| Summary: | 碩士 === 國立臺灣大學 === 分子醫學研究所 === 87 === Metabolic byproducts and exogenous compounds can produce oxidative damages to the genomic DNA. Among these damages, 7,8-dihydro-8-oxoguanine (GO) is one of the most stable and abundant products. In Escherichia coli, the GO system which is composed of MutM, MutT, and MutY proteins is responsible for repairing the GO lesion. In this study, we characterized mutY homologues from human (hMYH) and Mycobaterium tuberculosis (TbmutY). The hMYH cDNA has been cloned from a tumor cell line with RT-PCR in this labarotary. This cDNA was subcloned into the expression vector pET32a+ and the recombinant plasmid was transformed into the E. coli strain BL21(DE3)pLysS for overexpression of this protein. This recombinant protein can not be expressed in this host with the different induction conditions that we tried. However, the recombinant hMYH protein can be expressed in the E. coli strain RIL which is specifically engineered for the expression of mammalian genes. The majority of the overexpressed recombinant protein were found in the inclusion body. The recombinant protein was purified to near homogeneity with an affinity nickel column. In vivo complementation assay showed that expression of hMYH in the mutY mutant strain can revert the mutator phenotype of this mutant. Recombinant hMYH lysate isolated from the E. coli mutY strain displayed a glycosylase and an AP lyase activity on heteroduplex DNA containing A/G mismatch. In addition, endogenous hMYH in HeLa cell line was localized to the nucleus as shown by immunoflourescent staining experiment using different hMYH antibodies. This observation is consistent with the result of the transfection experiment with a flag-tagged hMYH. These results suggest that hMYH is involved in removing adenine from the A/G mismatch in the nucleus.
A putative mutY homologue (TbmutY) was identified in the Mycobaterium tuberculosis genome with homology comparison. The TbmutY gene encodes a protein that displays 35.5% similarity to E. coli mutY. It is highly conserved in the [4Fe-4S] cluster and the HhH motif. There are two putative initiation codons in the TbmutY DNA sequence. These two open reading frames were cloned with PCR and subcloned into several pET vectors for overexpression in the mutY-fpg- mutant strain. Only the proteins from the long ORF possessed a DNA glycosylase and an AP lyase activity. These results indicate that mutY throughout evolution is an important repair enzyme and is highly conserved among different organisms.
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