THERMOSTABLE REPAIR ENZYME FOR OXIDATIVE DNA-DAMAGE FROM EXTREMELY THERMOPHILIC BACTERIUM, THERMUS-THERMOPHILUS HB8

The mutM(fpg) gene, which encodes a DNA glycosylase that excises an ox idatively damaged form of guanine, was cloned from an extremely thermo philic bacterium, Thermus thermophilus HB8. Its nucleotide sequence en coded a 266 amino acid protein with a molecular mass of similar to 30 kDa, Its predicted amino acid sequence showed 42% identity with the Es cherichia coil protein, The amino acid residues Cys, Asn, Gln and Met, known to be chemically unstable at high temperatures, were decreased in number in T.thermophilus MutM protein compared to those of the E.co li one, whereas the number of Pro residues, considered to increase pro tein stability, was increased, The T.thermophilus mutM gene complement ed the mutability of the E.coli mutM mutY double mutant, suggesting th at T.thermophilus MutM protein was active in E.coli. The T.thermophilu s MutM protein was overproduced in E.coli and then purified to homogen eity. Size-exclusion chromatography indicated that T.thermophilus MutM protein exists as a more compact monomer than the E.coli MutM protein in solution, Circular dichroism measurements indicated that the alpha -helical content of the protein was similar to 30%, Thermus thermophil us MutM protein was stable up to 75 degrees C at neutral pH, and betwe en pH 5 and 11 and in the presence of up to 4 M urea at 25 degrees C, Denaturation analysis of T.thermophilus MutM protein in the presence o f urea suggested that the protein had at least two domains, with estim ated stabilities of 8.6 and 16.2 kcal/mol(-1), respectively, Thermus t hermophilus MutM protein showed 8-oxoguanine DNA glycosylase activity in vitro at both low and high temperatures.