Effect of pH on the Misincorporation Rate of DNA Polymerase η

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Eukaryotic DNA polymerases, crucial for DNA replication and repair, are classified into four families: A, B, X, and Y.
• DNA polymerase eta (Pol η), a Y-family enzyme, plays a role in translesional synthesis and somatic hypermutation, often with high accuracy despite its low fidelity.
• Previous studies suggest Pol η cannot exclude water from its active site, implying environmental factors like ions may affect its function.

Purpose of the Study:

• To investigate the impact of ambient conditions, specifically pH, on the misincorporation rate of DNA polymerase eta.
• To determine if pH influences the accuracy of translesional synthesis mediated by Pol η.

Main Methods:

• Assessed the misincorporation rate of deoxyguanosine triphosphate (dGTP) opposite a template T using a synthetic template-primer with purified DNA polymerase eta.
• Evaluated misincorporation rates across a range of pH levels (6.5-7.5 and 8.0-9.0).
• Performed kinetic analysis to understand the effect of pH on enzyme kinetics, specifically the Km value for dGTP misincorporation.

Main Results:

• The misincorporation rate of dGTP by DNA polymerase eta opposite template T significantly increased at alkaline pH (8.0-9.0) compared to neutral or slightly acidic conditions (6.5-7.5).
• Kinetic analysis demonstrated that pH markedly affected the Km value for dGTP misincorporation, indicating a direct influence on substrate binding or catalytic efficiency.
• Low fidelity DNA polymerase alpha showed no such drastic change in misincorporation rate across the tested pH range.

Conclusions:

• pH is a critical environmental factor that modulates the fidelity of DNA polymerase eta during translesional synthesis.
• The active site's sensitivity to pH may explain variations in Pol η's accuracy observed in different cellular environments.
• Understanding pH-dependent fidelity is crucial for comprehending DNA repair mechanisms and mutation frequencies in immunoglobulin genes and other cellular contexts.