Substrate-induced DNA polymerase β activation.

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• DNA polymerases undergo conformational changes upon binding substrates, influencing DNA synthesis and accuracy.
• Conserved residues in DNA polymerase β are crucial for forming active ternary complexes and catalysis.

Purpose of the Study:

• Investigate the roles of conserved side chains (Asp-192, Arg-258, Phe-272, Glu-295, Tyr-296) in DNA polymerase β.
• Characterize the impact of mutations on nucleotide insertion, fidelity, and conformational activation.

Main Methods:

• Site-directed mutagenesis of conserved residues in DNA polymerase β.
• Kinetic characterization of mutant enzymes.
• X-ray crystallography of binary and ternary complexes.

Main Results:

• Mutations affected catalytic efficiencies for correct nucleotide insertion, with R258A showing increased single-turnover rates.
• Fidelity was modestly affected (<5-fold) across mutants.
• The R258A mutant structure was similar to wild-type, but the E295K mutant revealed an open ternary complex structure.

Conclusions:

• Arg-258 in wild-type DNA polymerase β likely stabilizes non-productive ternary complexes, reducing catalysis.
• The E295K mutation disrupts N-subdomain closure, leading to an open ternary complex that binds metal ions but is catalytically impaired.
• Structural insights into intermediate conformations were gained, particularly for the R258A and E295K mutants.