Formation and Genotoxicity of Novel Oxidatively Generated Tandem DNA Lesions and N2-(1-carboxyethyl)-2'-deoxyguanosine
Exogenous and endogenous agents can induce the formation of both single- and tandem-nucleobase lesions in DNA. In this dissertation, we assessed the formation and genotoxicity of three different types of DNA lesions; a novel guanine-thymine intrastrand cross-link lesion (G[8-5m]T), two tandem single-nucleobase lesions consisting of a thymidine glycol and an 8-oxo-7,8-dihydro-2'-deoxyguanosine [5'-(8-oxodG)-Tg-3' and 5'-Tg-(8-oxodG)-3' ], and a single nucleobase lesion, N2-(1-carboxyethyl)-2'-deoxyguanosine (N2-CEdG).
In Chapter 2, We demonstrated the dose-dependent induction of the G[8-5m]T cross-link in human HeLa-S3 cells upon exposure to gamma rays by LC-MS/MS. The in-vitro replication studies on the lesion-bearing substrate showed that the Klenow fragment of Escherichia coli (E. coli) DNA polymerase I stopped synthesis mostly after incorporating one nucleotide opposite the 3'-thymine moiety of the lesion. Yeast polymerase eta, a translesion synthesis DNA polymerase, could replicate past the lesion with a markedly reduced efficiency. However, it could also induce nucleotide misincorporation (i.e., dAMP and dGMP) opposite the 5'-guanine moiety of the G[8-5m]T. In Chapters 3 and 4, we developed an LC-MS/MS-based strategy for quantitative analysis and demonstrated the efficient formation of a tandem lesion, 5'-Tg-(8-oxodG)-3', in calf thymus DNA upon exposure to Cu(II)/ascorbate along with H2O2 or gamma rays. Both in-vitro and in-vivo replication studies on two tandem lesions, 5'-(8-oxodG)-Tg-3' and 5'-Tg-(8-oxodG)-3', revealed that the tandem lesions blocked DNA replication mediated by the Klenow fragment and yeast pol eta; more readily than when the Tg or 8-oxodG was present alone and the mutagenicity of Tg or 8-oxodG differed while they were present alone or in tandem. Moreover, the activities of base excision repair enzymes were altered in substrates bearing the tandem lesions. The 5'-Tg-(8-oxodG)-3' could also give rise to a substantial frequency of TG to GT tandem double mutation. These results support that complex lesions could exert a greater cytotoxic effect than when the composing lesions are present alone and the mutagenic properties of the tandem lesions could be markedly affected by the spatial arrangement of the component lesions.
In Chapter 5, we analyzed the formation and genotoxic properties of N2-CEdG, demonstrated that this major stable DNA adduct could be induced by methylglyoxal (MG) in human cancer cells. We also found that N2-CEdG is weakly mutagenic, and DinB (i.e., polymerase IV in E. coli) is the major DNA polymerase responsible for bypassing the lesion in vivo; the E. coli pol IV- and human polymerase kappa-mediated nucleotide incorporation opposite this lesion is both accurate and efficient. Our results supported N2-CEdG may constitute an important endogenous substrate for DinB DNA polymerase.