Characterization of two DNA polymerases from the hyperthermophilic euryarchaeon Pyrococcus abyssi

Area of Science:

• Molecular Biology
• Biochemistry
• Genomics

Background:

• The genome of the hyperthermophilic archaeon Pyrococcus abyssi encodes two distinct DNA polymerases: family B (Pol I) and family D (Pol II).
• Understanding euryarchaeal DNA polymerases is crucial for comprehending DNA replication mechanisms in archaea.

Purpose of the Study:

• To clone, express, and purify Pyrococcus abyssi DNA polymerases I and II.
• To biochemically characterize these enzymes, including their optimal reaction conditions and associated activities.
• To investigate the structural and functional relationship between family D DNA polymerase and Mre11 nucleases.

Main Methods:

• Gene cloning and expression in Escherichia coli.
• Protein purification to homogeneity using standard biochemical techniques.
• Biochemical characterization including pH and Mg(2+) optima determination.
• Sequence analysis to identify conserved domains and catalytic residues.

Main Results:

• Purified Pol I (90 kDa) showed optimal activity at pH 8.5-9.0 and 3 mM Mg(2+).
• Purified Pol II, a two-subunit enzyme, had optimal activity at pH 6.5 and 15-20 mM Mg(2+).
• Both Pol I and Pol II possess 3'-->5' exonuclease activity, despite the absence of canonical motifs in Pol II.
• Sequence analysis suggested the phosphoesterase domain of the Pol II small subunit is responsible for its exonuclease activity, sharing conserved catalytic residues with Mre11 nucleases.

Conclusions:

• The characterization of P. abyssi Pol I and Pol II provides significant insights into euryarchaeal DNA polymerase function.
• The conserved phosphoesterase domain in family D DNA polymerase suggests a novel mechanism for its exonuclease activity.
• These findings contribute to a comprehensive understanding of DNA replication in Pyrococcus abyssi and related archaea.