Mechanisms of Chromosome Maintenance in Bacteria

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Of the two V. cholerae chromosomes, the larger one (Chr1) carries most of the housekeeping genes and is considered the primary chromosome. The smaller chromosome (Chr2) seems to have evolved from a plasmid. Plasmids, although prevalent as extrachromosomal elements in bacteria, are rarely found integrated into the chromosome and driving the chromosomal replication. One reason could be that the firing of plasmid origins is generally not restricted to a specific time in the cell cycle, whereas timely firing is the norm for chromosomal origins in all domains of life. Comparison of plasmid and Chr2 replication initiation mechanisms could thus be valuable to understand how the timing of a biological process has evolved from being random to be specific in the cell cycle. The timing of Chr2 replication depends on prior replication of a site, crtS, in Chr1. Our discovery of this site (in 2014), where the Chr2 initiator RctB also binds, demonstrated that chromosomes do actively communicate and encouraged studies to understand the mechanism of replication coordination between the two chromosomes in other labs. In eukaryotes, uncoordinated replication from different origins causes developmental abnormalities and cancer. Our progress in understanding replication of Chr2 and its coordination with that of Chr1 is reported below. 1. Communication between Chr1 and Chr2 for replication initiation: Identification of a novel check point control in bacteria. The location of crtS on Chr1 is such that it would replicate just before the time of Chr2 replication initiation. This affords a straight forward mechanism for communication: Chr1 replication initiates first. When the fork passes through crtS, it activates the bound RctB initiator molecules that triggers Chr2 replication. Replication of crtS thus relieves the check point that prevents Chr2 replication. The mechanistic details of crtS activation of Chr2 replication however remain enigmatic. The activity appears to be remodeling of RctB that improves specifically the initiator's origin binding activity. As expected, initiator binding to the origin is such a fundamental requirement that when RctB was mutated at random but selecting for its initiator function, the mutants remained responsive to crtS without exception. In other words, crtS is controlling some basic function of RctB that cannot be inactivated. It appears that origin binding is such an activity of the initiator. Last year, we showed that crtS activity depends on a global transcription regulator Lrp that also binds to crtS together with RctB. Our present studies indicate that there are at least two mechanisms by which Lrp could activate RctB binding: Direct protein-protein interaction by which Lrp bound to crtS can help load RctB there, and second by modifying crtS DNA structure which apparently increases RctB's affinity for crtS DNA. 2. Control of initiator function by dimerization. Protein function is often controlled by oligomerization. RctB dimerizes efficiently but several studies indicate that monomers are the active initiators. To get further insight on the role of dimerization, we made several structure-guided mutations in the dimerization domain of RctB. The mutants were variously defective in dimerization and the dimerization defect was inversely correlated with the initiator function. This indicated that dimerization is an inhibitory mechanism for replication initiation. These mutants have been rewarding for two reasons: They were no longer responsive to crtS, indicating that crtS could be activating RctB by dimer dissociation, and they were responsive to molecular chaperones, indicating that monomerization is not sufficient to relieve chaperone requirement. These results supports an alternate model that monomers need to be remodeled for them to be active in initiation. 3. Towards generation of Vibrio-specific antimicrobial agents: RctB, is conserved only in the Vibrio family and appears ideally suited for developing potential drugs specifically against Vibrios. In the case of cholera, although oral rehydration treatment is the mainstay, antimicrobial therapy becomes mandatory at times, and V. cholerae is no exception in developing resistance to multiple antibiotics. By systematic structure-function analysis of the protein we are trying to zeroing in on regions of the protein that are essential for replication initiation and, hence, survival of Vibrio. These regions can be specifically targeted for drug design. 4. Replication initiation of Chr2: Opening of the strands of replication origin. All transactions on DNA require strand-opening which is an energetically unfavorable reaction. How cells overcome this energy barrier for replication initiation is not clearly understood. The hypothesis is that initiator binding to the origin creates torsional stress on DNA that is released in a neighboring AT-rich region which is easier to melt. However, the melting needs to be stabilized by capturing at least one of the single strands, otherwise the stress would diffuse out of the origin. Evidence in favor of stabilization has been obtained in the Chr2 origin. We find that the Chr2 initiator RctB has single-stranded DNA binding activity, which is greatly stabilized in vitro by specific double stranded sites of the origin. The stability derives from the formation of ternary complexes of the initiator with the single and double stranded sites. Simultaneous binding to two kinds of sites in the origin appears to be a common mechanism by which bacterial replication initiators stabilize an open origin. Initiator binding to origin double-stranded sites thus plays a dual role by contributing to both in initiation of opening by DNA stressing and stabilization of the opening by capturing a single strand.