Functional Architecture of Molecular Complexes Involved in DNA Double-strand Break Repair

Homologous recombination, the exchange of strands between homologous DNA molecules, is a universal aspect of genome metabolism needed to repair DNA double strand breaks, to ensure proper replication and chromosome segregation and to create genetic diversity. The defining mechanistic steps of homologous recombination are homology search, strand invasion and joint molecule formation. These reactions are catalyzed by a class of proteins called recombinases, including bacterial RecA, the RadA homologs in archea and the Rad51 homologs in eukaryotes. The catalytic core of these reactions is the recombinase protein assembled into a helical nucleoprotein filament on the invading DNA. We described the effect of reaction conditions that influence in vitro recombination on the structure of human Rad51 nucleoprotein filaments by directly observing and characterizing filaments with scanning force microscopy. In all cases conditions that enhance in vitro recombination activity result in regular and stable filaments on dsDNA with elongated DNA. In addition, human Rad51 filaments are irregular and apparently unstable in the presence of ATP. Disassembly of filaments immobilized on mica was followed over time revealing that protein disassociation occurs all over the filament with no directionality or end effects. Preliminary single molecule dynamic studies of human Rad51 filament assembly and disassembly also showed that stable filaments formed in conditions that favour in vitro strand exchange reactions. As well, the kinetics of filament disassembly followed in solution in real time indicated disassociation of human Rad51 protomers form many points at once not coordinately from an end of the filament.