Substrate Specificity and Dynamic Instability of RAD51-Filament Assembly on Single- and Double-Stranded DNA

Abstract

Homologous recombination (HR) is an essential DNA-repair strategy present in all life forms. The core machinery catalyzing this transaction in human cells is the recombinase protein RAD51, bound as a helical filament on single-stranded DNA (ssDNA). Here we use a combination of single-molecule fluorescence microscopy, optical tweezers and micro-fluidics to directly visualize the assembly and disassembly of RAD51 filaments on ssDNA with single monomer resolution. This approach allows us to quantify rates of nucleation and growth as well as the size of the nucleation unit. Our findings show that RAD51 can select with high specificity between ssDNA and double-stranded DNA by sensing their different mechanical properties (see figure). We also observed that RAD51 nuclei are characterized by dynamic instabilities, whose occurrence depends on the actual size of the nascent RAD51 filament. Our findings provide the basis for a molecular description of the RAD51-ssDNA assembly mechanism, which is essential to understand the role of accessory proteins, such as BRCA2 that mediate and regulate RAD51 filament formation in vivo.View Large Image | View Hi-Res Image | Download PowerPoint Slide