RESUMEN
Replication is initiated bidirectionally in the three domains of life by the assembly of two replication forks at an origin of replication. This is made possible by the recruitment of two replicative helicases to a nucleoprotein platform built at the origin of replication with the initiator protein. The reason why replication is initiated bidirectionally has never been experimentally addressed due to the lack of a suitable biological system. Using genetic and genomic approaches, we show that upon depletion of DciA, replication is no longer initiated bidirectionally at the origin of replication of Vibrio cholerae chromosome 1. We show that following unidirectional replication on the left replichore, nascent DNA strands at ori1 anneal to each other to form a double-stranded DNA end. While this DNA end can be efficiently resected in recB+ cells, only a few cells use it to trigger replication on the right replichore. In most DciA-depleted cells, chromosome 1 is degraded leading to cell death. Our results suggest that DciA is essential to ensuring bidirectional initiation of replication in bacteria, preventing a cascade of deleterious events following unidirectional replication initiation.
RESUMEN
About 10% of bacteria have a multichromosome genome with a primary replicon of bacterial origin, called the chromosome, and other replicons of plasmid origin, the chromids. Studies on multichromosome bacteria revealed potential points of coordination between the replication/segregation of chromids and the progression of the cell cycle. For example, replication of the chromid of Vibrionales (called Chr2) is initiated upon duplication of a sequence carried by the primary chromosome (called Chr1), in such a way that replication of both replicons is completed synchronously. Also, Chr2 uses the Chr1 as a scaffold for its partition in the daughter cells. How many of the features detected so far are required for the proper integration of a secondary chromosome in the cell cycle? How many more features remain to be discovered? We hypothesized that critical features for the integration of the replication/segregation of a given chromid within the cell cycle program would be conserved independently of the species in which the chromid has settled. Hence, we searched for a chromid related to that found in Vibrionales outside of this order. We identified one in Plesiomonas shigelloides, an aquatic and pathogenic enterobacterium that diverged early within the clade of Enterobacterales. Our results suggest that the chromids present in P. shigelloides and Vibrionales derive from a common ancestor. We initiated in silico genomic and proteomic comparative analyses of P. shigelloides, Vibrionales, and Enterobacterales that enabled us to establish a list of features likely involved in the maintenance of the chromid within the host cell cycle.
Asunto(s)
Plesiomonas , Vibrio , Cromosomas Bacterianos/genética , Genoma Bacteriano , Plesiomonas/genética , Proteómica , Vibrio/genéticaRESUMEN
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.