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.
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.
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