The long-term objective of this project is to further elucidate the roles of mechanical forces for chromosomes and chromosomal reactions. Chromosome morphogenesis and dynamics will be examined from this perspective, comparatively for E.coli versus the prophase-to-prometaphase transition of eukaryotic chromosomes. Approaches will focus on high resolution 3D analysis, over time in the cell cycle, in wild-type and "mutant" situations, in living cells where ever possible. For E.coli, specific issues to be addressed will include the underlying physical and molecular basis for development of the nucleoid's helical ellipsoidal shape/density, the significance of this shape for dynamic chromatin movements, and the possibility that of cell division-related licensing of replication initiation. For eukaryotic chromosomes, the possibility of a meiosis-like intermediate will be explored. In complementary studies, our "beads and bottlenecks" magnetic micropiston will be used to probe the effects of compression and confinement on nucleoids/chromatin and DNA, including effects of real-time changes in buffer and molecular conditions. The roles of mechanical force for RecA- mediated homology recognition and strand exchange will be examined by parallel single molecule studies. Individual steps will be isolated and studied. The notion that reactions mediated by protein phosphatase PP2A are governed in important ways by the elasticity of its HEAT repeat scaffolding subunit will be further explored. Approaches to be used will include in vitro AFM analysis, in silico molecular dynamics, in vivo studies analysis of the PP2A-mediated response to spindle tension during the second division of meiosis, with budding yeast as an experimental system.

Public Health Relevance

All components of biological systems have intrinsic physical and mechanical properties. The long- term goal of our research is to understand the extent to which these properties play governing roles in directing the complex array of biochemical and structural changes that underlie life. Since strategies for public health depend upon, and are nucleated by, an understanding of basic processes, research from this relatively new perspective is likely to lead ultimately to new approaches and thus to improved human health.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM025326-34
Application #
8299561
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Reddy, Michael K
Project Start
1978-07-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
34
Fiscal Year
2012
Total Cost
$930,687
Indirect Cost
$376,706
Name
Harvard University
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138
Gladyshev, Eugene; Kleckner, Nancy (2014) Direct recognition of homology between double helices of DNA in Neurospora crassa. Nat Commun 5:3509
Danilowicz, Claudia; Peacock-Villada, Alexandra; Vlassakis, Julea et al. (2014) The differential extension in dsDNA bound to Rad51 filaments may play important roles in homology recognition and strand exchange. Nucleic Acids Res 42:526-33
Jiang, Lili; Prentiss, Mara (2014) RecA-mediated sequence homology recognition as an example of how searching speed in self-assembly systems can be optimized by balancing entropic and enthalpic barriers. Phys Rev E Stat Nonlin Soft Matter Phys 90:022704
Fisher, J K; Kleckner, N (2014) Magnetic force micropiston: an integrated force/microfluidic device for the application of compressive forces in a confined environment. Rev Sci Instrum 85:023704
Kleckner, Nancy; Fisher, Jay K; Stouf, Mathieu et al. (2014) The bacterial nucleoid: nature, dynamics and sister segregation. Curr Opin Microbiol 22:127-37
Kleckner, Nancy; Zickler, Denise; Witz, Guillaume (2013) Molecular biology. Chromosome capture brings it all together. Science 342:940-1
Kates-Harbeck, Julian; Tilloy, Antoine; Prentiss, Mara (2013) Simplified biased random walk model for RecA-protein-mediated homology recognition offers rapid and accurate self-assembly of long linear arrays of binding sites. Phys Rev E Stat Nonlin Soft Matter Phys 88:012702
Fisher, Jay K; Bourniquel, Aude; Witz, Guillaume et al. (2013) Four-dimensional imaging of E. coli nucleoid organization and dynamics in living cells. Cell 153:882-95
Danilowicz, Claudia; Feinstein, Efraim; Conover, Alyson et al. (2012) RecA homology search is promoted by mechanical stress along the scanned duplex DNA. Nucleic Acids Res 40:1717-27
Conover, Alyson J; Danilowicz, Claudia; Gunaratne, Ruwan et al. (2011) Changes in the tension in dsDNA alter the conformation of RecA bound to dsDNA-RecA filaments. Nucleic Acids Res 39:8833-43

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