A Novel ψ - χ Fusion Protein for Unravelling the Contributions of χ to DNA Replication and Repair

Faithful DNA replication in Escherichia coli requires the DNA polymerase III holoenzyme (DNA pol III HE) and its clamp loader complex, which couples processive DNA synthesis with $\text{β}$ -clamp loading. The clamp loader accessory subunits $\text{χ}$ and $\text{ψ}$ link single-stranded DNA-binding protein (SSB) to the replisome, stabilizing replication on SSB-coated templates through interactions with the SSB C-terminal tail. Chi has also been implicated in tolerance to the chain-terminating nucleoside analog azidothymidine (AZT), though whether this function depends on $\text{χ}$ within DNA pol III HE or on its independent interaction with the YoaA helicase remains unclear. To address this, we engineered $\text{ψ}-\text{χ}$ fusion proteins with flexible glycine-serine linkers to tether the two subunits while preserving folding and activity. Both fusions were biochemically competent, supporting ATP hydrolysis and clamp loading on SSB-coated DNA. In vivo, however, neither the $\text{ψ}$ -GS12- $\text{χ}$ fusion nor expression of a $\text{ψχ}$ operon restored AZT tolerance in ΔholC cells, whereas expression of $\text{χ}$ alone was sufficient. Fusion expression impaired growth in both wild-type (WT) and ΔholC backgrounds, a phenotype alleviated by disrupting $\text{χ}$ -SSB binding. These findings support a model in which $\text{χ}$ must dynamically engage SSB and YoaA outside of the clamp loader to promote AZT tolerance, highlighting the importance of regulated $\text{χ}$ -SSB interactions in genome maintenance.