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

Emphasis was placed in this work on the assessment of biological features o f 2,2,4-triaminooxazolone, a major one-electron and (OH)-O-.-mediated oxida tion product of guanine, For this purpose, two oligonucleotides that contai n a unique oxazolone residue were synthesized. Herein we report the mutagen ic potential of oxazolone during in vitro DNA synthesis and its behavior to wards DRIA repair enzymes. Nucleotide insertion opposite oxazolone, catalyz ed by Klenow fragment exo(-) and Taq polymerase indicates that the oxazolon e lesion induces mainly dAMP insertion. This suggests that the formation of oxazolone in DNA may lead to G-->T transversions, On the other hand, oxazo lone represents a blocking lesion when DNA synthesis is performed with DNA polymerase beta, Interestingly, DNA repair experiments carried out with for mamidopyrimidine DNA N-glycosylase (Fpg) and endonuclease III (endo ill) sh ow that oxazolone is a substrate for both enzymes. Values of k(cat)/K-m 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 t he case of endo III-mediated cleavage of modified bases, the present result s suggest that oxazolone is a better substrate than 5-OHC, an oxidized pyri midine base. Finally, MALDI-TOF-MS analysis of the DNA fragments released u pon digestion of an oxazolone-containing oligonucleotide by Fpg gave insigh ts into the enzymatic mechanism of oligonucleotide cleavage.