ZIA BC 010277 (ZIA) | |||
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Title | Mechanisms of Chromosome Maintenance in Bacteria | ||
Institution | NCI, Bethesda, MD | ||
Principal Investigator | Chattoraj, Dhruba | NCI Program Director | N/A |
Cancer Activity | N/A | Division | CCR |
Funded Amount | $569,301 | Project Dates | 01/01/1997 - 00/00/0000 |
Fiscal Year | 2015 | Project Type | Intramural |
Research Topics w/ Percent Relevance | Cancer Types w/ Percent Relevance | ||
Cancer (100.0%) Gene Therapy (10.0%) |
N/A | ||
Research Type | |||
Normal Functioning Cancer Initiation: Alterations in Chromosomes |
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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 in bacteria, rarely serve as the source of replicons that drive chromosomal replication. One reason could be that the firing of plasmid origins is generally not restricted to a specific time of the cell cycle, whereas timely firing is the norm for chromosomal origins in all domains of life. Comparison of plasmid and Chr2 systems could thus be valuable to understand how the timing of a biological process is regulated in the cell cycle. Chr2 replication seems also to depend on ChrI replication. How the two chromosomes communicate to coordinate replication is largely unknown and this knowledge is basic to our understanding of how multipartite genomes are maintained in bacteria. In eukaryotes, in addition to incomplete and over-replication, uncoordinated replication causes developmental abnormalities and cancer. Finally, Chr2 replication is controlled by a segregation protein. The discovery of the influence of segregation on replication is a recent development in the field of chromosome dynamics in bacteria, and cross-talk between replication and segregation has been demonstrated recently in yeast and human cells. Our progress in understanding these processes is reported below. Transition from random to cell-cycle regulated replication initiation: The initial characterization of Chr2 replication suggested that its control would be more complex than that of its presumed progenitor plasmid. Our past studies identified three new features of the chr2 origin: 1) Requirement for methylation of initiator binding sites in the origin for initiator binding. Several bacterial origins have methylation sites and timely firing of origins depends on methylation; 2) Presence of a new kind of initiator binding site (39-mers) in the origin exclusively for inhibiting over-replication; and 3) Presence of an extra regulatory feedback loop to control initiator synthesis more tightly. In 2013-2014, we discovered three additional regulators in the control of chr2 replication, which we are continuing to characterize. The cell-cycle regulation of replication initiation thus appears to be considerably more involved than the mechanisms that regulate plasmid replication. The mechanisms of action of the new regulators are discussed below. 1. Participation of initiator dimers in the control of chr2 replication: The chr2 initiator does not have any enzymatic activity but uses homodimerization to regulate its activity. Only monomer binding to the replication origin leads to initiation. The dimers compete with monomers for binding to the origin and thus hinders initiation. The dimerization also precludes initiator binding to replication inhibitory 39-mer, which binds only the monomers. Controlling the association state of the initiator is thus critical for controlling Chr2 replication. We have determined the domains of the initiator involved in origin and 39-mer DNA binding and in dimerization. Important residues for all three activities are found to reside within 71 out of the 658 residue long protein. The domains for the three activities also overlap explaining how changes in one domain can influence the activities of the other two domains. The interplay between dimerization and DNA binding is well-known in gene regulation but it seems to play a more profound role in controlling Chr2 replication. 2. Control of Chr2 replication by a site in Chr1: We hypothesize that the timely replication and segregation of the two V. cholerae chromosomes would require communication between them, so that both the processes can complete prior to cell division. Preliminary evidence for inter-chromosomal communication has been obtained. We identified a locus on Chr1 that can significantly stimulate Chr2 replication, likely by remodeling the Chr2 initi |