Overexpression of human NTH1 DNA glycosylase and its catalysis-defective mutant in HeLa cells: Effect on sensitivity to oxidative and alkylation DNA damage

Ikeda, S., Tabata, R., and Kasuu E

Human NTH1 (hNTH1) is a DNA glycosylase that acts on oxidatively damaged pyrimidine bases such as thymine glycol, at an initial step of base excision repair. In the present study, we established HeLa cell lines overexpressing wild-type hNTH1 and a catalysis-defective mutant hNTH1, to study the roles of hNTH1 in thymine glycol repair in vivo. Expression of hNTH1 in these cells is inducible under a Tet-On system, and is turned on by addition of doxycycline. Mutation of Lys-212 to Gln (K212Q) destroys catalytic activity of hNTH1, but the mutant protein retains damage recognition and will bind to thymine glycol on DNA. Therefore, K212Q proteins overexpressed in cells probably interfere with normal hNTH1 function by dominant negative effect. Sensitivity of the stable cell lines against methylation and oxidative stresses was measured in the presence or absence of doxycycline. Overexpression of wild-type hNTH1 did not alter the viability of the cells, indicating that the amount of hNTH1 in normal cells is sufficient to repair all thymine glycol. Dominant negative interference of hNTH1 function had no effect, implying the presence of a backup enzyme for hNTH1 and an alternative repair system for thymine glycol.

The Bulletin of Okayama University of Science

岡山理科大学

39A

57

62