DUF-721 and N-terminal extension of the helicase loader DciA bind ssDNA to promote replicative DnaB helicase loading in Caulobacter crescentus.

The establishment of replication forks relies on a dedicated molecular system by which a ring-shaped replicative DNA helicase is loaded onto the chromosome DNA, facilitating the unwinding of duplex DNA into single-stranded DNA. In most bacteria, the DnaB helicase coevolved with its accessory protein DciA loader, a member of the domain of unknown function (DUF)-721-containing protein family. In the model bacterium Caulobacter crescentus, DciA promotes helicase loading and the C-terminal extension of DUF-721 serves as a specific binding site for the cognate DnaB helicase. However, the mechanistic role of DUF-721 in DnaB helicase loading remains unknown. Here, we provide evidence that the ssDNA binding activity of DUF-721 is crucial for DnaB helicase loading in C. crescentus. Using plasmid complementation assays, we identified DciA Arg106 and Leu119 in DUF-721 as essential residues for in vivo DciA functions. Biochemical analyses revealed that both residues are essential for helicase loading in vitro. Specifically, Arg106 is important for ssDNA binding, with this activity being supported directly or indirectly by Leu119. Yet, these residues are dispensable for DnaB binding. Additionally, we reveal the N-terminal extension of DUE-721 is crucial for ssDNA binding and helicase loading. Given the conservation of Arg106 and Leu119 among DciA family proteins, these results suggest that ssDNA binding via DciA DUF-721 domain plays a specific and conserved role in helicase loading. Providing a molecular insight into how DciA stimulates helicase loading, our findings highlight a conserved mechanism of ssDNA binding among DciA-family proteins.