A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Replicative DNA polymerases exhibit varied, polymerase-specific mechanisms for high processivity.
• Generalizing processivity enhancement strategies across different DNA polymerases is challenging.

Purpose of the Study:

• To develop a broadly applicable protein engineering strategy to enhance DNA polymerase processivity.
• To investigate the role of dsDNA binding proteins in improving polymerase function.

Main Methods:

• Covalently linking DNA polymerase domains to a sequence non-specific dsDNA binding protein (Sso7d).
• Utilizing protein engineering and point mutations in Sso7d to assess dsDNA binding's role.
• Evaluating the impact of fusion on polymerase processivity, catalytic activity, and enzyme stability.

Main Results:

• Significant enhancement of processivity in both family A and family B DNA polymerases via fusion with Sso7d.
• Demonstrated that the dsDNA binding capability of Sso7d is crucial for processivity enhancement.
• Fusion enzymes maintained catalytic activity and enzyme stability while exhibiting increased processivity.

Conclusions:

• Heterologous dsDNA binding protein fusion enhances DNA polymerase processivity without compromising enzyme function.
• Increased polymerase processivity is critical for PCR efficiency, with fusion enzymes showing superior performance.
• This technology offers a versatile approach to broadly improve nucleic acid modifying enzymes.