Combined Solution and Crystal Methods Reveal the Electrostatic Tethers That Provide a Flexible Platform for Replication Activities in the Bacteriophage T7 Replisome

We use Illumina sequencing to monitor mutations in the bacteriophage T7 genome in the presence of T7 DNA polymerase that has an altered exonuclease active site. These alterations include mutation of key residues in the exonuclease active site. Bacteriophage T7 that is missing the gene 5 DNA polymerase (T7Δ5) was passaged ten times in the presence of either wild-type or exonuclease-deficient T7 DNA polymerase provided on a plasmid DNA. The genomic DNA from the passaged virus was purified, sequenced, then compared to a T7 genome deposited in GenBank (NC_001604.1-2). Mutations that accumulated during the passages were mapped to specific regions of the bacteriophage genome. We also sequenced our starting passage zero virus as a reference control.