Single amino acid substitution in DNA Polymerase I dramatically alters infection dynamics of bacteriophage T7

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ABSTRACT Viruses constitute a significant proportion of Earth’s genetic diversity, yet most remain uncharacterized beyond their sequences in viral metagenomes. Linking viral genotypes to phenotypes—especially enzyme function to phage infection dynamics—is challenging due to the lack of cultured virus–host systems. DNA polymerase I (PolA), essential for genome replication in ∼25% of dsDNA phages, provides an opportunity to explore these connections. In phage T7, residue 526 is critical for nucleotide incorporation, with previous in vitro evidence indicating impacts on enzyme efficiency and fidelity. Previous analyses identified three substitutions at this position (Tyr/Y, Phe/F, Leu/L) linked with deeply rooted viral PolA clades. Mutation impacts at residue 526 were tested in vitro and in vivo . The Y526F protein exhibited a 50% reduction in specific activity, and when introduced via High Complexity Golden Gate Assembly into T7 demonstrated a 53% decrease in burst size and significantly longer latent period compared to wild type. The Y526L protein exhibited a 97% decrease in activity, and the Y526L phage was incapable of completing its lifecycle. These findings confirm historical biochemical data, provide in vivo context for these mutations in the T7– E. coli system, and offer experimental support for genotype-to-phenotype associations in viral PolA, informing viral metagenomics studies. GRAPHICAL ABSTRACT Created in BioRender. Keown, R. (2026) https://BioRender.com/mhrmup3

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