In vitro DNA synthesis opposite oxazolone and repair of this DNA damage using modified oligonucleotides

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Oxford University Press

RESUMO

Emphasis was placed in this work on the assessment of biological features of 2,2,4-triaminooxazolone, a major one-electron and ·OH-mediated oxidation product of guanine. For this purpose, two oligonucleotides that contain a unique oxazolone residue were synthesized. Herein we report the mutagenic potential of oxazolone during in vitro DNA synthesis and its behavior towards DNA repair enzymes. Nucleotide insertion opposite oxazolone, catalyzed by Klenow fragment exo– and Taq polymerase indicates that the oxazolone lesion induces mainly dAMP insertion. This suggests that the formation of oxazolone in DNA may lead to G→T transversions. On the other hand, oxazolone represents a blocking lesion when DNA synthesis is performed with DNA polymerase β. Interestingly, DNA repair experiments carried out with formamidopyrimidine DNA N-glycosylase (Fpg) and endonuclease III (endo III) show that oxazolone is a substrate for both enzymes. Values of kcat/Km for the Fpg-mediated removal of oxidative guanine lesions revealed that 8-oxo-7,8-dihydroguanine is only a slightly better substrate than oxazolone. In the case of endo III-mediated cleavage of modified bases, the present results suggest that oxazolone is a better substrate than 5-OHC, an oxidized pyrimidine base. Finally, MALDI-TOF-MS analysis of the DNA fragments released upon digestion of an oxazolone-containing oligonucleotide by Fpg gave insights into the enzymatic mechanism of oligonucleotide cleavage.

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