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The loading of the bacterial replicative helicase DnaB is an essential step for genome replication and depends on the assistance of accessory proteins. Several of these proteins have been identified across the bacterial phyla. DciA is the most common loading protein in bacteria, yet the one whose mechanism is the least understood. We have previously shown that DciA from Vibrio cholerae is composed of a globular domain followed by an unfolded extension and demonstrated its strong affinity for DNA. Here, we characterize the condensates formed by VcDciA upon interaction with a short single-stranded DNA substrate. We demonstrate the fluidity of these condensates using light microscopy and address their network organization through electron microscopy, thereby bridging events to conclude on a liquid-liquid phase separation behavior. Additionally, we observe the recruitment of DnaB in the droplets, concomitant with the release of DciA. We show that the well-known helicase loader DnaC from Escherichia coli is also competent to form these phase-separated condensates in the presence of ssDNA. Our phenomenological data are still preliminary as regards the existence of these condensates in vivo, but open the way for exploring the potential involvement of DciA in the formation of non-membrane compartments within the bacterium to facilitate the assembly of replication players on chromosomal DNA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

During the initiation step of bacterial genome replication, replicative helicases depend on specialized proteins for their loading onto oriC. DnaC and DnaI were the first loaders to be characterized. However, most bacteria do not contain any of these genes, which are domesticated phage elements that have replaced the ancestral and unrelated loader gene dciA several times during evolution. To understand how DciA assists the loading of DnaB, the crystal structure of the complex from Vibrio cholerae was determined, in which two VcDciA molecules interact with a dimer of VcDnaB without changing its canonical structure. The data showed that the VcDciA binding site on VcDnaB is the conserved module formed by the linker helix LH of one monomer and the determinant helix DH of the second monomer. Interestingly, DnaC from Escherichia coli also targets this module onto EcDnaB. Thanks to their common target site, it was shown that VcDciA and EcDnaC could be functionally interchanged in vitro despite sharing no structural similarity. This represents a milestone in understanding the mechanism employed by phage helicase loaders to hijack bacterial replicative helicases during evolution., (open access.)

DciA is the ancestral bacterial replicative helicase loader, punctually replaced during evolution by the DnaC/I loaders of phage origin. DnaC helps the helicase to load onto DNA by cracking open the hexameric ring, but the mechanism of loading by DciA remains unknown. We demonstrate by electron microscopy, nuclear magnetic resonance (NMR) spectroscopy, and biochemistry experiments that DciA, which folds into a KH-like domain, interacts with not only single-stranded but also double-stranded DNA, in an atypical mode. Some point mutations of the long α-helix 1 demonstrate its importance in the interaction of DciA for various DNA substrates mimicking single-stranded, double-stranded, and forked DNA. Some of these mutations also affect the loading of the helicase by DciA. We come to the hypothesis that DciA could be a DNA chaperone by intercalating itself between the two DNA strands to stabilize it. This work allows us to propose that the direct interaction of DciA with DNA could play a role in the loading mechanism of the helicase.

To enable chromosomal replication, DNA is unwound by the ATPase molecular motor replicative helicase. The bacterial helicase DnaB is a ring-shaped homo-hexamer whose conformational dynamics are being studied through its different 3D structural states adopted along its functional cycle. Our findings describe a new crystal structure for the apo-DnaB from Vibrio cholerae, forming a planar hexamer with pseudo-symmetry, constituted by a trimer of dimers in which the C-terminal domains delimit a triskelion-shaped hole. This hexamer is labile and inactive. We suggest that it represents an intermediate state allowing the formation of the active NTP-bound hexamer from dimers., (© 2022 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The ComFC protein is essential for natural transformation, a process that plays a major role in the spread of antibiotic resistance genes and virulence factors across bacteria. However, its role remains largely unknown. Here, we show that Helicobacter pylori ComFC is involved in DNA transport through the cell membrane, and is required for the handling of the single-stranded DNA once it is delivered into the cytoplasm. The crystal structure of ComFC includes a zinc-finger motif and a putative phosphoribosyl transferase domain, both necessary for the protein's in vivo activity. Furthermore, we show that ComFC is a membrane-associated protein with affinity for single-stranded DNA. Our results suggest that ComFC provides the link between the transport of the transforming DNA into the cytoplasm and its handling by the recombination machinery., (© 2022. The Author(s).)