Abstract
RAD51 recombinase assembles on single-stranded (ss)DNA substrates exposed by DNA end-resection to initiate homologous recombination (HR), a process fundamental to genome integrity. RAD51 assembly has been characterized using purified proteins, but its ultrastructural topography in the cell nucleus is unexplored. Here, we combine cell genetics with single-molecule localization microscopy and a palette of bespoke analytical tools, to visualize molecular transactions during RAD51 assembly in the cellular milieu at resolutions approaching 30–40 nm. In several human cell types, RAD51 focalizes in clusters that progressively extend into long filaments, which abut—but do not overlap—with globular bundles of replication protein A (RPA). Extended filaments alter topographically over time, suggestive of succeeding steps in HR. In cells depleted of the tumor suppressor protein BRCA2, or overexpressing its RAD51-binding BRC repeats, RAD51 fails to assemble at damage sites, although RPA accumulates unhindered. By contrast, in cells lacking a BRCA2 carboxyl (C)-terminal region targeted by cancer-causing mutations, damage-induced RAD51 assemblies initiate but do not extend into filaments. We suggest a model wherein RAD51 assembly proceeds concurrently with end-resection at adjacent sites, via an initiation step dependent on the BRC repeats, followed by filament extension through the C-terminal region of BRCA2.
Highlights
homologous recombination (HR) preserves genomic integrity through its role in DNA double-strand break (DSB) repair
Our observations suggest a model for HR in which (a) DNA end-resection and RAD51 filament assembly at each cellular DSB occur concurrently at adjacent but non-overlapping sites, (b) BRC repeats are necessary for initial RAD51 filament formation at sites of damage and (c) RAD51 filament extension is critically dependent on the C-terminal region of BRCA2
We have used direct STochastic Optical Reconstruction Microscopy (d-STORM), a well-established super-resolution optical nanoscopy technique, to dissect cellular events leading to HR. d-STORM is a powerful tool to study molecular complexes inside fixed cells at resolution approaching tens of nanometers [62,63,64,65,66]. d-STORM provides coordinates of molecular localization, but usually d-STORM data are represented as pixelated images, often limiting analysis of the spatial organization of the molecules being visualized
Summary
HR preserves genomic integrity through its role in DNA double-strand break (DSB) repair (reviewed in [1,2]). Biochemical [12,13,14,15,16] and cytological studies carried out in meiotic [17] or mitotic [18] yeast cells suggest that these tracts are rapidly bound by Replication protein A (RPA), an abundant ssDNA-binding protein, which regulates the activity of end-resecting nucleases, and protects ssDNA [11,19,20]. Human RAD51 assembly has been characterized in vitro with biochemical and structural studies using purified proteins [22,23,24,25,26], which suggest that the tumor suppressor protein, BRCA2, is an essential mediator that targets RAD51 to appropriate DNA substrates to correctly order the biochemical reactions leading to HR [27,28,29,30,31,32]
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