Abstract
Coupling interactions between the alpha (α) subunit of the polymerase III core (α-Pol III core) and the tau (τ) subunit of the clamp loader complex (τ-CLC) are vital for efficient and rapid DNA replication in Escherichia coli. Specific and targeted mutations in the C-terminal τ-interaction region of the Pol III α-subunit disrupted efficient coupled rolling circle DNA synthesis in vitro and caused significant genomic defects in CRISPR-Cas9 dnaE edited cell strains. These α-Pol III mutations eliminated the interaction with τ-CLC but retained WT polymerase and exonuclease activities. The most severely affected mutant strains, dnaE:Y1119A and dnaE:L1097/8S, had significantly reduced doubling times, reduced fitness, and increased cellular length phenotypes as a result of this targeted decoupling of the replisome and the generation of replication stress. Those strains also showed significant SOS induction from unwound but unreplicated regions within the genome. In support, significant ssDNA gaps were detected by fluorescence microscopy and quantified by fluorescence activated cytometry using an in situ PLUG assay for those dnaE:mut strains. By comparing the biochemical and genomic consequences of disrupting the τ-CLC-α-Pol III coupling contacts, we have unveiled a more cohesive picture and mechanistic understanding of replisome dynamics and the essential interactions required to maintain overall fitness through a stable genome.