Biochemical characterization of uracil processing activities in the hyperthermophilic archaeon Pyrobaculum aerophilum

Deamination of cytosine to uracil and 5-methyleytosine 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 similar to 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.