Title

Mutagenesis studies of single and tandem DNA damages by site-specific approach

Date of Completion

January 2008

Keywords

Chemistry, Biochemistry

Degree

Ph.D.

Abstract

Comparative mutagenesis of 1,N6 -ethenoadenine (ϵA) and 8-oxoguanine (G8-oxo) was evaluated in extracts of HeLa and Xeroderma pigmentosum variant (XPV) cells. In HeLa extracts, ϵA was weakly mutagenic, inducing all three base substitutions in approximately equal frequencies, whereas G8-oxo was 10-fold more mutagenic, inducing primarily G8-oxo→T transversions. In XPV extracts, the frequencies of ϵA→G and ϵA→C remained approximately the same, but ϵA→T events increased five-fold relative to HeLa extract. However, the G8-oxo mutagenesis was not significantly affected in extracts of XPV cells. ^ The cytotoxic and mutagenic properties of a model tandem lesion of 8-oxoguanine and tetrahydrofuran (Z), a stable abasic site analog, were studied in uninduced and SOS-induced Escherichia coli cells. Constructs containing tandem lesions showed comparable viability as that of single abasic site in both uninduced and SOS-induced cells. However, relative to uninduced cells, viability of the constructs increased by 5–17 times in SOS-induced cells. Targeted single base deletions (Z→Δ) were the major mutations determined from the constructs containing a single abasic site or tandem lesions, in uninduced cells. In SOS-induced cells, Z→T was the dominant mutation for the GZGTC and GZG8-oxoTC sequences while Z→Δ remained the primary mutation in the GTGZC and GTG8-oxoZC sequences, suggesting a probable role of sequence context. The frequency of targeted G8-oxo→T transversions was approximately the same when either T or Z was located 5' to G8-oxo, in uninduced cells. However, G8-oxo→T mutation was seven times greater when Z was located 3' to G8-oxo compared to when Z was a T. For the SOS-induced cells, a significant increase in the targeted G8-oxo →T transversions was observed when Z was at either adjacent side of G8-oxo relative to when Z was a T. ^ The same tandem lesions studied in E. coli were investigated further in human cells (HEK 293T/17). The major mutations induced by all four sequences were single, targeted Z→T mutation. Relative to the single G8-oxo-containing construct, the frequency of targeted G 8-oxo→T transversions in the tandem lesions increased by 1.9 times when Z was located 5' of G8-oxo while, surprisingly, it decreased by 0.56 times when Z was at the 3' position. ^