Source: https://irp.nih.gov/pi/dhruba-chattoraj
Timestamp: 2019-04-23 08:23:36+00:00

Document:
The faithful replication of chromosomes and their segregation to incipient daughter cells before cell division are required for the stable transmission of the genome. A host of intricate control mechanisms allow chromosome duplication once and only once per cell cycle, and ensure that a full complement of chromosomes is passed into each daughter cell. Defects in any aspect of chromosome replication or segregation pose significant threats to genome stability, and underlie the development of many human diseases, foremost among them being cancer.
While most bacterial genomes consist of a single circular chromosome, it is becoming clear from genome sequencing projects that about 10% of bacterial genomes are divided into multiple chromosomes, as in eukaryotes. These bacteria provide a unique opportunity to probe the mechanisms involved in the maintenance of multiple chromosomes. We study how replication and segregation are controlled and coordinated in a model organism with a divided genome, Vibrio cholerae. It has two chromosomes (chrI and chrII) but otherwise it is closely related to the paradigmatic model organism Escherichia coli. V. cholerae is also a potent human pathogen. A better understanding of the mechanisms operating in bacterial models will enhance our general understanding of chromosome replication and segregation, and can guide the rational development of therapeutics for many human diseases.
Cell cycle control of V. cholerae replication. The chrI of V. cholerae, like the E. coli chromosome are controlled by the highly conserved initiator protein DnaA. The initiator is also homologous to one of the ORC proteins that initiate replication in eukaryotes. ChrII is controlled differently, but retains the salient features of bona fide chromosomal replication: initiation of replication once per cell cycle replication and at a particular time of the cell cycle. Understanding replication control of chrII thus is likely to provide a fresh perspective on the basic regulatory principles that govern chromosomal replication. We are determining the genetic basis of the regulation and, where identified, the mechanisms behind the functioning of the regulators.
The chrII initiator protein, RctB, as a therapeutic target. An added value of studying chrII replication is that the knowledge gained has the potential to contribute to anti-Vibrio therapy. V. cholerae causes significant human suffering world-wide, as recently evidenced in Haiti. The initiator protein specific to chrII, RctB, is essential for Vibrio survival and is unique to the Vibrio family, offering a specific target for chemotherapy. The protein has several distinct essential functions, offering the opportunities to develop drug inhibitors specific to the different functions. These considerations have prompted us to embark on a systematic structure-function analysis of the protein.
Inter-chromosome communication in V. cholerae. One of the advantages afforded in studying a bacterium with a divided genome is to be able to ask: Do the chromosomes communicate to improve stability of their maintenance? We are approaching this question by developing systems in which replication or segregation is specifically blocked one chromosome at a time. When chrI replication is selectively blocked, both chrII replication and cell division are inhibited. However, when chrII replication is blocked, replication and segregation of chrI and cell division remain unaffected. Apparently, there is no checkpoint to ensure completion of replication and segregation of both chromosomes before cell division. It also appears that there has been a one-way adaptation of chrII to the maintenance of the main chromosome (chrI), and not the reverse. An understanding of chromosome coordination is expected to bear on the evolutionary process of chromosome acquisition and cooption.
Communication between replication and segregation in V. cholerae. The processes that contribute to chromosome segregation in bacteria are still poorly understood. In addition to the range of mechanisms proposed, which includes chromosome replication, there are clearly additional mechanisms that anchor the centromere analog to the cell pole in some bacteria, including V. cholerae. We are specifically interested in studying the role of chromosome replication in the segregation process and how the centromeres are retained at the pole after they have segregated.
Most sequenced bacterial genomes have orthologs of genes that help in segregation/partition of plasmids (par genes). Studies in several bacteria indicate that par genes, in addition to their role in chromosome segregation, contribute to other cellular processes such as chromosome replication and cell division. We have found that one of the proteins encoded by the par locus of chrI both promotes chrI segregation and regulates chrI replication. The results are strikingly similar to those found in Bacillus subtilis. It is remarkable that the replication phenotype of par has been retained or has independently evolved in a convergent fashion in the two bacteria that are believed to have diverged more than a billion years ago. We are interested in determining whether par genes in general modulate chromosomal replication.
Dr. Chattoraj received his Ph.D. in Biophysics in 1970 from the University of Calcutta, India, where he studied chromatin structure. As a postdoctoral associate, he studied nucleic acid-protein interactions involved in phage DNA replication and recombination at the University of Wisconsin with Dr. Ross Inman and at the University of Oregon with Dr. Frank Stahl. His current research interest is on replication and segregation of chromosomes in the bacterial cell cycle. He is a fellow of the American Academy of Microbiology and the President of the International Society of Plasmid Biology.
Baek JH, Chattoraj DK. Chromosome I controls chromosome II replication in Vibrio cholerae. PLoS Genet. 2014;10(2):e1004184.
Hill NS, Kadoya R, Chattoraj DK, Levin PA. Cell size and the initiation of DNA replication in bacteria. PLoS Genet. 2012;8(3):e1002549.
Das N, Valjavec-Gratian M, Basuray AN, Fekete RA, Papp PP, Paulsson J, Chattoraj DK. Multiple homeostatic mechanisms in the control of P1 plasmid replication. Proc Natl Acad Sci U S A. 2005;102(8):2856-61.
Chattoraj DK, Mason RJ, Wickner SH. Mini-P1 plasmid replication: the autoregulation-sequestration paradox. Cell. 1988;52(4):551-7.
Chattoraj DK, Gosule LC, Schellman A. DNA condensation with polyamines. II. Electron microscopic studies. J Mol Biol. 1978;121(3):327-37.

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