Replication, transcription, repair and maintenance of the genome are processes critical for maintaining cellular viability. In eukaryotes, these functions are carried out on DNA that is organized into highly condensed chromatin. Specialized proteins must be employed to modify the structure of chromatin before other essential factors can access the DNA. With the identification of an increasing number of disease-associated genes, the importance of chromatin in human disorders has become abundantly clear, and several diseases have been linked to defects in chromatin remodeling. For example, mutations associated with the Snf2 family of chromatin remodeling proteins can cause debilitating genetic disorders and are associated with cancers. Understanding the basic properties of the proteins involved in these processes will be essential for revealing the underlying defects that produce the disease phenotypes. However, the mechanistic details of chromatin remodeling have remained elusive. This is because most methods used for studying remodeling can only probe the initial and final states of nucleosomes, and often don't reveal dynamic aspects of the reactions. Our hypothesis is that Snf2 proteins destabilize nucleosomes and other nucleoprotein complexes via a mechanism coupled to DNA translocation, thereby allowing other cellular components to gain unimpeded access to the DNA. To test this hypothesis we will use a single-molecule optical microscopy assay to directly visualize the Snf2 remodeling proteins Rdh54 and RSC as they interact with both naked DNA and also with chromatin substrates. These experiments will rely on a new technology developed in the Greene laboratory, which allows us to directly visualize hundreds of individual protein-DNA interactions in real time. The benefits of this approach are that we can probe the dynamics of chromatin remodeling in a format that allows us to rapidly gather statistically relevant information from hundreds of individual biomolecules and we can study these reactions with an unprecedented level of detail.

Public Health Relevance

Defects in chromatin remodeling enzymes can result in extremely severe human diseases, which is consistent with their roles as global regulators of genome structure. As a first step towards developing targeted therapies that can be used to effectively prevent or cure these debilitating disorders it is essential to understand the basic biochemical properties of the chromatin remodeling proteins themselves. To help extend our understanding of chromatin remodeling we have developed fluorescence-based approaches for directly observing the interactions between proteins and individual DNA molecules, and our emphasis is placed on understanding biochemical reactions relevant to human biology and disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM082848-02
Application #
7649463
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Preusch, Peter C
Project Start
2008-07-15
Project End
2012-06-30
Budget Start
2009-07-01
Budget End
2010-06-30
Support Year
2
Fiscal Year
2009
Total Cost
$305,172
Indirect Cost
Name
Columbia University (N.Y.)
Department
Biochemistry
Type
Schools of Medicine
DUNS #
621889815
City
New York
State
NY
Country
United States
Zip Code
10032
Erdel, Fabian; Kratz, Katja; Willcox, Smaranda et al. (2017) Telomere Recognition and Assembly Mechanism of Mammalian Shelterin. Cell Rep 18:41-53
Stigler, Johannes; Çamdere, Gamze Ö; Koshland, Douglas E et al. (2016) Single-Molecule Imaging Reveals a Collapsed Conformational State for DNA-Bound Cohesin. Cell Rep 15:988-998
Erdel, Fabian; Greene, Eric C (2016) Generalized nucleation and looping model for epigenetic memory of histone modifications. Proc Natl Acad Sci U S A 113:E4180-9
Qi, Zhi; Greene, Eric C (2016) Visualizing recombination intermediates with single-stranded DNA curtains. Methods 105:62-74
Greene, Eric C (2016) On the influence of protein-DNA register during homologous recombination. Cell Cycle 15:172-5
Qi, Zhi; Redding, Sy; Lee, Ja Yil et al. (2015) DNA sequence alignment by microhomology sampling during homologous recombination. Cell 160:856-69
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Lee, Ja Yil; Finkelstein, Ilya J; Arciszewska, Lidia K et al. (2014) Single-molecule imaging of FtsK translocation reveals mechanistic features of protein-protein collisions on DNA. Mol Cell 54:832-43
Silverstein, Timothy D; Gibb, Bryan; Greene, Eric C (2014) Visualizing protein movement on DNA at the single-molecule level using DNA curtains. DNA Repair (Amst) 20:94-109
Duzdevich, Daniel; Redding, Sy; Greene, Eric C (2014) DNA dynamics and single-molecule biology. Chem Rev 114:3072-86

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