The long-term objectives of this research are to understand the mechanism of chromosome cohesion in bacteria, to determine what role cohesion plays in maintenance and organization of bacterial chromosomes, and to develop a general model for bacterial chromosome segregation. The research will impact three important areas relevant to human health: (i) It will fill major voids in our understanding of how bacterial chromosomes are maintained, and will directly impact many areas of bacterial genetics and molecular biology that are important for human health, including antibiotic resistance and mechanisms of gross chromosomal instability (GCI). (ii) It is expected to reveal essential and unknown roles of Topo IV protein, target of the most highly prescribed class of antibiotics in the world, the fluoroquinolones. (iii) We also predict that it will illuminate parallel cohesion mechanisms that occur in eukaryotes, and enable new strategies to detect and prevent disease caused by defects in cohesion, including human aneuploidies and cancer.
Three specific aims will be pursued:
(Aim1) Identify the molecular mechanism of chromosome cohesion in E. coli. We hypothesize that cohesion is caused by topological knotting of sister chromosomes, eventually resolved by Topo IV.
This aim will develop a molecular picture of the structure, assembly and removal of sister cohesion linkages.
(Aim2) Determine how cohesion is regulated within the cell cycle. Activities to be examined include the regulatory effects of proteins that bid newly replicated DNA, either stabilizing cohesion directly or by mediating Topo IV.
(Aim3) Define the role of cohesion in promoting efficient sister chromosome separation and development of spatially defined daughter nucleoids. We hypothesize that controlled removal of cohesion is an underlying driver of chromosome segregation in all cells. Experimental approach: Innovative genomic and single-locus assays will be used to develop a picture of cohesion-relevant activities across the chromosome in E. coli. High temporal resolution will be achieved by synchronizing cell populations by baby machine method. Select mutants will then be assayed for defects in these activities, and protein-DNA and protein-protein relationships will be determined. Lastly, chromosome dynamics will be examined in cohesion-defective cells using live cell fluorescent reporter operator systems (FROS) and a novel whole-genome fluorescence method, chromosome painting. Understanding the mechanisms of cohesion in E. coli will provide important definitions of bacterial chromosome organization, maintenance and antibiotic action, and will illuminate general mechanisms of avoidance of disease-promoting GCI.
This research will investigate how replicated DNA is paired in bacterial cells, and how this pairing affects maintenance, organization and separation of DNA before cell division. This research will provide critical missing information for antibiotic resistance and will reveal related mechanisms of DNA pairing in humans, defects in which lead to devastating genetic disease such as Down syndrome and cancer.
|Kleckner, Nancy; Fisher, Jay K; Stouf, Mathieu et al. (2014) The bacterial nucleoid: nature, dynamics and sister segregation. Curr Opin Microbiol 22:127-37|
|Cambridge, Joshua; Blinkova, Alexandra; Magnan, David et al. (2014) A replication-inhibited unsegregated nucleoid at mid-cell blocks Z-ring formation and cell division independently of SOS and the SlmA nucleoid occlusion protein in Escherichia coli. J Bacteriol 196:36-49|
|Joshi, Mohan C; Magnan, David; Montminy, Timothy P et al. (2013) Regulation of sister chromosome cohesion by the replication fork tracking protein SeqA. PLoS Genet 9:e1003673|
|Shee, Chandan; Cox, Ben D; Gu, Franklin et al. (2013) Engineered proteins detect spontaneous DNA breakage in human and bacterial cells. Elife 2:e01222|