Characterization of two DNA polymerases from the hyperthermophilic euryarchaeon Pyrococcus abyssi.
The complete genome sequence of the hyperthermophilic archaeon Pyrococcus abyssi revealed the presence of a family B DNA polymerase (Pol I) and a family D DNA polymerase (Pol II). To extend our knowledge about euryarchaeal DNA polymerases, we cloned the genes encoding these two enzymes and expressed them in Escherichia coli. The DNA polymerases (Pol I and Pol II) were purified to homogeneity and characterized. Pol I had a molecular mass of approximately 90 kDa, as estimated by SDS/PAGE. The optimum pH and Mg(2+) concentration of Pol I were 8.5-9.0 and 3 mm, respectively. Pol II is composed of two subunits that are encoded by two genes arranged in tandem on the P. abyssi genome. We cloned these genes and purified the Pol II DNA polymerase from an E. coli strain coexpressing the cloned genes. The optimum pH and Mg(2+) concentration 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 archaeal family D DNA polymerase sequences. Sequence analysis has revealed that the small subunit of family D DNA polymerase and the Mre11 nucleases belong to the calcineurin-like phosphoesterase superfamily and that residues involved in catalysis and metal coordination in the Mre11 nuclease three-dimensional structure are strictly conserved in both families. 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 importance to push forward the complete understanding of the DNA replication in P. abyssi.
Gueguen
Y
Y
IFREMER, Centre de Brest, DRV-VP-LBMH, Plouzané, France. ygueguen@ifremer.fr
Rolland
J L
JL
Lecompte
O
O
Azam
P
P
Le Romancer
G
G
Flament
D
D
Raffin
J P
JP
Dietrich
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D019711:Pyrococcus / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D004259:DNA-Directed DNA Polymerase