title: Biochemical characterization of uracil processing activities in the hyperthermophilic archaeon Pyrobaculum aerophilum.
abstract: Deamination of cytosine to uracil and 5-methylcytosine to thymine represents a major mutagenic threat particularly at high temperatures. In double-stranded DNA, these spontaneous hydrolytic reactions give rise to G.U and G.T mispairs, respectively, that must be restored to G.C pairs prior to the next round of DNA replication; if left unrepaired, 50% of progeny DNA would acquire G.C --> A.T transition mutations. The genome of the hyperthermophilic archaeon Pyrobaculum aerophilum has been recently shown to encode a protein, Pa-MIG, a member of the endonuclease III family, capable of processing both G.U and G.T mispairs. We now show that this latter activity is undetectable in crude extracts of P. aerophilum. However, uracil residues in G.U mispairs, in A.U pairs, and in single-stranded DNA were efficiently removed in these extracts. These activities were assigned to a approximately 22-kDa polypeptide named Pa-UDG (P. aerophilum uracil-DNA glycosylase). The recombinant Pa-UDG protein is highly thermostable and displays a considerable degree of homology to the recently described uracil-DNA glycosylases from Archaeoglobus fulgidus and Thermotoga maritima. Interestingly, neither Pa-MIG nor Pa-UDG was inhibited by UGI, a generic inhibitor of the UNG family of uracil glycosylases. Yet a small fraction of the total uracil processing activity present in crude extracts of P. aerophilum was inhibited by this peptide. This implies that the hyperthermophilic archaeon possesses at least a three-pronged defense against the mutagenic threat of hydrolytic deamination of cytosines in its genomic DNA.
authors.affiliation: Institute of Medical Radiobiology, University of Zürich and the Paul Scherrer-Institute, August Forel-Strasse 7, CH-8008 Zürich, Switzerland.
authors.forename: A A
authors.initials: AA
authors.lastname: Sartori
authors.forename: P
authors.initials: P
authors.lastname: Schär
authors.forename: S
authors.initials: S
authors.lastname: Fitz-Gibbon
authors.forename: J H
authors.initials: JH
authors.lastname: Miller
authors.forename: J
authors.initials: J
authors.lastname: Jiricny
mesh_terms: D000595:Amino Acid Sequence; D001105:Archaea / Q000378:metabolism*; D019843:Archaeal Proteins; D003001:Cloning, Molecular; D003596:Cytosine / Q000378:metabolism; D045647:DNA Glycosylases; D004277:DNA, Single-Stranded / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism; D004720:Endonucleases / Q000737:chemistry* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000494:pharmacology; D010802:Phylogeny; D010957:Plasmids / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors; D014498:Uracil / Q000378:metabolism*; D051981:Uracil-DNA Glycosidase; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D004277:DNA, Single-Stranded; D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C027619:uracil-DNA glycosylase inhibitor protein, B. subtilis bacteriophage; D014498:Uracil; D003596:Cytosine; D004706:Endodeoxyribonucleases; D004720:Endonucleases; C434059:Pa-MIG protein, Pyrobaculum aerophilum; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase