Enzymes have applications in a wide array of fields due to their ability to catalyze specific reactions at high rates. Enzymes have advantages over conventional catalytic processes, but the lack of natural enzymes that have the desired capabilities limits their use. Enzyme engineering seeks to create an enzyme, either <i>de novo</i> or as a modification of a known protein, with the desired function. In this work, engineering efforts are focused on developing a high-fidelity, damage-tolerant DNA polymerase for replication of damaged DNA, with an eye toward forensic applications. While DNA polymerases can typically replicate DNA at high speed and low error rates, damage to the bases can cause problems for these replicative polymerases, either by halting their progress or increasing the likelihood of misincorporation. Since DNA damage is ubiquitous, organisms have developed specialized polymerases to bypass damaged bases and allow for replication to continue. However, they can help drive mutagenesis as their bypasss ability is associated with a decrease in fidelity. A polymerase that can retain the accuracy and speed of the replicative polymerases, while incorporating the lesion bypass ability of others, would be a useful biochemical tool. Computational methods are being utilized to understand the fidelity of the natural polymerase that is our starting point for engineering and its engineered variants against the DNA lesion, specifically through molecular dynamics and docking to analyze conformations and binding modes. Our computational method, THEMATICS, helps identify which residues are contributing to the activity of the polymerase and whether variants retain the electrostatic properties of the wild-type polymerase that provide conditions for catalysis. The combination of these computational methods are being used to identify specific variants for biochemical characterization, such as thermal stability, catalytic activity, and fidelity on both undamaged and damaged DNA substrates. Supported by NSF-MCB-1517290 and the National Institute of Justice.

Engineering a polymerase for accurate bypass of damaged DNA samples

253rd National Meeting (2017)

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