title: Characterization of a thermostable DNA glycosylase specific for U/G and T/G mismatches from the hyperthermophilic archaeon Pyrobaculum aerophilum.
abstract: 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.
authors.affiliation: Department of Microbiology and Molecular Genetics and the Molecular Biology Institute, University of California, Los Angeles, California 90095, USA.
authors.forename: H
authors.initials: H
authors.lastname: Yang
authors.forename: S
authors.initials: S
authors.lastname: Fitz-Gibbon
authors.forename: E M
authors.initials: EM
authors.lastname: Marcotte
authors.forename: J H
authors.initials: JH
authors.lastname: Tai
authors.forename: E C
authors.initials: EC
authors.lastname: Hyman
authors.forename: J H
authors.initials: JH
authors.lastname: Miller
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D020137:Base Pair Mismatch; D019757:Carbon-Oxygen Lyases / Q000378:metabolism; D045647:DNA Glycosylases; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D019624:Thermoproteaceae / Q000201:enzymology* / Q000235:genetics; D044502:Thymine DNA Glycosylase; D051981:Uracil-DNA Glycosidase
chemical_list: D019843:Archaeal Proteins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; C024829:8-hydroxyguanine; D006147:Guanine; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D044502:Thymine DNA Glycosylase; D051981:Uracil-DNA Glycosidase; C113921:mismatch-specific thymine uracil-DNA glycosylase; C061738:mutY adenine glycosylase; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase