A novel ψ–χ fusion protein for unravelling the contributions of χ to DNA replication and repair

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Abstract
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 β-clamp loading. The clamp loader accessory subunits χ and 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 χ within DNA pol III HE or on its independent interaction with the YoaA helicase remains unclear. To address this, we engineered ψ–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 ψ–GS12–χ fusion nor expression of a ψχ operon restored AZT tolerance in ΔholC cells, whereas expression of χ alone was sufficient. Fusion expression impaired growth in both wild-type (WT) and ΔholC backgrounds, a phenotype alleviated by disrupting χ–SSB binding. These findings support a model in which χ must dynamically engage SSB and YoaA outside of the clamp loader to promote AZT tolerance, highlighting the importance of regulated χ–SSB interactions in genome maintenance.
Funding Information
  • National Institutes of Health (R01GM140166)
  • National Institutes of General Medical Science (R01GM051753)
  • National Institute of Allergy and Infectious Diseases (T32AI007110)

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