DnaB mutants suppress DciA essentiality in Thermus thermophilus and uncover its role in recombination-dependent replication

Bacterial DNA replication is initiated by assembling two hexameric replicative DnaB helicases at the DnaA-unwound oriC region, a process that requires a cognate helicase loader, DciA, DnaC, or DnaI. Members of the phylum Deinococcota encode a DciA-type helicase loader composed of an N-terminal DUF721 domain, an intermediate lasso-like region (IR), and two specific domains: a Zn-finger (ZnF) and a replication-recombination domain (RRD). Here, we show that deletion of the Thermus thermophilus dciA gene is lethal, but viability is restored by a dnaB M375I extragenic suppressor. DnaB M375I expression is necessary and sufficient to sustain replication in otherwise wild-type Δ dciA cells. In vitro, DciA interacts with DnaB and its mutant variants. DnaB efficiently unwinds 3’-forked DNA and this activity is restrained by DciA through inhibition of DnaB’s ATPase and helicase functions. Cells lacking DciA, or specifically the RRD, fail to grow at the optimal temperature (70 °C) but grow at lower temperatures (60–65 °C). Notably, the combined loss of the RRD and the recombination proteins RecO or RecR is lethal. Together, these results establish DciA as the replicative helicase loader in Deinococcota and reveal a functional link between DNA replication and homologous recombination.