Study of the DnaB:DciA interplay reveals insights into the primary mode of loading of the bacterial replicative helicase

Abstract

Replicative helicases are essential proteins that unwind DNA in front of replication forks. Their loading depends on accessory proteins and in bacteria, DnaC and DnaI are well characterized loaders. However, most bacteria do not express either of these two proteins. Instead, they are proposed to rely on DciA, an ancestral protein unrelated to DnaC/I. While the DciA structure from Vibrio cholerae shares no homology with DnaC, it reveals similarities with DnaA and DnaX, two proteins involved during replication initiation. As other bacterial replicative helicases, VcDnaB adopts a toroid-shaped homo-hexameric structure, but with a slightly open dynamic conformation in the free state. We show that VcDnaB can load itself on DNA in vitro and that VcDciA stimulates this function, resulting in an increased DNA unwinding. VcDciA interacts with VcDnaB with a 3/6 stoichiometry and we show that a determinant residue, which discriminates DciA- and DnaC/I-helicases, is critical in vivo. Our work is the first step toward the understanding of the ancestral mode of loading of bacterial replicative helicases on DNA. It sheds light on the strategy employed by phage helicase loaders to hijack bacterial replicative helicases and may explain the recurrent domestication of dnaC/I through evolution in bacteria.

DciA helicase operators exhibit diversity across bacterial phyla

“…Similar to the structure predicted for B. fragilis DciA ( Fig. 2B ), other Group 4 DciA homologs from thermotogae ( Thermotoga maritima , AAD35914.1 ) and α-proteobacteria ( Rickettsia conorii , AAL03818.1 ) only contain the folds previously reported for the DciA domain in V. cholerae (36) ( Fig. 5 ) .…”

“…For Groups 1–3, we also compared the structures of homologs with and without IDRs predicted in the sequence extensions. Regardless of Group designation, all DciA domains were predicted to fold into a structure similar to that previously predicted for M. tuberculosis DciA (34) and experimentally validated for V. cholerae DciA (36) ( Fig. 5 ) .…”

“…We also verified that these two eukaryotic DciA proteins were part of annotated ORFs in complete genomes, suggesting that they are truly eukaryotic proteins. Further, using AlphaFold structure prediction, we found that the DciA domains within the K. uniflora and H. bicolor E proteins resembled a truncated version of the V. cholerae DciA domain, solved previously using NMR (36) (Fig. S1B) .…”

“…An example of a DciA protein where the total protein length is similar to the size of the single DciA structural domain is the Bacteroides fragilis DciA homolog ( AUI45441.1 ), which is 96aa long with the DciA domain annotated from amino acids 8-95. The AlphaFold structural prediction tool predicts that the entire B. fragilis DciA protein is comprised of the predicted DciA domain structure, consisting of one alpha-helix followed by a two beta-sheet motif, one kinked alpha-helix, and a third beta-sheet (34,36) ( Fig. 2B ) .…”

“…Based on studies in M. tuberculosis , the DciA domain was predicted to contain a region of structural homology to the N-terminus of DnaA (34), which was subsequently confirmed in V. cholerae DciA (35,36). The DciA domain in the M. tuberculosis DciA homolog is essential for protein function (34).…”