Date of Award
Doctor of Philosophy (PhD)
Biochemistry and Molecular Biology
DNA that stores genetic information is frequently damaged in cells. The DNA bases carrying an exocyclic amino group [adenine (A), guanine (G), cytosine (C)] encounter deamination even under physiological conditions. Xanthine (X) and the newly discovered oxanine (O) are derived from deamination of guanine; they are potentially cytotoxic and mutagenic lesions. However, in yeast and eukaryotes, studies on the enzymatic repair of these lesions are limited.
In the yeast, Schizosaccharomyces pombe thymine-DNA glycosylase (Spo TDG) is homologous to human thymine-DNA glycosylase (hTDG), an enzyme that removes thymine from T/G pair. It was reported that Spo TDG contains uracil DNA glycosylase and hypoxanthine DNA glycosylase activity (Nucl. Acids Res. 31: 2261). In the present study, we examined the repair activity of the Spo TDG for four deaminated lesions: xanthine, oxanine, uracil (U) and inosine (I) by using synthetic lesion-containing oligonucleotide substrates. The cleavage analyses with the double and single oligonucleotide substrates revealed that Spo TDG could remove X, I and U relatively efficiently with no preference to opposite base. The order of the Spo TDG glycosylase activity for these deaminated lesions was found to be X>I>U>O. Spo TDG is the first glycosylase in the yeast that catalyses the removal of xanthine and oxanine. To investigate the mechanism for this broader substrate specificity from Spo TDG, we performed mutational analysis in motifs of Spo TDG and revealed functional role of L157, S163, G288, I289, and S291 in determining substrate specificity.
So far, AAG is the only glycosylase that was reported to repair oxanine and xanthine in mammalian system. Here, we report our recent findings on addition oxanine DNA glycosylase (ODG) and xanthine DNA glycosylase (XDG) activity in Aag knockout mice tissues and other mammalian tissues. Data obtained from DNA cleavage assay using the purified glycosylases demonstrated that hSMUG1 and hNEIL1 contained weak but detectable ODG activities. hSMUG1 also showed weak XDG activity. Moreover, during the purification of the new XDG from pig tissues, we found major additional XDG activity from unknown protein(s), the protein(s) are being identified in our group.
Dong, Liang, "NOVEL XANTHINE AND OXANINE DNA GLYCOSYLASE ACTIVITIES IN YEAST AND MAMMALIAN SYSTEMS" (2008). All Dissertations. 311.