
Characterization of two DNA polymerases from the hyperthermophilic euryarchaeon Pyrococcus abyssi
Author(s) -
Gueguen Yannick,
Rolland Jeanluc,
Lecompte Odile,
Azam Philippe,
Le Romancer Gisèle,
Flament Didier,
Raffin JeanPaul,
Dietrich Jacques
Publication year - 2001
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.0014-2956.2001.02550.x
Subject(s) - dna polymerase , dna polymerase ii , biology , polymerase , microbiology and biotechnology , processivity , dna polymerase mu , rna polymerase iii , dna polymerase i , genetics , exonuclease , dna , gene , circular bacterial chromosome , reverse transcriptase , polymerase chain reaction , rna dependent rna polymerase
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 ≈ 90 kDa, as estimated by SDS/PAGE. The optimum pH and Mg 2+ concentration of Pol I were 8.5–9.0 and 3 m m , 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 m m , 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 .