Nucleases play important roles in all DNA transactions, including replication, repair, and recombination. Many different nucleases from bacterial and eukaryotic organisms have been identified and functionally characterized. However, our knowledge about the nucleases from Archaea, the third domain of life, is still limited. We searched for 3′-5′ exonuclease activity in the hyperthermophilic archaeon, Pyrococcus furiosus, and identified a protein with the target activity. The purified protein, encoded by PF2046, is composed of 229 amino acids with a molecular weight of 25,596, and displayed single-strand specific 3′-5′ exonuclease activity. The protein, designated as PfuExo I, forms a stable trimeric complex in solution and excises the DNA at every two nucleotides from the 3′ to 5′ direction. The amino acid sequence of this protein is conserved only in Thermococci, one of the hyperthermophilic classes in the Euryarchaeota subdomain in Archaea. The newly discovered exonuclease lacks similarity to any other proteins with known function, including hitherto reported 3′-5′ exonucleases. This novel nuclease may be involved in a DNA repair pathway conserved in the living organisms as a specific member for some hyperthermophilic archaea.
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