Tau-mediated coupling between Pol III synthesis and DnaB helicase unwinding helps maintain genomic stability

Abstract

The τ-subunit of the clamp loader complex (CLC) physically interacts with both the DnaB helicase and the polymerase III (Pol III) core α-subunit through Domains IV and V, respectively. This interaction is proposed to help maintain rapid and efficient DNA synthesis rates with high genomic fidelity and plasticity, facilitating enzymatic coupling within the replisome. To test this hypothesis, CRISPR-Cas9 editing was used to create site-directed genomic mutations within the dnaX gene at the C-terminus of τ predicted to interact with the α-subunit of Pol III. Perturbation of the α-τ binding interaction in vivo resulted in cellular and genomic stress markers that included reduced growth rates, fitness, and viabilities. Specifically, dnaX:mut strains showed increased cell filamentation, mutagenesis frequencies, and activated SOS. In situ fluorescence flow cytometry and microscopy quantified large increases in the amount of single-stranded DNA (ssDNA) gaps present. Removal of the C-terminus of τ (I618X) still maintained its interactions with DnaB and stimulated unwinding but lost its interaction with Pol III, resulting in significantly reduced rolling circle DNA synthesis. Intriguingly, dnaX:L635P/D636G had the largest induction of SOS, high mutagenesis, and the most prominent ssDNA gaps, which can be explained by an impaired ability to regulate the unwinding speed of DnaB resulting in a faster rate of in vitro rolling circle DNA replication, inducing replisome decoupling. Therefore, τ stimulated DnaB unwinding and physical coupling with Pol III acts to enforce replisome plasticity to maintain an efficient rate of synthesis and prevent genomic instability.