Abstract
Repair of DNA double-stranded breaks is essential for maintaining genome integrity and is executed by homologous recombination (HR). In humans, the core catalyst of HR is RAD51, a bacterial RecA-like ATP-dependent recombinase. It forms nucleoprotein filaments around single-stranded DNA (ssDNA) that catalyze strand exchange between regions with homologous DNA sequences. At the RAD51 monomer-monomer interface, ATP binds, is hydrolyzed and dictates the structure and conformation of the filaments. Using a combination of optical trapping and fluorescence microscopy, we have studied how modifications in the monomer-monomer interface may influence filament dynamics at the single molecule level.We observed that two naturally occurring polymorphic variants of RAD51 (RAD51-Q313 and RAD51-K313) behave dramatically different. RAD51-Q313 nuclei bind statically to ssDNA, with small nuclei bound for a shorter time than larger ones. RAD51-K313, on the other hand, can slide and hop along ssDNA. More specifically, we show that RAD51-K313 monomers predominantly move in a diffusive way, while larger nuclei are more likely to bind statically to ssDNA. These results indicate that subtle differences in the local environment of the monomer-monomer interface can have a dramatic impact on filament dynamics, making this interface a likely target for control by mediators or post-translational modifications.In addition, we have analyzed how RAD51-ssDNA filaments respond to tension. We were able to separate the effects of ATP hydrolysis from filament disassembly, revealing a force-induced structural change in RAD51-ssDNA filaments. If exposed to forces above 9 pN, ADP-bound RAD51, naturally in a compressed state, switches to an extended conformation, resembling the one active for strand exchange. We reveal that the energy needed for this transition is between 3 and 4 kBT. Thermal fluctuations can therefore supply sufficient energy to drive this molecular transition, possibly playing a vital role in strand exchange.
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