Programmed Allelic Mutagenesis of a DNA Polymerase with Single Amino Acid Resolution.

ACS Synthetic Biology
Ali NikoomanzarJohn C Chaput

Abstract

Most DNA polymerase libraries sample unknown portions of mutational space and are constrained by the limitations of random mutagenesis. Here we describe a programmed allelic mutagenesis (PAM) strategy to comprehensively evaluate all possible single-point mutations in the entire catalytic domain of a replicative DNA polymerase. By applying the PAM strategy with ultrafast high-throughput screening, we show how DNA polymerases can be mapped for allelic mutations that exhibit enhanced activity for unnatural nucleic acid substrates. We suggest that comprehensive missense mutational scans may aid the discovery of specificity determining residues that are necessary for reprogramming the biological functions of natural DNA polymerases.

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Jul 13, 2019·Journal of the American Chemical Society·Jen-Yu LiaoJohn C Chaput

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Citations

Jul 28, 2020·Quarterly Reviews of Biophysics·Ali NikoomanzarJohn C Chaput
Feb 4, 2021·Accounts of Chemical Research·John C Chaput
May 25, 2021·ACS Synthetic Biology·Esau MedinaJohn C Chaput
Oct 12, 2021·Nucleic Acids Research·Yajun WangJohn C Chaput
Nov 6, 2021·ACS Synthetic Biology·Cailen M McCloskeyJohn C Chaput

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