A pre-equilibrium before nucleotide binding limits fingers subdomain closure by Klentaq1

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• DNA polymerases are crucial for DNA replication and repair.
• Nucleotide selection is a key step in DNA synthesis, involving a rate-limiting step before chemical bond formation.
• Conformational changes in DNA polymerases, particularly the fingers subdomain, are implicated in nucleotide selection.

Purpose of the Study:

• To investigate the role of DNA polymerase conformational changes in nucleotide selection.
• To characterize the kinetics of DNA binding and nucleotide incorporation by Klentaq1.
• To elucidate the influence of the templating base on the initial selection of deoxynucleoside triphosphates (dNTPs).

Main Methods:

• Utilized a fluorescence resonance energy transfer (FRET) system to monitor the open-to-closed conformational transition of the Klentaq1 fingers subdomain.
• Performed kinetic experiments to measure DNA binding and dNTP incorporation rates.
• Analyzed the effect of different templating bases on these processes.

Main Results:

• Fingers subdomain closure in Klentaq1 is faster than the rate-limiting step for nucleotide incorporation.
• DNA binding occurs via a two-step association, with slower dissociation from closed ternary complexes.
• A distinct pre-binding step for nucleotide incorporation was identified, varying for each of the four dNTPs.

Conclusions:

• The templating base plays a significant role in the initial selection of dNTPs, likely influencing the ground state of the enzyme-ternary complex.
• The conformational dynamics of the fingers subdomain are not the sole determinant of the rate-limiting step in dNTP selection.
• Understanding these mechanisms is vital for comprehending DNA replication fidelity and developing polymerase inhibitors.