Nucleases play important roles in nucleic acid processes, such as replication, repair and recombination.Recently, we identified a novel single-strandspecific 3′-5′ exonuclease, PfuExo I, from the hyperthermophilicarchaeon Pyrococcus furiosus, whichmay be involved in the Thermococcales-specific DNArepair system. PfuExo I forms a trimer and cleavessingle-stranded DNA at every two nucleotides. Here, we report the structural basis for the cleavage mechanismof this novel exonuclease family. A structuralanalysis of PhoExo I, the homologous enzyme from P. horikoshii OT3, showed that PhoExo I utilizesan RNase H-like active site and possesses a 3′-OHrecognition site ∼9 Å away from the active site, whichenables cleavage at every two nucleotides. Analysesof the heterotrimeric and monomeric PhoExo I activitiesshowed that trimerization is indispensable for itsprocessive cleavage mechanism, but only one active site of the trimer is required.
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