Single-Molecule Assembly of Protein-DNA Complexes

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DESCRIPTION (provided by applicant): The major long-term objective of this grant proposal is to understand the behavior of proteins involved in recombinational DNA repair at the level single protein-DNA complexes. This proposal takes advantage of a novel single-molecule approach that can literally visualize the dynamics and function of individual complexes of proteins and DNA. Several different protein-DNA complexes will be examined; each is an essential component of the DNA recombination process. One class of proteins that will be examined is the DNA strand exchange proteins, RecA and Rad51; and the second is the class of proteins that modify RecA/Rad51 nucleoprotein filament dynamics; and the third is the nucleoprotein- and chromatin-remodeling translocase, Rad54 protein. The specific aims are to: 1) Visualize and measure the assembly, disassembly, and polarity of RecA and Rad51 nucleoprotein filament formation; 2) Observe the role of mediator proteins such as SSB/RPA, RecFOR, Rad52, Rad51 paralogs, and BRCA2 on RecA/Rad51 filament assembly; and 3) Define the function and consequences of Rad54 protein translocation along dsDNA. Each of these proteins is involved in the repair of DNA breaks by recombination, a process whose mechanism is not fully understood. Left unrepaired, DNA breaks result in genomic instabilities that give rise to cancers. Mutations in the human counterparts of these proteins result in predispositions to cancer, aberrant meiosis, and embryonic lethality. Consequently, a detailed molecular understanding of recombinational DNA is necessary to understand the functions of the many proteins involved. Recently, new methods of visualizing the action of these repair enzymes on single-molecules of DNA have been developed. These methods can provide an unprecedented level of understanding of the real-time behavior of these intricate processes. These single-molecule methods will be used to define some of the molecular events comprising increasingly complicated biochemical processes involved in the repair of DNA by recombination.