DNA replication, transcription, repair, epigenetic inheritance, and chromosome segregation are all processes critical for maintaining cellular viability. In eukaryotes, these functions must be carried out on DNA that is organized into highly condensed chromatin. 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 biology. To help understand how different aspects of DNA metabolism are influenced by chromatin we have developed unique technologies that allow us to directly visualize hundreds of individual DNA molecules at the single molecule level using optical microscopy. Here we will assess how nucleosomes influence the spatial and temporal progression of reactions related to DNA metabolism by visualizing these processes in real time using single molecule optical microscopy. We will analyze factors that influence nucleosome positioning, we will determine how nucleosomes affect DNA repair proteins, we will ask how nucleosomes are affected by interactions with DNA motor proteins, and we will begin working towards a mechanistic understanding of epigenetic phenomena. We will seek to determine detailed mechanistic information related to these questions, and part of the significance of this project lies in the depth of the answers we strive to obtain.
Defects in chromatin biology can result in extremely severe human diseases, and represent a hallmark of cancer. As a first step towards developing targeted therapies that can be used to effectively prevent or cure these disorders it is essential to understand the basic biochemical properties of chromatin and its impact on DNA metabolism. To help extend our understanding the relationship between chromatin and DNA metabolism we have developed optical microscopy-based approaches for directly observing the interactions between proteins and individual DNA molecules, which allows us to address questions that cannot be tackled with more traditional approaches, and our emphasis is placed on understanding biochemical reactions relevant to human biology and disease.
|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|
|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|
|Collins, Bridget E; Ye, Ling F; Duzdevich, Daniel et al. (2014) DNA curtains: novel tools for imaging protein-nucleic acid interactions at the single-molecule level. Methods Cell Biol 123:217-34|
|Duzdevich, Daniel; Redding, Sy; Greene, Eric C (2014) DNA dynamics and single-molecule biology. Chem Rev 114:3072-86|
|Finkelstein, Ilya J; Greene, Eric C (2013) Molecular traffic jams on DNA. Annu Rev Biophys 42:241-63|
|Redding, Sy; Greene, Eric C (2013) How do proteins locate specific targets in DNA? Chem Phys Lett 570:|
|Wang, Feng; Greene, Eric C (2011) Single-molecule studies of transcription: from one RNA polymerase at a time to the gene expression profile of a cell. J Mol Biol 412:814-31|
|Finkelstein, Ilya J; Visnapuu, Mari-Liis; Greene, Eric C (2010) Single-molecule imaging reveals mechanisms of protein disruption by a DNA translocase. Nature 468:983-7|
|Gorman, Jason; Plys, Aaron J; Visnapuu, Mari-Liis et al. (2010) Visualizing one-dimensional diffusion of eukaryotic DNA repair factors along a chromatin lattice. Nat Struct Mol Biol 17:932-8|
|Visnapuu, Mari-Liis; Greene, Eric C (2009) Single-molecule imaging of DNA curtains reveals intrinsic energy landscapes for nucleosome deposition. Nat Struct Mol Biol 16:1056-62|