Single-molecule Visualization and Mechanisms of DNA Recombination and Intersecting DNA Transactions

Project Description The broad objective of this proposal is to understand the mechanism of homologous recombination and its relationship to chromosome maintenance in both Bacteria and Eukarya. Our approach is to reconstitute increasingly more complex reactions that recapitulate the steps of the homologous recombination process, using purified proteins from Escherichia coli and from Saccharomyces cerevisiae, and to use sophisticated biochemical and biophysical methods of analysis. In particular, we will use single-molecule visualization to image key steps and intermediates of these complex biological processes. We have developed new experimental strategies for studying protein-DNA interactions at the single-molecule level. We devised several approaches wherein we can easily manipulate an individual DNA molecule and expose it to a variety of different proteins, enzymes, and buffers. Using fluorescent detection, we can image individual proteins on DNA or detect their action on individual DNA molecules, typically in real-time. We propose to continue to use these approaches to study unresolved aspects of recombinational DNA repair and its interface with the important biological processes of chromatin remodeling and DNA replication. We seek to continue to address unresolved questions regarding the dynamic behavior of RecA/Rad51 nucleoprotein filaments and their regulation by protein mediators and modulators; the mechanism by which RecA and Rad51 find DNA sequence homology; the reconstitution increasingly more complex recombination processes using purified proteins; the mechanism and function of DNA helicases and their partner proteins; and understanding the mechanism of DNA transactions that intersect with recombination, namely, chromatin remodeling and DNA replication.