Biological organisms must compactly store and yet efficiently read the huge amounts of genetic information contained in their DNA. Many DNA-based enzymes involved in these processes function as highly processive molecular motors capable of translocating over thousands of base pairs without detaching from the DNA template. These motors face mechanical obstacles to their movement, especially in the highly packed DNA of chromatin, and many of these obstacles are known to be important regulators of gene expression. The broad goal of this proposal is to address the question of how these DNA-based motors deal with these obstacles. DNA in chromatin is highly compact as compared to naked DNA. The primary packing unit of chromatin, the nucleosome, consists of roughly two turns of DNA wrapped around a core histone octamer. The molecular mechanism by which RNA polymerase deals with nucleosomes during transcription is not fully understood at the molecular level and remains one of the most fundamental questions in biology. We propose a unique, single-molecule, biophysical approach to address the question of how RNA polymerase deals with nucleosomes. The proposed method combines optical trapping with nanometer-precision position detection techniques, and complements ongoing biochemical and structural studies. This approach, which has proven to be a powerful tool for the study of transcription on naked DNA, will provide direct measurements and visualization of individual molecular events of transcription in chromatin in vitro.
Two specific aims are proposed: (1) mechanical stability of DNA associated with nucleosomes, and (2) transcription through nucleosomes.
Aim number 1 determines the strength of histone-DNA interactions by stretching a nucleosomal DNA from end-to-end and measuring the tension required to disrupt the nucleosomes.
Aim number 2 makes a direct observation of the fate of a nucleosome during an encounter with a transcribing RNA polymerase by monitoring the movement of single molecules of RNA polymerase during transcription and simultaneously detecting possible nucleosome disruption events. Using these methods, we will determine the effects of histone acetylation and chromatin remodeling complexes on transcription through nucleosomal DNA. This proposed basic research will help to elucidate the mechanisms of transcription in eukaryotes, and will further establish the technical foundation for mechanical studies of other nucleic acid-based molecular motors at the single molecule level.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM059849-03
Application #
6386596
Study Section
Molecular Biology Study Section (MBY)
Program Officer
Lewis, Catherine D
Project Start
1999-07-01
Project End
2004-06-30
Budget Start
2001-07-01
Budget End
2002-06-30
Support Year
3
Fiscal Year
2001
Total Cost
$262,506
Indirect Cost
Name
Cornell University
Department
Physics
Type
Schools of Arts and Sciences
DUNS #
City
Ithaca
State
NY
Country
United States
Zip Code
14850
Brennan, Lucy D; Forties, Robert A; Patel, Smita S et al. (2016) DNA looping mediates nucleosome transfer. Nat Commun 7:13337
Nodelman, Ilana M; Horvath, Kyle C; Levendosky, Robert F et al. (2016) The Chd1 chromatin remodeler can sense both entry and exit sides of the nucleosome. Nucleic Acids Res 44:7580-91
Sun, Bo; Pandey, Manjula; Inman, James T et al. (2015) T7 replisome directly overcomes DNA damage. Nat Commun 6:10260
Li, Ming; Hada, Arjan; Sen, Payel et al. (2015) Dynamic regulation of transcription factors by nucleosome remodeling. Elife 4:
Inman, James T; Smith, Benjamin Y; Hall, Michael A et al. (2014) DNA Y structure: a versatile, multidimensional single molecule assay. Nano Lett 14:6475-80
Soltani, Mohammad; Lin, Jun; Forties, Robert A et al. (2014) Nanophotonic trapping for precise manipulation of biomolecular arrays. Nat Nanotechnol 9:448-52
Brennan, Lucy D; Roland, Thibault; Morton, Diane G et al. (2013) Small molecule injection into single-cell C. elegans embryos via carbon-reinforced nanopipettes. PLoS One 8:e75712
Sheinin, Maxim Y; Li, Ming; Soltani, Mohammad et al. (2013) Torque modulates nucleosome stability and facilitates H2A/H2B dimer loss. Nat Commun 4:2579
Dame, Remus T; Hall, Michael A; Wang, Michelle D (2013) Single-molecule unzipping force analysis of HU-DNA complexes. Chembiochem 14:1954-7
Ma, Jie; Bai, Lu; Wang, Michelle D (2013) Transcription under torsion. Science 340:1580-3

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