Accurate chromosome segregation is crucial to ensure that each daughter cell receives a complete copy of the genetic information. Molecular mechanisms that drive these processes are well understood in eukaryotes, however, we know little about these events in bacteria. Recent advances in the application of cell biological techniques have revealed that the spatial distribution of the bacterial chromosome is highly ordered and that chromosome segregation is likely to be a progressive process. The force for DNA segregation has been ascribed to replication itself, transcription, transertion (the coupling of transcription and co-translational insertion of proteins into the membrane], and entropy. And the role of the recently discovered MreB cytoskeleton remains unclear. The long term goal of this grant is to understand the events necessary for proper segregation of the newly duplicated Escherichia coli sister chromosomes to a new daughter cell. Our focus in these investigations is topoisomerase IV (Topo IV), which is responsible for unlinking the catenated sister chromosomes, and its interactions with other proteins involved in chromosome dynamics and cell division. In the previous grant period we have: 1) shown that Topo IV activity is regulated by the oligomeric state of the cytoskeletal element MreB, monomeric MreB inhibits whereas filamentous MreB stimulates, possibly accounting for the temporal regulation of Topo IV activity in the cell;2) discovered and characterized an interaction between the ParC subunit of Topo IV and MukB, the bacterial condensin, that stimulates Topo IV activity;3) demonstrated that stimulation of Topo IV by FtsK, the molecular motor required for final sorting of the chromosomes, does not require FtsK DNA translocation; 4) identified a nucleoid associated protein, YejK, that interacts with Topo IV and demonstrated that yejK cells have a cell cycle defect;5) demonstrated biochemically that RecQ and topoisomerase III (Topo III) can resolve convergent replication forks, a reaction that may be important at the terminal stages of DNA replication;and 6) discovered that regulation of cell division is coupled to the condensation state of the nucleoid, possibly via a checkpoint that, when engaged, inhibits Min protein oscillation, required for proper placement of the division septum. We will proceed to use a combination of biochemical, cell biologic, and molecular genetic approaches to answer the following questions: What is the role of Topo IV in chromosome dynamics and how is this role modulated by the interactions between Topo IV and MukB and Topo IV and FtsK? How is Topo IV activity regulated in the cell and what is the role of the Topo IV-MreB interaction in this process? What is the nature of the coupling between cell division and the condensation state of the nucleoid? And, do RecQ and Topo III support an alternate pathway of sister chromosome decatenation in vivo?

Public Health Relevance

Resistance of bacteria to treatment with antibiotic and anti-microbial drugs is a persistent public health problem that is increasingly of concern. This proposal investigates emerging new paradigms in the bacterium Escherichia coli that are involved in the accurate transmission of the genetic information to the daughter cells. We anticipate that as we understand more about these pathways and more of the participants are revealed, potential new targets for anti- microbial chemotherapy will be presented.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034558-27
Application #
8247764
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Janes, Daniel E
Project Start
1984-07-01
Project End
2013-12-31
Budget Start
2012-04-01
Budget End
2013-12-31
Support Year
27
Fiscal Year
2012
Total Cost
$486,956
Indirect Cost
$230,123
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
Nurse, Pearl; Marians, Kenneth J (2013) Purification and characterization of Escherichia coli MreB protein. J Biol Chem 288:3469-75
Lee, Chong; Marians, Kenneth J (2013) Characterization of the nucleoid-associated protein YejK. J Biol Chem 288:31503-16
Hayama, Ryo; Bahng, Soon; Karasu, Mehmet E et al. (2013) The MukB-ParC interaction affects the intramolecular, not intermolecular, activities of topoisomerase IV. J Biol Chem 288:7653-61
Hayama, Ryo; Marians, Kenneth J (2010) Physical and functional interaction between the condensin MukB and the decatenase topoisomerase IV in Escherichia coli. Proc Natl Acad Sci U S A 107:18826-31
Bigot, Sarah; Marians, Kenneth J (2010) DNA chirality-dependent stimulation of topoisomerase IV activity by the C-terminal AAA+ domain of FtsK. Nucleic Acids Res 38:3031-40
Madabhushi, Ram; Marians, Kenneth J (2009) Actin homolog MreB affects chromosome segregation by regulating topoisomerase IV in Escherichia coli. Mol Cell 33:171-80
Suski, Catherine; Marians, Kenneth J (2008) Resolution of converging replication forks by RecQ and topoisomerase III. Mol Cell 30:779-89
Espeli, Olivier; Marians, Kenneth J (2004) Untangling intracellular DNA topology. Mol Microbiol 52:925-31
Nurse, Pearl; Levine, Cindy; Hassing, Heide et al. (2003) Topoisomerase III can serve as the cellular decatenase in Escherichia coli. J Biol Chem 278:8653-60
Espeli, Olivier; Lee, Chong; Marians, Kenneth J (2003) A physical and functional interaction between Escherichia coli FtsK and topoisomerase IV. J Biol Chem 278:44639-44

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