Engineering a DNA polymerase from Pyrobaculum calidifontis for improved activity, processivity and extension rate

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Positively charged amino acids in DNA polymerase are crucial for DNA interaction.
• The palm domain of DNA polymerases plays a key role in enzyme function.

Purpose of the Study:

• To investigate the role of specific residues in the palm domain of Pca-Pol.
• To engineer a Pca-Pol mutant with improved enzymatic properties for potential applications.

Main Methods:

• Site-directed mutagenesis was used to introduce arginine substitutions (G522R, E555R) in Pca-Pol.
• Mutant proteins were expressed heterologously in Escherichia coli.
• Biochemical characterization assessed enzyme activity, processivity, extension rate, and fidelity.

Main Results:

• Mutant Pca-Pol enzymes showed no significant differences in pH, metal ion, or buffer preferences compared to wild-type.
• 3'-5' exonuclease activity and error rates remained comparable between wild-type and mutant enzymes.
• Specific activity, processivity, and extension rate were significantly increased in the mutant enzymes, with G522R-E555R exhibiting a nearly 9-fold higher specific activity.

Conclusions:

• The identified mutations (G522R-E555R) in Pca-Pol enhance key enzymatic properties without compromising fidelity.
• Engineered Pca-Pol mutants, particularly G522R-E555R, are promising candidates for biotechnological and commercial applications requiring high DNA polymerase performance.