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Engineering a Replicative DNA Polymerase for Accurate Bypass of Damaged DNA

NCJ Number
Date Published
8 pages
Since 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, the research reported in this article focuses on the common oxidative DNA lesion 8-oxoguanine, a small modification yet often mutagenic.
Enzyme engineering seeks to create an enzyme, either de novo or by modification of a known protein, with a new desired function. While biocatalysis has advantages over conventional catalytic processes, there most often are not natural enzymes with the desired properties many commercial applications. For example, many DNA polymerases can replicate DNA accurately at high speeds but are inhibited by bases damaged by environmental mutagens, oxidative stress, or UV light. 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 efficient, even on undamaged DNA. This accuracy tradeoff allows genomic replication, and life, to continue, but can also contribute to antibiotic resistance or oncogenesis. The additional hydrogen bond donor allows formation of a Hoogsteen pair with adenosine when 8-oxo-dG resides in the syn conformation. To identify variants for accurate bypass, multiple criteria were used to identify positive mutations. Residue scanning was used to assess mutations effects on affinity towards both the preferred anti conformation and the mutagenic syn conformation. The researchers' computational method THEMATICS was then used to filter out mutations that affect the electrostatic properties of catalytic residues, and therefore are more likely to affect the polymerase activity negatively. These methods are being utilized to identify specific variants for biochemical characterization, including thermal stability, catalytic activity, lesion bypass capability, and fidelity. (publisher abstract modified)

Date Published: January 1, 2018