Characterization of the full length uracil-DNA glycosylase in the extreme thermophile Thermotoga maritima

A full length (192 amino acids) uracil-DNA glycosylase (TMUDG) has been exp ressed and purified from the extreme thermophile Thermotoga maritima. This protein is active up to 85 degreesC. The enzyme is product inhibited by aba sic sites in DNA and weakly inhibited by uracil. TMUDG was originally clone d from an ORF which encoded a protein of 185 amino acids. This shorter prot ein was stable up to 70-75 degreesC and it seemed unusual that this enzyme had an optimal activity temperature below the growth temperature of the org anism (80-90 degreesC). Following the publication of the complete genomic s equence of T. maritima, it was shown that the gene contains an additional s even amino acids (LYTREEL) at the N-terminal end of the protein. It is sugg ested that these seven residues are important in maintaining proper protein folding that results in increased temperature stability. We have also demo nstrated that TMUDG can substitute for the Escherichia coli uracil-DNA glyc osylase and initiate base excision repair using a closed circular DNA subst rate containing a unique U:G base pair. (C) 2001 Elsevier Science B.V. All rights reserved.