Enzyme engineering seeks to create an enzyme, either de novo or by modification of a known protein, with a new desired function. Biocatalysis has advantages over conventional catalytic processes, but there are not always natural enzymes with the desired properties and activity. For example, many DNA polymerases can replicate DNA accurately at high speeds but can be inhibited by bases that have been damaged by environmental mutagens, oxidative stress, or UV light. Depending on the type of damage, polymerases may not be able to insert nucleotides opposite a lesion or may do so with reduced accuracy. Specialized polymerases may be well suited to bypass the damage, but are typically less accurate and less efficient, even on undamaged DNA. This accuracy tradeoff allows genomic replication, and life, to continue, but can contribute to problems such as antibiotic resistance or oncogenesis.<br/>A hybrid DNA polymerase that retains the accuracy and speed of replicative polymerases, while incorporating specific lesion-bypass abilities, would be a useful biochemical tool. This research focuses on the common oxidative DNA lesion 8-oxoguanine, which is quite mutagenic despite the small size of the damage. The additional hydrogen bond donor allows formation of a Hoogsteen pair with adenosine when 8-oxo-dG resides in the syn conformation. Molecular dynamics and docking are being utilized to understand the lesion conformation and relative fidelity of model polymerases and engineered variants. Our computational methods, THEMATICS and POOL, identify functional residues and assess whether variants retain the electrostatic properties of the natural polymerase for catalysis. The combination of these computational methods is being used to identify specific variants for biochemical characterization, including thermal stability, catalytic activity, lesion bypass capability, and fidelity. Supported by NSF-MCB-1517290 and the National Institute of Justice.

Engineering a replicative DNA polymerase for specific damage bypass capability

254th National Meeting (2017)

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