Enterobacteria phage T7 DNA Polymerase I point mutation greatly affects enzyme activity

• Owen Donnelly, Biological Sciences, University of Delaware

• K. Wommack, Plant and Soil Sciences, University of Delaware

Viruses are the most abundant biological entities on the planet. DNA polymerase I (polA) is present in 25% of dsDNA viruses that infect bacteria (phages) and is the sole replication enzyme for these phages. The 762 residue (E. coli numbering) is responsible for dNTP incorporation, and amino acid substitutions at this single position can have profound effects on enzyme replication speed and accuracy. When compared to the phenylalanine (Phe) amino acid (wild type in E. coli), tyrosine (Tyr) substitutions increase enzyme speed and are associated with lytic viral life cycles, while leucine (Leu) substitutions greatly increase fidelity at the expense of speed and are associated with temperate life cycles. Mutagenesis experiments on Enterobacteria phage T7 PolA, which originally possessed a tyrosine 762 residue (526 in T7 numbering), produced two mutants; one with a phenylalanine 526 residue, and another with a leucine 526 residue. Strengthening the connection between the 762 residue and the biochemical characteristics of polA can improve life-cycle predictions of mutated phages. This study sought to investigate how the 526 residue of T7 PolA impacts enzyme biochemistry and ultimately predicts phage phenotype. After producing and purifying all three T7 PolA stocks (Tyr, Phe, Leu), contaminating nucleases such as DNase and RNase were found to be negligible enough to not affect DNA manipulation. Primer extension activity was observed in a wide range of temperatures for T7 Phe, while T7 Leu primer extension was weak or absent at all temperatures tested. Enzyme speed differed greatly between the two mutant T7 enzymes; T7 Phe was similar to that of the wild type, while T7 Leu was around 27x slower. The difference in performance between these mutants and the wild type were consistent with the change in amino acid structure; Phe substitution is a minor structural difference while Leu is a substantial change in structure.