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Small Rad51 and Dmc1 Complexes Often Co-occupy Both Ends of a Meiotic DNA Double Strand Break.

Brown MS, Grubb J, Zhang A, Rust MJ, Bishop DK - PLoS Genet. (2015)

Bottom Line: Previous studies showed that Rad51 and Dmc1 form partially overlapping co-foci.Here we show these Rad51-Dmc1 co-foci are often arranged in pairs separated by distances of up to 400 nm.The data support a model in which the two tracts of ssDNA formed by a single DSB separate from one another by distances of up to 400 nm, with both tracts often bound by one or more short (about 100 nt) Rad51 filaments and also by one or more short Dmc1 filaments.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Cell Biology, University of Chicago, Cummings Life Science Center, Chicago, Illinois, United States of America.

ABSTRACT
The Eukaryotic RecA-like proteins Rad51 and Dmc1 cooperate during meiosis to promote recombination between homologous chromosomes by repairing programmed DNA double strand breaks (DSBs). Previous studies showed that Rad51 and Dmc1 form partially overlapping co-foci. Here we show these Rad51-Dmc1 co-foci are often arranged in pairs separated by distances of up to 400 nm. Paired co-foci remain prevalent when DSBs are dramatically reduced or when strand exchange or synapsis is blocked. Super-resolution dSTORM microscopy reveals that individual foci observed by conventional light microscopy are often composed of two or more substructures. The data support a model in which the two tracts of ssDNA formed by a single DSB separate from one another by distances of up to 400 nm, with both tracts often bound by one or more short (about 100 nt) Rad51 filaments and also by one or more short Dmc1 filaments.

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The paired architecture of Rad51-Dmc1 co-foci is independent of strand exchange and synapsis.(A,B) Micrographs of (A)mnd1 and (B)zip1 mutant nuclei in the spo11 hypomorphic tetraploid background. Scale bar = 1 μm, 400 nm for the inset. Rad51 staining is shown in green, Dmc1 in red, and DAPI in blue. (C-F) Accompanying distribution of nearest neighbor measurements as in Fig 1 for (C) Rad51→Rad51, (D) Dmc1→Dmc1, (E) Rad51→Dmc1, and (F) Dmc1→Rad51. Micrographs from cultures 2.5 hours after meiotic induction. Wild type data repeated from Fig 1 for comparison. Scoring of 70 and 44 focus-positive nuclei; 6,364 and 2,824 Rad51 foci; and 6,114 and 2,655 Dmc1 foci are included in histograms for mnd1 and zip1 mutants, respectively.
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pgen.1005653.g003: The paired architecture of Rad51-Dmc1 co-foci is independent of strand exchange and synapsis.(A,B) Micrographs of (A)mnd1 and (B)zip1 mutant nuclei in the spo11 hypomorphic tetraploid background. Scale bar = 1 μm, 400 nm for the inset. Rad51 staining is shown in green, Dmc1 in red, and DAPI in blue. (C-F) Accompanying distribution of nearest neighbor measurements as in Fig 1 for (C) Rad51→Rad51, (D) Dmc1→Dmc1, (E) Rad51→Dmc1, and (F) Dmc1→Rad51. Micrographs from cultures 2.5 hours after meiotic induction. Wild type data repeated from Fig 1 for comparison. Scoring of 70 and 44 focus-positive nuclei; 6,364 and 2,824 Rad51 foci; and 6,114 and 2,655 Dmc1 foci are included in histograms for mnd1 and zip1 mutants, respectively.

Mentions: To probe the relationship between the paired co-focus architecture and both the progression of recombination reactions and transitions in global chromosome structure, nearest neighbor distributions were determined in strand exchange-defective (mnd1) and synapsis-defective (zip1) mutants. When strand exchange was blocked in an mnd1 mutant[32–34], the paired character of Rad51 (and Dmc1) foci was maintained (Fig 3A and 3C and 3D). The pairing of Rad51 (and Dmc1) foci was also unaltered in the zip1 mutant which blocks the progress of recombination reactions after strand exchange (Fig 3B–3D) [36–38]. In zip1 mutants, nascent post-strand exchange intermediates promote homolog co-alignment at a distance of 400 nm or less, but the closer 100 nm alignment of lateral elements resulting from elongation of the synaptonemal complex does not occur[38]. Together, these mutants suggest that the ≤400 nm paired Rad51-Dmc1 co-focus architecture does not require strand exchange and is retained until synapsis. Further analysis demonstrated that the nearest neighbor distributions of Rad51-to-Dmc1 and Dmc1-to-Rad51 distances are also unaltered in strand exchange-defective and synapsis-defective mutants (Fig 3E and 3F), suggesting that the side-by-side Rad51-Dmc1 configuration is also established prior to, and persists after, strand exchange.


Small Rad51 and Dmc1 Complexes Often Co-occupy Both Ends of a Meiotic DNA Double Strand Break.

Brown MS, Grubb J, Zhang A, Rust MJ, Bishop DK - PLoS Genet. (2015)

The paired architecture of Rad51-Dmc1 co-foci is independent of strand exchange and synapsis.(A,B) Micrographs of (A)mnd1 and (B)zip1 mutant nuclei in the spo11 hypomorphic tetraploid background. Scale bar = 1 μm, 400 nm for the inset. Rad51 staining is shown in green, Dmc1 in red, and DAPI in blue. (C-F) Accompanying distribution of nearest neighbor measurements as in Fig 1 for (C) Rad51→Rad51, (D) Dmc1→Dmc1, (E) Rad51→Dmc1, and (F) Dmc1→Rad51. Micrographs from cultures 2.5 hours after meiotic induction. Wild type data repeated from Fig 1 for comparison. Scoring of 70 and 44 focus-positive nuclei; 6,364 and 2,824 Rad51 foci; and 6,114 and 2,655 Dmc1 foci are included in histograms for mnd1 and zip1 mutants, respectively.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4697796&req=5

pgen.1005653.g003: The paired architecture of Rad51-Dmc1 co-foci is independent of strand exchange and synapsis.(A,B) Micrographs of (A)mnd1 and (B)zip1 mutant nuclei in the spo11 hypomorphic tetraploid background. Scale bar = 1 μm, 400 nm for the inset. Rad51 staining is shown in green, Dmc1 in red, and DAPI in blue. (C-F) Accompanying distribution of nearest neighbor measurements as in Fig 1 for (C) Rad51→Rad51, (D) Dmc1→Dmc1, (E) Rad51→Dmc1, and (F) Dmc1→Rad51. Micrographs from cultures 2.5 hours after meiotic induction. Wild type data repeated from Fig 1 for comparison. Scoring of 70 and 44 focus-positive nuclei; 6,364 and 2,824 Rad51 foci; and 6,114 and 2,655 Dmc1 foci are included in histograms for mnd1 and zip1 mutants, respectively.
Mentions: To probe the relationship between the paired co-focus architecture and both the progression of recombination reactions and transitions in global chromosome structure, nearest neighbor distributions were determined in strand exchange-defective (mnd1) and synapsis-defective (zip1) mutants. When strand exchange was blocked in an mnd1 mutant[32–34], the paired character of Rad51 (and Dmc1) foci was maintained (Fig 3A and 3C and 3D). The pairing of Rad51 (and Dmc1) foci was also unaltered in the zip1 mutant which blocks the progress of recombination reactions after strand exchange (Fig 3B–3D) [36–38]. In zip1 mutants, nascent post-strand exchange intermediates promote homolog co-alignment at a distance of 400 nm or less, but the closer 100 nm alignment of lateral elements resulting from elongation of the synaptonemal complex does not occur[38]. Together, these mutants suggest that the ≤400 nm paired Rad51-Dmc1 co-focus architecture does not require strand exchange and is retained until synapsis. Further analysis demonstrated that the nearest neighbor distributions of Rad51-to-Dmc1 and Dmc1-to-Rad51 distances are also unaltered in strand exchange-defective and synapsis-defective mutants (Fig 3E and 3F), suggesting that the side-by-side Rad51-Dmc1 configuration is also established prior to, and persists after, strand exchange.

Bottom Line: Previous studies showed that Rad51 and Dmc1 form partially overlapping co-foci.Here we show these Rad51-Dmc1 co-foci are often arranged in pairs separated by distances of up to 400 nm.The data support a model in which the two tracts of ssDNA formed by a single DSB separate from one another by distances of up to 400 nm, with both tracts often bound by one or more short (about 100 nt) Rad51 filaments and also by one or more short Dmc1 filaments.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Cell Biology, University of Chicago, Cummings Life Science Center, Chicago, Illinois, United States of America.

ABSTRACT
The Eukaryotic RecA-like proteins Rad51 and Dmc1 cooperate during meiosis to promote recombination between homologous chromosomes by repairing programmed DNA double strand breaks (DSBs). Previous studies showed that Rad51 and Dmc1 form partially overlapping co-foci. Here we show these Rad51-Dmc1 co-foci are often arranged in pairs separated by distances of up to 400 nm. Paired co-foci remain prevalent when DSBs are dramatically reduced or when strand exchange or synapsis is blocked. Super-resolution dSTORM microscopy reveals that individual foci observed by conventional light microscopy are often composed of two or more substructures. The data support a model in which the two tracts of ssDNA formed by a single DSB separate from one another by distances of up to 400 nm, with both tracts often bound by one or more short (about 100 nt) Rad51 filaments and also by one or more short Dmc1 filaments.

Show MeSH
Related in: MedlinePlus