A DNA ligase from a hyperthermophilic archaeon with unique cofactor specificity

Area of Science:

• Molecular Biology
• Biochemistry
• Extremophile Research

Background:

• DNA ligases are crucial enzymes for DNA replication, repair, and recombination.
• Understanding DNA ligases from hyperthermophilic archaea provides insights into enzyme stability and function at extreme temperatures.
• The DNA ligase from Thermococcus kodakaraensis KOD1 (lig(Tk)) has not been biochemically characterized.

Purpose of the Study:

• To clone, sequence, and characterize the DNA ligase (lig(Tk)) from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1.
• To investigate the enzymatic properties, including optimal conditions and cofactor specificity, of recombinant Lig(Tk).
• To determine the phylogenetic relationship of Lig(Tk) with other known DNA ligases.

Main Methods:

• Gene cloning and sequencing of lig(Tk) from Thermococcus kodakaraensis KOD1.
• Expression of recombinant Lig(Tk) in Escherichia coli and subsequent protein purification.
• Biochemical assays to determine DNA ligase activity, optimal pH, Mg(2+), and K(+) concentrations, and cofactor specificity (ATP and NAD(+)).
• Phylogenetic analysis using conserved motifs and sequence comparison with known DNA ligases.

Main Results:

• The lig(Tk) gene encodes a 562-amino acid polypeptide with a predicted molecular mass of 64,079 Da.
• Recombinant Lig(Tk) exhibited DNA ligase activity in the presence of ATP and Mg(2+), with optimal activity at pH 8.0, 14-18 mM Mg(2+), and 10-30 mM K(+).
• Lig(Tk) demonstrated significant DNA ligase activity even at 100°C and unexpectedly showed activity with NAD(+) as a cofactor, indicating unique cofactor specificity.

Conclusions:

• Lig(Tk) is an ATP-dependent DNA ligase from a hyperthermophilic archaeon, closely related to other archaeal DNA ligases.
• The enzyme is stable and active at high temperatures (100°C) and exhibits unique cofactor flexibility, functioning with both ATP and NAD(+).
• This study represents the first biochemical characterization of a DNA ligase from a hyperthermophilic archaeon, offering valuable insights into extremolytes and enzyme engineering.