Assembly and disassembly of Rad51 filaments on single-stranded DNA: A novel assay to study the dynamics of protein-ssDNA interactions at the single-molecule level

Eukaryotic recombinase protein Rad51 is the key player in homologous recombination, an essential DNA repair mechanism used for the repair of double-strand breaks. Double-strand breaks can lead to chromosome fragmentation and are particularly hazardous during and shortly after DNA replication. The mechanism of homologous recombination is highly conserved between species and recombinase proteins are expressed in a wide range of prokaryotic and eukaryotic cells. The primary event in homologous recombination is the formation of a helical nucleoprotein filament on single- stranded DNA overhangs around double-strand breaks. The nucleoprotein filament mediates all subsequent steps of homologous recombination and is capable of performing strand exchange reactions unassisted in vitro. Dynamic assembly and disassembly interactions between the nucleoprotein filament and its DNA substrate are essential for strand exchange. Investigating the Rad51 – single-stranded DNA complex presents numerous challenges and it has never been observed directly. This study presents a novel assay to investigate these interactions at the single- molecule level. A combination of dual-beam optical trapping and high-resolution fluorescence microscopy allows in situ creation and manipulation of single-stranded DNA molecules, while at the same time binding of fluorescently labeled Rad51 proteins can be observed directly. Using this novel single-molecule approach, it is possible to distinguish between the different steps of filament formation: nucleation, extension and disassembly. We are able to simultaneously interrogate the molecular mechanisms of filament assembly and disassembly, and determine nucleation, extension and disassembly rates as well as binding unit sizes without any a priori assumptions. Furthermore, we are able to investigate the effects of tension on the various stages of filament formation and observe Rad51-induced forces in real time. INSTITUTION: VU University Amsterdam, Faculty of Sciences, Department of Physics and Astronomy, Physics of Living Systems