Single-Molecule Assembly of Protein-DNA Complexes

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 DESCRIPTION (provided by applicant): The broad objective of this proposal is to visualize and quantify the dynamic behavior of biological processes at the single-molecule level. We have developed new experimental strategies for studying protein-DNA interactions at the single-molecule level. We devised several approaches wherein we could easily manipulate an individual DNA molecule and expose it to a variety of different proteins, enzymes, and buffers. Using fluorescent detection, we could image individual proteins on DNA or detect their action on individual DNA molecules, typically in real-time. We propose to continue to use this approach to study unresolved aspects of recombinational DNA repair, chromatin remodeling, and DNA replication. We seek to continue to address unresolved questions regarding the assembly and disassembly of RecA/Rad51 nucleoprotein filaments, their control and function in homology-directed DNA repair. In addition, we propose to expand our interests and success in the area of chromatin remodeling by motor proteins. And finally, we have initiated studies on single-molecule imaging of DNA replication, and propose to address important issues that intersect with recombinational DNA repair.