Mechanisms of Chromosome Maintenance in Bacteria

Division Of Basic Sciences - Nci

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Abstract

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 in Chr1. Our discovery of this site (in 2014) demonstrated that chromosomes do 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. Replication initiation of Chr2: The initial characterization of Chr2 replication suggested that its regulation is more complex than that of its presumed progenitor plasmid. Many, if not all, of these regulations are mediated through the Chr2 specific initiator protein, RctB. We have embarked on a systematic structure-function analysis of the protein not only to understand Chr2 replication control in greater depth but also for drug design against V. cholerae (Section 2 below). In the past year, we succeeded in identifying a new domain of RctB that interacts with the molecular chaperone DnaK (Hsp70). DnaK increases RctB binding to the replication origin of Chr2 and this observation suffices to explain why DnaK is required for Chr2 replication. DnaK also helps RctB binding to regions outside of the replication origin for regulatory purposes. Identification of the DnaK interacting domain of RctB (K-domain) provided valuable insights on how DnaK helps in controlling the two different kinds of DNA binding activity of RctB. Mutations in the K-domain inactivates the replication initiator activity of RctB and intragenic suppressors that allow initiator activity seem to have done so by reducing replication inhibitory activities of the protein. These results have been rewarding in identifying the major negative regulatory mechanisms of Chr2 replication. At present, we are trying to identify the RctB domain for interaction with the DnaK co-chaperone, DnaJ. 2. Towards generation of Vibrio-specific antimicrobial agents: The Chr2 initiator, 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. Design of new drugs is greatly facilitated when the 3-D structural information of target proteins are available. Replication initiators in general have proven refractory to structural studies most likely because they have unstructured regions and require remodeling by chaperone proteins for activity, which seems to be the case for RctB. In collaboration with Alex Wlodawer (CCR), Lisa Jenkins (CCR) and Rodolfo Ghirlando (NIDDK), we succeeded in recent months solving the structure of one half of RctB. Many of the regulatory mutations are mapped in this region. Determination of the structure of the remaining regions that serve essential functions has so far proven refractory, possibly because such regions could be intrinsically unstructured (from NMR evidence obtained in collaboration with Yawen Bai, CCR). The folding of these regions may require the presence of their interacting partners. At present we are gearing up to try cryo-EM to solve the structure of RctB with its partner DNA binding site in the NCI core facility in Frederick. 3. Replication coordination between Chr1 and Chr2: Identification of a novel check point control in bacteria. We hypothesized that the timely replication and segregation of the two V. cholerae chromosomes would require communication between them, so that both the processes are completed prior to cell division. An evidence for inter-chromosomal communication was obtained when we identified a site on Chr1 (crtS for Chr2 replication triggering site) that can bind RctB and significantly stimulate Chr2 replication. 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 Chr2 initiator that triggers Chr2 replication. Replication of crtS thus relieves the check point that prevents Chr2 replication. We have now shown that blocking of Chr1 replication also blocks Chr2 replication, demonstrating that Chr2 replication indeed depends on Chr1 replication. Strikingly, two unreplicated copies of crtS could also allow Chr2 replication, suggesting that crtS has significant activity without itself being replicated and a role of replication is to increase this activity by doubling the crtS gene dosage. The activity appears to be remodeling of RctB, since crtS could relieve the dependence of the initiator on molecular chaperones. The study provides a novel example of how doubling of gene dosage by replication is utilized for other regulatory purposes. At present, we are trying to understand how DNA-protein interactions at crtS remodels RctB. crtS site is minimally about 70 bp long, which is too large for a protein binding site. The part of this mystery is solved by our discovery in 2018 that a well conserved global transcription factor Lrp (Leucine responsive protein) also binds to crtS and significantly stimulates simultaneous RctB binding to crtS. How crtS stimulates RctB binding and how the binding remodels RctB to activate its initiator function are the questions that we are currently addressing. 4. In 2018, we initiated addressing an age-old yet unsolved question on how opening of the strands of DNA comes about. All transactions on DNA require strand-opening which is an energetically unfavorable reaction. How cells overcome this energy barrier is not clearly understood. We are focussing on how strands of replication origin opens. 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. 5. In collaboration with Ling Chin Hwang of Sheffield U (UK), we are trying to understand how Chr2 segregates using two proteins, ParA and ParB. This is a collaboration where we create strains and do some cell biology whereas Ling does the biochemistry and biophysics. The challenge here is to understand how sister chromosomes move in a directed fashion without the presence of spindle or mitotic motors.