sb0c00236_si_001.pdf (2.87 MB)
Programmed Allelic Mutagenesis of a DNA Polymerase with Single Amino Acid Resolution
journal contribution
posted on 2020-06-30, 15:07 authored by Ali Nikoomanzar, Derek Vallejo, Eric J. Yik, John C. ChaputMost 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.