Modulating the DNA polymerase β reaction equilibrium to dissect the reverse reaction

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• DNA polymerases synthesize DNA with high fidelity.
• DNA polymerases also catalyze pyrophosphorolysis, a reverse reaction that removes DNA primer termini.
• Pyrophosphorolysis impacts polymerase fidelity and sensitivity to chain-terminating nucleosides.

Purpose of the Study:

• To analyze the pyrophosphorolysis reaction catalyzed by human DNA polymerase β.
• To investigate the factors influencing the rate and mechanism of pyrophosphorolysis.

Main Methods:

• Enzyme kinetics assays were performed using human DNA polymerase β.
• Thio-elemental effect studies were conducted to probe the rate-determining step.
• A pyrophosphate analog (PNP) was used to study the effect of the bridging atom.
• Time-lapse crystallography was employed to capture enzyme-intermediate structures.

Main Results:

• Human DNA polymerase β exhibited inefficient pyrophosphorolysis.
• A lack of thio-elemental effect indicated a non-chemical rate-limiting step.
• Use of the PNP analog accelerated the reverse reaction and showed a significant thio-elemental effect, suggesting chemistry became rate-determining.
• Crystallography revealed structures favoring the reverse reaction equilibrium with PNP.

Conclusions:

• The bridging atom between the β- and γ-phosphates is crucial for DNA polymerase reaction chemistry.
• Enzyme conformational changes and overall reaction equilibrium are influenced by this bridging atom.
• Understanding pyrophosphorolysis provides insights into DNA polymerase mechanisms and fidelity.