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Abstract

DNA double strand break (DSB) repair is essential to meiotic chromosome segregation. Rad51 and Dmc1 are DNA strand exchange proteins that cooperate in meiotic homologous recombination. Both proteins share very similar capabilities in vitro, but are functionally differentiated in vivo. Dmc1 is the catalytic recombinase; Rad51 plays a non-enzymatic accessory role, promoting Dmc1 assembly and directing recombination partner choice. The molecular architecture underlying this cooperation and functional differentiation is unknown. To better understand the mechanistic relationship between these meiotic recombinases, the structures of Rad51 and Dmc1 complexes were extensively characterized cytologically. Rad51-Dmc1 co-foci commonly occur in pairs, separated by distances of up to 400 nm. Observations from spo11 hypomorphic tetraploids, spo11 mutants heterozygous for a VDE cut site, cytologically marked DSB hotpots, and spatial simulations suggest that a pair of Rad51-Dmc1 co-foci represent a single meiotic DSB. Co-focus pairing occurs at distances similar to that which separates sister chromatids but requires neither strand exchange nor synapsis. These results suggest that: 1. both Rad51 and Dmc1 co-occupy both ends of a meiotic DSB and 2. the two ends of a DSB are spatially separated. Super-resolution direct stochastic optical reconstruction microscopy (dSTORM) reveals that Rad51 and Dmc1 filaments are extremely short and clustered in vivo. Meiotic Rad51 and Dmc1 super-resolution (sr) foci are little more than 100 nm long, corresponding to filaments a mere 40 protomers long or shorter. Additionally, multiple Rad51 and Dmc1 filaments likely occupy a single ssDNA tract. Careful controls demonstrated that these structural attributes are not artifacts of the imaging procedure and led to the correction of an artifact generated during super-resolution image reconstruction. These results suggest that multiple, short Rad51 and Dmc1 filaments co-occupy a single tract of ssDNA. In an effort to observe DNA recombination complexes in living cells, Rad51, Dmc1, and Rad52 were fused to fluorescent proteins or tetracysteine tags. The functionality of these proteins was characterized in mitotic and meiotic cells. Comparison of different cytological preparations suggests that the majority of recombination complexes are too small to be observed above background fluorescence in live cells. These results call into question the “recombination factory” paradigm and provide tools for future live cell imaging studies.

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