In situ enzymatic reclosure of opened imidazole rings of purines in DNA damaged by gamma-irradiation.

Abstract

When aqueous solutions of DNA were treated with 10-500 grays of frays, the imidazole rings of some adenine and guanine residues underwent scission, resulting in the conversion of these purines to formamidopyrimidines. It was found that formamidopyrimidine-DNA glycosylase, known to remove imidazole-ring-opened 7-methylguanine from DNA, did not excise the radiation-induced non-alkylated formamidopyrimidines formed from adenine and guanine. The repair of these ring-opened purines was found to involve an enzymatic recyclizing of the opened imidazole ring that effects a restoration of the C-8 to N-9 bond. The enzyme, purine imidazole-ring cyclase reclosed the imidazole rings of 90% of ring-opened adenine or guanine, but did not close the opened imidazole ring of 7-methylguaninederived formamidopyrimidine in DNA.The destruction of nucleic acid constituents by ionizing radiation (1-3) includes the cleavage of the imidazole rings of purine nucleosides and nucleotides, resulting in the conversion of adenine to 4,6-diamino-5-formamidopyrimidine (4, 5) and of guanine to 2,6-diamino-4-oxo-5-formamidopyrimidine (6). These two radiation products are generally referred to as formamidopyrimidines (FAPyr). The radiation-induced conversion of guanine to FAPyrGua (7) and adenine to FAPyrAde (8) in DNA has been reported. We have recently confirmed the production of FAPyrGua and FAPyrAde in aqueous solutions of DNA exposed to t-irradiation (9).The mutagenic/carcinogenic potential of FAPyr lesions has recently been highlighted by the demonstration that FAPyr residues derived from ring-opened 7-MeGua in DNA block DNA chain elongation (10). It remains to be shown whether the non-alkylated FAPyr residues have a similar effect on DNA synthesis.When DNA containing either FAPyrAde or FAPyrGa was used as a substrate for formamidopyrimidine-DNA (FAPyr-DNA) glycosylase, an enzyme that excises imidazole-ringopened 7-MeGua (MeFAPyr) (11,12) and phosphoramide mustard-FAPyr (13) from DNA, there was no removal of either type of FAPyr residue from DNA. (Phosphoramide mustard is a metabolite of the antineoplastic agent, cyclophosphamide.) It is possible that the enzyme failed to remove these FAPyr residues because they were components of denatured DNA, which is not a good substrate for FAPyr-DNA glycosylase. We report here the discovery of a mechanism by which these radiation-induced FAPyr lesions are repaired in DNA. Rather than an excision repair of the lesions, the mechanism involves an enzymatic in situ reclosure of the imidazole ring to restore the C-8 to N-9 linkage previously cleaved by ionizing radiation. The imidazole ring-recyclizing activity has been named purine imidazole-ring (PurIR) cyclase. A short report of this work has been published elsewhere (14). 25 ,ug/ml. In doseresponse studies, N2-saturated solutions of DNA (500 ,ug/ml) in 10 mM sodium phosphate, pH 7.0/1 mM EDTA were irradiated, under continuous flushing with N2, with 0.1-10,000 grays (Gy, 1 Gy = 100 rads) with the University of Michigan 'Co irradiator. MATERIALS A...

Mechanism of Degradation of Purine Nucleosides by Formamide. Implications for Chemical DNA Sequencing Procedures

“…2,6-Diamino-4-oxo-5-formamidopyrimidine 7. Compound 7 was identical to that reported by Chetsanga and Grigorian . IR (KBr) 3230 (NH), 1780 (CO), 1679 (CO), 1640 (CC), 1290, 970 cm -1 .…”

“…In the latter case, 2,6-diamino-4-oxo-5-formamido-6-[N-(2′-deoxy-β-D-ribofuranosyl)]pyrimidine 6 and 2,6-diamino-4-oxo-5-formamidopyrimidine 7 were obtained (Scheme 2) after purification by TLC in reverse phase (butanol saturated by water) or by flashchromatography (after distillation of the excess formamide). Formamidopyrimidine derivatives (FAPyr) 4, 5, 6, and 7 are usually referred to as products of imidazole C(8)-ring cleavage of purine nucleosides and nucleotides by ionizing radiation [17][18][19][20] or by basic treatment after heterocyclic nitrogen alkylation. 21 On the basis of the structural analogies among the isolated degradation products it is reasonable to suggest that the degradation pathway of 2′-deoxyguanosine is similar to that described for 2′-deoxyadenosine, even if, in the first case, other degradation pathways cannot be completely excluded because of the lack of characterization of all possible intermediates.…”

“…In the latter case, 2,6-diamino-4-oxo-5-formamido-6-[ N -(2‘-deoxy-β - d - ribofuranosyl)]pyrimidine 6 and 2,6-diamino-4-oxo-5-formamidopyrimidine 7 were obtained (Scheme ) after purification by TLC in reverse phase (butanol saturated by water) or by flash-chromatography (after distillation of the excess formamide). Formamidopyrimidine derivatives (FAPyr) 4 , 5 , 6 , and 7 are usually referred to as products of imidazole C(8)-ring cleavage of purine nucleosides and nucleotides by ionizing radiation − or by basic treatment after heterocyclic nitrogen alkylation …”