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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM082848-05A1
Application #
8443645
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Preusch, Peter C
Project Start
2008-07-15
Project End
2016-11-30
Budget Start
2013-01-01
Budget End
2013-11-30
Support Year
5
Fiscal Year
2013
Total Cost
$303,665
Indirect Cost
$112,264
Name
Columbia University (N.Y.)
Department
Biochemistry
Type
Schools of Medicine
DUNS #
621889815
City
New York
State
NY
Country
United States
Zip Code
10032
Qi, Zhi; Greene, Eric C (2016) Visualizing recombination intermediates with single-stranded DNA curtains. Methods 105:62-74
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
Greene, Eric C (2016) On the influence of protein-DNA register during homologous recombination. Cell Cycle 15:172-5
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 15:988-98
Duzdevich, Daniel; Warner, Megan D; Ticau, Simina et al. (2015) The dynamics of eukaryotic replication initiation: origin specificity, licensing, and firing at the single-molecule level. Mol Cell 58:483-94
Qi, Zhi; Redding, Sy; Lee, Ja Yil et al. (2015) DNA sequence alignment by microhomology sampling during homologous recombination. Cell 160:856-69
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
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
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
Duzdevich, Daniel; Redding, Sy; Greene, Eric C (2014) DNA dynamics and single-molecule biology. Chem Rev 114:3072-86

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