Single-Molecule Studies of Homologous Recombination
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Eric Greene, Ph.D.
Professor Department of Biochemistry & Molecular Biophysics
Columbia University Medical Center
Our group uses single-molecule optical microscopy to study fundamental interactions between proteins and nucleic acids. Our overall goal is to reveal the molecular mechanisms that cells use to repair, maintain, and decode their genetic information. This research combines aspects of biochemistry, physics, and nanoscale technology to answer questions about complex biological problems that cannot be easily addressed through traditional biochemical approaches. As part of our work, we have established robust experimental platforms that enable single molecule imaging of biochemical reaction mechanisms in a “high throughput” experimental format that can be applied to the study of protein-nucleic acid interactions. The advantages of our approaches are that we can see what proteins are bound to DNA, where they are bound, how they move, and how they interact with and influence other components of the system – all in real-time, at the level of a single reaction. We are applying this technology towards determining the physical basis for the mechanisms that proteins use to maintain genome integrity, with particular emphasis on reactions related to homologous DNA recombination.
Summary
Homologous recombination (HR) is essential for maintenance of genome integrity. Rad51 paralogs fulfill a conserved but undefined role in HR, and their mutations are associated with increased cancer risk in humans. We have used single-molecule imaging to reveal that the Saccharomyces cerevisiae Rad51 paralog complex Rad55-Rad57 promotes assembly of Rad51 recombinase filament through transient interactions, providing evidence that it acts like a classical molecular chaperone. Srs2 is an ATP-dependent anti-recombinase that downregulates HR by actively dismantling Rad51 filaments. Contrary to the current model, we find that Rad55-Rad57 does not physically block the movement of Srs2. Instead, Rad55-Rad57 promotes rapid re-assembly of Rad51 filaments after their disruption by Srs2. Our findings support a model in which Rad51 is in flux between free and single-stranded DNA (ssDNA)-bound states, the rate of which is controlled dynamically though the opposing actions of Rad55-Rad57 and Srs2.
This page was last updated on Wednesday, May 18, 2022