ein- enzyme is apparently ubiquitous supports the suggestions that stein cm-2 min-1. Photoalkylated DNA was extensively di- this is its prime function (1, 4, 6). alyzed into 10 mM Tris HCl/1 mM EDTA, pH 8.0. DNA was The presence of UV photoproducts results in alteration of methylated with 10 mM Me2SO4 for 1 hr (11) and purified by dUra(DNA) glycosylase activity toward phage PBS-2 DNA, passage through a Sephadex G-50 column in 10 mM Tris HCI/ which contains uracil in place ofthymine. The rate of enzymic 1 mM EDTA, pH 8.0 (12). In some experiments, methylated release ofuracil from such UV-irradiated DNA decreases as the PBS-2 DNA was partially depurinated by heat (13) followed by number of photodimers increases (7). This indicates that the dialysis into the above buffer. presence of pyrimidine dimers in DNA may affect excision of Analysis of DNA Damage. The degree of purine modifica- uracil. tion in DNA was assessed by quantitating labeled purines lib- We investigated whether this alteration occurs with other erated by acid hydrolysis (14) utilizing paper chromatography types of DNA modifications. Three different types of purine (14) and HPLC. All HPLC for modified purines used a Waters damage were introduced into PBS-2 DNA, which was then used chromatographic system and a Bio-Rad Aminex A-9 column (250 as substrate for dUra(DNA) glycosylase. Photoalkylation pro- x 4 mm) maintained at 600C. The column was eluted with am- duced the modified purines 8-(2-hydroxy-2-propyl)guanine monium formate pH 3.0 buffer at 0.8 ml/min; sample volume (HPG) and 8-(2-hydroxy-2-propyl)adenine (HPA) in DNA. 7- was 500 In runs for the quantitation of Methylguanine (mGua) was introduced by chemical alkylation ,jL. chromatography DNA sites were of by dimethylsulfate (Me2SO4), and apurinic Abbreviations: HPA, 8-(2-hydroxy-2-propyl)adenine; HPG, 8-(2-hy- produced by gentle heating of such alkylated DNA. The rate droxy-2-propyl)guanine; dUra(DNA) glycosylase, uracil-DNA glycosy- of enzymic release of uracil was markedly decreased when the lase; Me2SO4, dimethylsulfate; mGua, 7-methylguanine. * Presented in part at the 72nd Annual Meeting of the American As- The publication costs ofthis article were defrayed in part by page charge sociation for Cancer Research, Apr. 27, 1981 (abstract no. 348), and payment. This article must therefore be hereby marked "advertise- at the 66th Annual Meeting of the Federation of American Societies ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. for Experimental Biology, Apr. 19, 1982 (abstract no. 3840). 4878 Biochemistry: Duker et aL Proc. NatL Acad. Sci. USA 79 (1982) 4879 mGua yields in Me2SO4-treated DNA, the elution buffer was However, the yield as determined by paper chromatography 0.5 M and the guanine, mGua, and adenine peaks were de- was consistently greater than that obtained on HPLC. In all tected at 23, 32, and 38 min after sample application, respec- cases, the discrepancy in HPA radioactivity was accounted for tively. In the analysis for HPG, the buffer was 0.8 M and HPG, by two uncharacterized peaks. Neither xanthine nor hypoxan- guanine and adenine peaks were detected at 14, 21, and 36 min, thine was formed. The yield of HPG was higher than that of respectively. In the analysis for HPA, the elution buffer was 0.4 HPA. Five minutes ofphotoalkylation modified 0.7% ofguanine M and guanine, HPA, and adenine peaks were detected at 47, to HPG; 10 and 20 min of photoalkylation yielded 1.6% and 53, and 85 min, respectively. The assessment of radioactivity 3.9% guanine modification, respectively. There were no sig- and quantitation of concentrations in column elution fractions nificant differences in recovery between the two methods of were as described (15). HPA marker was purchased from CDS analysis, and no other type of modified guanine was detected. Laboratories (Durango, CO). HPG marker was made by pho- The relative yields of photoalkylated purines here differ from toalkylation of guanine (16). Identities of both were confirmed those reported for E. coli and PM2 phage DNAs, in which HPG bypaper chromatography (9, 14). mGuawas obtained from Vega and HPA are formed in equal quantities (9, 14). Biochemicals (Tucson, AZ). The number of modified adducts Uracil-containing photodimers were also formed during per molecule were calculated from the known size and com- photoalkylation. After 5 min of irradiation, 0.2% of the radio- position of PBS-2 DNA (17). activity ofthe DNA labeled in uracil was present as dimers. This Photoalkylated DNA was assayed for uracil-containing di- increased to 0.5%, 1.0%, and 1.5% with 10, 20, and 40 min of mers as described (7). Other pyrimidine damage was investi- photoalkylation, respectively. The low level of uracil dimers gated by enzymic digestion of DNA to deoxynucleosides (18) produced by 10 min ofphotoalkylation has no detectable effect and precipitation of the enzymes with a% trichloroacetic acid. on dUra(DNA) glycosylase activity (7). All nondimer material The supernatant was neutralized and analyzed by TLC (19) and in ['4C]uridine-labeled DNA was identified as deoxyuridine by HPLC, performed on a Waters C18 uBondapack column (250 enzymic hydrolysis followed by HPLC and TLC. No photoal- X 4 mm) at room temperature; sample volume was 200 A1. A kylated derivatives ofcytosine have been reported (27). There- 60-min 0-10% methanol gradient in 10 mM K2HPO4 (pH 7.4) fore, the major products ofphotoalkylation ofPBS-2 DNA were was applied by using curve 10 on a Waters model 660 solvent photoalkylated purines. As with native thymine-containing programmer; the elution rate was 0.3 ml/min. The deoxyuri- DNAs, no photoalkylated pyrimidines were detected (9, 14). dine peak appeared at 25 min into the gradient. This is consistent with the suppression of photoalkylation of Enzymology. dUra(DNA) glycosylase was purified from B. pyrimidines by equimolar purines, reported for both uracil and subtilis according to Cone et al (20).
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