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

• Published in: Journal of bacteriology Vol. 182; no. 5; pp. 1272 - 1279
• Main Authors: Yang, H, Fitz-Gibbon, S, Marcotte, E M, Tai, J H, Hyman, E C, Miller, J H
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 01.03.2000
• Subjects: 5-Methylcytosine; 7,8-Dihydro-oxoguanine; Aeropyrum pernix; Amino Acid Sequence; Amino acids; Archaeal Proteins - genetics; Archaeal Proteins - metabolism; Bacteriology; Base Pair Mismatch; Carbon-Oxygen Lyases - metabolism; cytosine; Deoxyribonuclease IV (Phage T4-Induced); Deoxyribonucleic acid; DNA; DNA glycosylase; DNA Glycosylases; DNA Repair; DNA-(Apurinic or Apyrimidinic Site) Lyase; Enzyme Stability; Enzymes and Proteins; Escherichia coli - genetics; Escherichia coli Proteins; Guanine - analogs & derivatives; Guanine - metabolism; Methanobacterium thermoautotrophicum; Mig protein; Molecular Sequence Data; MutY protein; N-Glycosyl Hydrolases - genetics; N-Glycosyl Hydrolases - metabolism; Nth protein; Phylogeny; Proteins; Pyrobaculum aerophilum; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; Sequence Alignment; Sequence Homology, Amino Acid; Temperature; Thermoproteaceae - enzymology; Thermoproteaceae - genetics; Thymine DNA Glycosylase; Uracil-DNA Glycosidase
• ISSN: 0021-9193; 1098-5530
• DOI: 10.1128/JB.182.5.1272-1279.2000

U/G and T/G mismatches commonly occur due to spontaneous deamination of cytosine and 5-methylcytosine in double-stranded DNA. This mutagenic effect is particularly strong for extreme thermophiles, since the spontaneous deamination 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 reading frame was first identified through a genome sequencing project in our laboratory. The predicted product of 230 amino acids shares significant sequence homology to [4Fe-4S]-containing Nth/MutY DNA glycosylases. The histidine-tagged recombinant protein was expressed in Escherichia coli and purified. It is thermostable and displays DNA glycosylase activities specific to U/G and T/G mismatches with an uncoupled AP lyase activity. It also processes U/7,8-dihydro-oxoguanine and T/7,8-dihydro-oxoguanine mismatches. We designate it Pa-MIG. Using sequence comparisons among complete bacterial and archaeal genomes, we have uncovered a putative MIG protein from another hyperthermophilic archaeon, Aeropyrum pernix. The unique conserved amino acid motifs of MIG proteins are proposed to distinguish MIG proteins from the closely related Nth/MutY DNA glycosylases.