Characterization of two DNA polymerases from the hyperthermophilic euryarchaeon Pyrococcus abyssi

The complete genome sequence of the hyperthermophilic archaeon Pyrococcus a byssi revealed the presence of a family B DNA polymerase (Pol I) and a fami ly D DNA polymerase (Pol II). To extend our knowledge about euryarchaeal DN A polymerases, we cloned the genes encoding these two enzymes and expressed them in Escherichia coli. The DNA polymerases (Pol I and Pol II) were puri fied to homogeneity and characterized. Pot I had a molecular mass of approx imate to 90 kDa, as estimated by SDS/PAGE. The optimum pH and Mg2+ concentr ation of Pol I were 8.5-9.0 and 3 mM, respectively. Pol II is composed of t wo subunits that are encoded by two genes arranged in tandem on the P. abys si genome. We cloned these genes and purified the Pol II DNA polymerase fro m an E. coli strain coexpressing the cloned genes. The optimum pH and Mg2concentration of Pol II were 6.5 and 15-20 mM, respectively. Both P. abyssi Pol I and Pol II have associated 3'-->5' exonuclease activity although the exonuclease motifs usually found in DNA polymerases are absent in the arch aeal family D DNA polymerase sequences. Sequence analysis has revealed that the small subunit of family D DNA polymerase and the Mre11 nucleases belon g to the calcineurin-like phosphoesterase superfamily and that residues inv olved in catalysis and metal coordination in the Mre11 nuclease three-dimen sional structure are strictly conserved in both fan-lilies. One hypothesis is that the phosphoesterase domain of the small subunit is responsible for the 3'-->5' exonuclease activity of family D DNA polymerase. These results increase our understanding of euryarchaeal DNA polymerases and are of impor tance to push forward the complete understanding of the DNA replication in P. abyssi.