Reconstructed evolutionary adaptive paths give polymerases accepting reversible terminators for sequencing and SNP...

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Understanding biological systems requires analyzing both history and structure.
• DNA polymerases are crucial enzymes in molecular biology, with applications in diagnostics and research.
• Existing DNA sequencing methods often rely on chain-terminating nucleotides.

Purpose of the Study:

• To engineer DNA polymerases capable of accepting a new class of reversible terminators (dNTP-ONH2s).
• To explore the relationship between protein structure, evolutionary history, and enzyme function.
• To develop novel tools for DNA analysis, including parallel sequencing and SNP detection.

Main Methods:

• Combined Reconstructed Evolutionary Adaptive Path (REAP) analysis with structural analyses of DNA polymerases.
• Utilized evolutionary and functional history to guide amino acid replacements.
• Created and screened a library of Taq polymerase variants for improved substrate acceptance.
• Employed crystallographic and modeling techniques to understand enzyme-substrate interactions.

Main Results:

• Identified 35 potential amino acid sites for modification in Taq polymerase.
• Generated 93 Taq variants, with eight showing improved acceptance of dNTP-ONH2 substrates.
• Developed a Taq variant (E520G, K540I, L616A) that efficiently and faithfully incorporates both dNTP-ONH2s and ddNTPs.
• Discovered a novel mutation (L616A) enabling the incorporation of both reversible and irreversible terminators.

Conclusions:

• Evolutionary and structural analyses are powerful tools for protein engineering.
• The engineered Taq polymerases provide new capabilities for DNA analysis.
• The developed extension-cleavage-extension strategy has significant implications for DNA sequencing and SNP detection.