Cloning and Characterization of Mycobacterium tuberculosis MutT2 Protein

• Main Authors: Hsiu-Lin Huang, 黃秀琳
• Other Authors: C. H. Herbert Wu
• Format: Others
• Language: en_US
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/18024988114983026498

碩士 === 國立臺灣大學 === 分子醫學研究所 === 88 === Deoxyribonucleotides in the cells are subjected to attacks by the reactive oxygen species. Unrepaired oxidative DNA damage is a source of mutation and plays a critical role in carcinogenesis and aging. Among different damaged nucleotides, 8-oxo-2’-deoxyguanosine 5’-triphosphate (8-oxo-dGTP) is one of the most stable products. In Escherichia coli, such a lesion is prevented and corrected by a system involving functions of three genes: mutT, fpg, and mutY. In this thesis, we have cloned and characterized the M. tuberculosis mutT2 gene. M. tuberculosis MutT2 is a 15.1-kD protein that shares 23% identity to E. coli MutT protein. MutT2 contains a MutT motif, which is highly conserved in the MutT-related proteins of both prokaryotes and eukaryotes. A DNA fragment of the mutT2 gene was amplified with PCR from the genomic DNA of M. tuberculosis strain H37Rv. This fragment was subcloned into the expression vector pET28a(+) and the recombinant plasmid was transformed into E. coli strain BL21(DE3) and MK602(DE3) for overexpression of this protein. In vivo complementation assay showed that expression of MutT2 in the mutT mutant could revert the mutator phenotype of this mutant. Purified and refolded MutT2 possesses a dGTPase activity, which is enhanced by an increasing concentration of magnesium and inhibited by an increasing concentration of nickel or EDTA. Endogenous MutT2 protein in M. tuberculosis lysate displayed a smear pattern in the Western blot suggesting instability of this protein in the bacteria. In summary, our data strongly suggest that the cloned M. tuberculosis mutT2 gene is a functional homologue of E. coli mutT.