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 #
5R01GM025326-35
Application #
8496054
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
2013-07-01
Budget End
2014-06-30
Support Year
35
Fiscal Year
2013
Total Cost
$898,115
Indirect Cost
$363,523
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
Kleckner, Nancy (2016) Questions and Assays. Genetics 204:1343-1349
Gladyshev, Eugene; Kleckner, Nancy (2016) Recombination-Independent Recognition of DNA Homology for Repeat-Induced Point Mutation (RIP) Is Modulated by the Underlying Nucleotide Sequence. PLoS Genet 12:e1006015
Liu, Chenli; Danilowicz, Claudia; Kleckner, Nancy et al. (2016) Single molecule identification of homology-dependent interactions between long ssRNA and dsDNA. Nucleic Acids Res :
Yoon, Sang-Wook; Lee, Min-Su; Xaver, Martin et al. (2016) Meiotic prophase roles of Rec8 in crossover recombination and chromosome structure. Nucleic Acids Res 44:9296-9314
Gladyshev, Eugene; Kleckner, Nancy (2016) Recombination-independent recognition of DNA homology for repeat-induced point mutation. Curr Genet :
Liang, Zhangyi; Zickler, Denise; Prentiss, Mara et al. (2015) Chromosomes Progress to Metaphase in Multiple Discrete Steps via Global Compaction/Expansion Cycles. Cell 161:1124-37
Yang, Darren; Boyer, Benjamin; Prévost, Chantal et al. (2015) Integrating multi-scale data on homologous recombination into a new recognition mechanism based on simulations of the RecA-ssDNA/dsDNA structure. Nucleic Acids Res 43:10251-63
Kleckner, Nancy; Fisher, Jay K; Stouf, Mathieu et al. (2014) The bacterial nucleoid: nature, dynamics and sister segregation. Curr Opin Microbiol 22:127-37
Gladyshev, Eugene; Kleckner, Nancy (2014) Direct recognition of homology between double helices of DNA in Neurospora crassa. Nat Commun 5:3509
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

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