title: A thermostable endonuclease III homolog from the archaeon Pyrobaculum aerophilum.
abstract: Pyrimidine adducts in cellular DNA arise from modification of the pyrimidine 5,6-double bond by oxidation, reduction or hydration. The biological outcome includes increased mutation rate and potential lethality. A major DNA N:-glycosylase responsible for the excision of modified pyrimidine bases is the base excision repair (BER) glycosylase endonuclease III, for which functional homologs have been identified and characterized in Escherichia coli, yeast and humans. So far, little is known about how hyperthermophilic Archaea cope with such pyrimidine damage. Here we report characterization of an endonuclease III homolog, PaNth, from the hyperthermophilic archaeon Pyrobaculum aerophilum, whose optimal growth temperature is 100 degrees C. The predicted product of 223 amino acids shares significant sequence homology with several [4Fe-4S]-containing DNA N:-glycosylases including E.coli endonuclease III (EcNth). The histidine-tagged recombinant protein was expressed in E.coli and purified. Under optimal conditions of 80-160 mM NaCl and 70 degrees C, PaNth displays DNA glycosylase/ss-lyase activity with the modified pyrimidine base 5,6-dihydrothymine (DHT). This activity is enhanced when DHT is paired with G. Our data, showing the structural and functional similarity between PaNth and EcNth, suggests that BER of modified pyrimidines may be a conserved repair mechanism in Archaea. Conserved amino acid residues are identified for five subfamilies of endonuclease III/UV endonuclease homologs clustered by phylogenetic analysis.
authors.affiliation: Department of Microbiology, IGPP Center for Astrobiology, University of California, Los Angeles, CA 90095, USA.
authors.forename: H
authors.initials: H
authors.lastname: Yang
authors.forename: I T
authors.initials: IT
authors.lastname: Phan
authors.forename: S
authors.initials: S
authors.lastname: Fitz-Gibbon
authors.forename: M K
authors.initials: MK
authors.lastname: Shivji
authors.forename: R D
authors.initials: RD
authors.lastname: Wood
authors.forename: W M
authors.initials: WM
authors.lastname: Clendenin
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; D019757:Carbon-Oxygen Lyases / Q000378:metabolism; D045647:DNA Glycosylases; D004274:DNA, Recombinant / Q000235:genetics; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043224:Deoxyribonuclease (Pyrimidine Dimer); D043223:Deoxyribonuclease IV (Phage T4-Induced); D004305:Dose-Response Relationship, Drug; D004706:Endodeoxyribonucleases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000378:metabolism; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D019624:Thermoproteaceae / Q000201:enzymology*
chemical_list: D004274:DNA, Recombinant; D029968:Escherichia coli Proteins; D009841:Oligonucleotides; D012965:Sodium Chloride; D004706:Endodeoxyribonucleases; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase