Characterization of a thermostable DNA glycosylase specific for U/G and T/G mismatches from the hyperthermophilic archaeon Pyrobaculum aerophilum

U/G and T/G mismatches commonly occur due to spontaneous deamination of cyt osine and 5-methylcytosine in double-stranded DNA. This mutagenic effect is particularly strong for extreme thermophiles, since the spontaneous deamin ation reaction is much enhanced at high temperature. Previously, a U/G and T/G mismatch-specific glycosylase (Mth-MIG) was found on a cryptic plasmid of the archaeon Methanobacterium thermoautotrophicum, a thermophile with an optimal growth temperature of 65 degrees C. We report characterization of a putative DNA glycosylase from the hyperthermophilic archaeon Pyrobaculum aerophilum, whose optimal growth temperature is 100 degrees C. The open rea ding frame was first identified through a genome sequencing project in our laboratory. The predicted product of 230 amino acids shares significant seq uence homology to [4Fe-4S]-containing Nth/MutY DNA glycosylases. The histid ine-tagged recombinant protein was expressed in Escherichia coli and purifi ed. It is thermostable and displays DNA glycosylase activities specific to U/G and TIG mismatches with an uncoupled AP lyase activity. It also process es U/7,8-dihydro-oxoguanine and T/7,8-dihydro-oxoguanine mismatches. We des ignate it Pa-MIG. Using sequence comparisons among complete bacterial and a rchaeal genomes, we have uncovered a putative MIG protein from another hype rthermophilic archaeon, Aeropyrum pernix. The unique conserved amino acid m otifs of MIG proteins are proposed to distinguish MIG proteins from the clo sely related Nth/MutY DNA glycosylases.