Title

Mutagenesis studies of selective lesions derived from purines under oxidative stress

Date of Completion

January 2005

Keywords

Biology, Molecular|Chemistry, Biochemistry

Degree

Ph.D.

Abstract

In vivo mutagenesis studies of selective oxidative DNA lesions derived from adenine and guanine have been investigated in simian kidney (COS7) cells. Replication of a shuttle vector modified with Fapy•dG and 8-oxodG showed preferential incorporation of dCMP opposite each lesion. However, ∼8% and ∼30% progeny in the 5'-TXA and 5'-TXT sequences, respectively, were mutants (where X represents the lesion). Most mutations were targeted G→T transversions (i.e., dAMP incorporation opposite the lesion), though small population (1-2%) of progeny containing G→C substitutions (i.e., dGMP incorporation) was detected in the 5'-TXT sequence. 8-oxodG was found to be 25-30% less mutagenic than Fapy•dG in each sequence. In similar studies, unlike the guanine derivatives, Fapy•dA and 8-oxodA exhibited weak mutagenicity in COS cells. In order to explore how tandem lesions may influence mutagenicity, in another study 8-oxodG and uracil residue (U) were incorporated in the sequence 5'--GNG*N'C--3' (where N or N' = T or U; G* = G or G8-oxo), and mutagenicity of each lesion was evaluated. The uracil residue would be excised by uracil DNA glycosylase in vivo , generating an abasic site (AP site). An isolated uracil in both GUGTC and GTGUC sequence contexts provided >60% progeny containing GTGTC and >30% population in which the misincorporation of C, G or T occurred. The trend of misincorporations at the GUG site was G >T >C, whereas the same at the GUC site was T >G >C. When U was part of the tandem lesions, the misincorporation of T became predominant in each case. For 8-oxoG, compared to 23-24% G→T mutants from a single 8-oxoG in a TG8-oxoT sequence context, in UG8-oxoT and TG8-oxoU sequences it generated ∼60% and >85% mutant progeny, respectively. A significant fraction of the mutations involved tandem mutations. In UG8-oxoT sequence about 17% of total mutation involved tandem mutations, whereas in TG8-oxoU it was about 45%. In order to rationalize the mutagenesis data, base-pairing modes of the tandem lesions were investigated. Thermal melting experiments showed that 8-oxoG:C pair in each sequence was more stable than the 8-oxoG:A pair. However, the ΔΔG of 1.6 kcal/mol in (AP*)G8-oxoT dropped to 0.4 kcal/mol in TG8-oxo(AP*). This indicates that the polymerase discrimination to incorporate appropriate nucleotides might be less efficient in the TG8-oxo(AP*) sequence relative to (AP*)G8-oxoT resulting in more misincorporations. ^