Coordinating the structural organization of chromosomes is essential for DNA replication, transcription, and chromosome segregation during cell division. Failure to achieve proper chromosomal organization during separation can result in DNA breakage, leading to an uneven distribution of the genetic material to the next generation. Chromosomal organization involves two principal mechanisms: topological maintenance and protein-mediated packaging of the DNA. The former prevents entanglement by regulating the topology of the DNA, resolving unwanted catenanes and knots. The latter shapes the conformation of chromosomes, increasing the efficiency of any particular macromolecular transaction. In this proposal, we investigate these fundamental processes of chromosome organization by testing two hypotheses that center on the role of the interaction of topoisomerase IV (Topo IV), the enzyme that unlinks the daughter chromosomes, with the bacterial condensin, MukB;the mechanism by which the condensin itself acts on DNA;and the role of topoisomerase III (Topo III) in chromosome segregation in Escherichia coli. We showed that the interaction between the ParC subunit of Topo IV and MukB results in stimulation of only intramolecular reactions (such as superhelical DNA relaxation and DNA knotting) and not intermolecular reactions (such as DNA decatenation) catalyzed by Topo IV, suggesting that the interaction does not play a role in separation of the linked chromosomes directly. We will test our hypothesis that MukB and Topo IV act to condense the chromosome by bringing distal segments of the DNA together by examining the effects of ablating this interaction on chromosome dynamics in vivo, characterizing biochemically the effects of ParC on formation of the complete MukBEF condensin and on MukB-modulation of DNA topology, and characterizing a new MukB-mediated reaction that we have discovered: catenation of gapped DNA rings in the presence of Topo III. This latter reaction is more likely to accurately reflect the action of MukB to condense DNA in vivo than any of the other MukB-mediated, DNA topology-altering reactions that have been described. We showed that deletion of Topo III sensitizes cells to the type II topoisomerase inhibitor novobiocin, even when DNA gyrase is resistant to the drug, and that ?topB mutations combined with temperature-sensitive mutations in the Topo IV genes were synthetically lethal and showed chromosome segregation defects at semi-permissive temperatures. We have also shown that Topo III co-localizes with replisome components in vivo. We propose that Topo III participates in chromosome segregation by unlinking precatenanes (windings of the two partially replicated sister duplexes about each other) as they form at the replication fork. We will test this hypothesis by asking whether Topo III tracks with the replication fork, determining the manner by which it associates with the replisome, and the consequences on sister chromosome cohesion (which is thought to be mediated by precatenation) of deleting Topo III from the cell.

Public Health Relevance

The chromosomal DNA inside a cell is condensed by about 1000-fold compared to its relaxed length. In order to ensure that the chromosomes can be duplicated and distributed properly during cell growth and division, this compacted structure has to be managed actively by classes of enzymes called topoisomerases and condensins. Failure to execute proper management can result in chromosome breakage and loss of genetic information, leading to the accumulation of mutations that can cause diseases such as cancer. In our proposal we study how these two classes of enzymes cooperate to accomplish their task.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM034558-28
Application #
8625549
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Janes, Daniel E
Project Start
1984-07-01
Project End
2017-12-31
Budget Start
2014-01-01
Budget End
2014-12-31
Support Year
28
Fiscal Year
2014
Total Cost
$472,366
Indirect Cost
$206,544
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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