Single-molecule localization microscopy reveals molecular transactions during RAD51 filament assembly at cellular DNA damage sites.

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Haas, Kalina T 
Lee, MiYoung 
Esposito, Alessandro  ORCID logo
Venkitaraman, Ashok R 

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.

BRCA2 Protein, Cell Line, DNA Damage, DNA Repair, DNA, Single-Stranded, HeLa Cells, Humans, Kinetics, Microscopy, Rad51 Recombinase, Replication Protein A
Journal Title
Nucleic Acids Res
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Oxford University Press (OUP)
Wellcome Trust (090340/Z/09/Z)
MRC (unknown)
MRC (4050551988)
Medical Research Council (G1001522)
Medical Research Council (MC_UU_12022/1)
MRC (MC_UU_12022/8)