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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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Rad51 and Dmc1 sr foci are extremely small and clustered at sub-diffraction distances.(A-D) dSTORM micrographs of Rad51 filaments assembled on a linear 2.7 kbp dsDNA in vitro in the presence of the meiotic protein Hed1. Corresponding widefield micrographs are inset at top right and the scale bars are 1 μm wide (E-H) Example dSTORM and widefield (bottom right insets) micrographs of wild type nuclei stained for (E) Rad51 and (F) for Dmc1; mnd1 nuclei stained for (G) Rad51 and (H) Dmc1. Arrowheads indicate examples of 100 nm paired Rad51 sr foci, magnified in the upper right insets. Scale bar is 1 μm for dSTORM and widefield micrographs, 100 nm in top right insets. (I-K) Distributions of Rad51→Rad51 sr foci nearest neighbor measurements for (I) wild type, (J)mnd1, and (K)zip1 mutants. Micrographs from cultures 3.5 hours after meiotic induction. (L-O) Each Rad51 focus in a pair of Rad51 foci imaged under widefield can contain more than one Rad51 sr focus. (L-N) Micrographs of paired Rad51 foci observed at widefield resolution (left) and with dSTORM (right) containing 1 and 1 (L); 1 and 2 (M); and 2 and 2 (N) Rad51 sr foci. Scale bar is 400 nm. (O) Distribution of the frequencies of Rad51 sr foci in one focus (blue) or both foci (red) in paired Rad51 foci at low resolution. 48 widefield pairs of Rad51 foci were scored. (P) Characterization of the dimensions of Rad51 and Dmc1 sr foci. An ellipse was fit to each sr focus. Lmajor is the length of the ellipse’s long axis; aspect ratio equals Lmajor/Lminor. Scoring of 432, 212, and 304 sr foci from 5 wild type, 5 mnd1, and 5 zip1 nuclei for Rad51 scoring; 359 and 592 sr foci from 3 wild type and 6 mnd1 nuclei for Dmc1 scoring. Errors are S.D.
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pgen.1005653.g004: Rad51 and Dmc1 sr foci are extremely small and clustered at sub-diffraction distances.(A-D) dSTORM micrographs of Rad51 filaments assembled on a linear 2.7 kbp dsDNA in vitro in the presence of the meiotic protein Hed1. Corresponding widefield micrographs are inset at top right and the scale bars are 1 μm wide (E-H) Example dSTORM and widefield (bottom right insets) micrographs of wild type nuclei stained for (E) Rad51 and (F) for Dmc1; mnd1 nuclei stained for (G) Rad51 and (H) Dmc1. Arrowheads indicate examples of 100 nm paired Rad51 sr foci, magnified in the upper right insets. Scale bar is 1 μm for dSTORM and widefield micrographs, 100 nm in top right insets. (I-K) Distributions of Rad51→Rad51 sr foci nearest neighbor measurements for (I) wild type, (J)mnd1, and (K)zip1 mutants. Micrographs from cultures 3.5 hours after meiotic induction. (L-O) Each Rad51 focus in a pair of Rad51 foci imaged under widefield can contain more than one Rad51 sr focus. (L-N) Micrographs of paired Rad51 foci observed at widefield resolution (left) and with dSTORM (right) containing 1 and 1 (L); 1 and 2 (M); and 2 and 2 (N) Rad51 sr foci. Scale bar is 400 nm. (O) Distribution of the frequencies of Rad51 sr foci in one focus (blue) or both foci (red) in paired Rad51 foci at low resolution. 48 widefield pairs of Rad51 foci were scored. (P) Characterization of the dimensions of Rad51 and Dmc1 sr foci. An ellipse was fit to each sr focus. Lmajor is the length of the ellipse’s long axis; aspect ratio equals Lmajor/Lminor. Scoring of 432, 212, and 304 sr foci from 5 wild type, 5 mnd1, and 5 zip1 nuclei for Rad51 scoring; 359 and 592 sr foci from 3 wild type and 6 mnd1 nuclei for Dmc1 scoring. Errors are S.D.

Mentions: To characterize recombination complexes in more detail we used direct stochastic optical reconstruction microscopy (dSTORM), a method with higher resolution than standard widefield microscopy[39,40]. We validated our experimental system by imaging long Rad51 filaments prepared by the same method as that used previously for analysis by electron microscopy[41–43]. The Rad51 filaments were assembled on dsDNA in vitro, deposited on a coverslip, immunostained, and imaged. As expected, reconstructed dSTORM micrographs clearly show long Rad51 filaments often exceeding one micron in length (Fig 4A–4D). Following indirect immunostaining, the structures observed are 70 nm wide, 60 nm wider than the underlying protein filament as a consequence of both antibody decoration and the finite resolution of the imaging method. Given this apparent filament width, only filaments longer than about 70 nm have readily identifiable long axes in dSTORM reconstructions. Such elongated filaments were readily identified. After validating our dSTORM imaging procedure, we utilized the methodology to interrogate the molecular structures underlying widefield Rad51 and Dmc1 foci in vivo.


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)

Rad51 and Dmc1 sr foci are extremely small and clustered at sub-diffraction distances.(A-D) dSTORM micrographs of Rad51 filaments assembled on a linear 2.7 kbp dsDNA in vitro in the presence of the meiotic protein Hed1. Corresponding widefield micrographs are inset at top right and the scale bars are 1 μm wide (E-H) Example dSTORM and widefield (bottom right insets) micrographs of wild type nuclei stained for (E) Rad51 and (F) for Dmc1; mnd1 nuclei stained for (G) Rad51 and (H) Dmc1. Arrowheads indicate examples of 100 nm paired Rad51 sr foci, magnified in the upper right insets. Scale bar is 1 μm for dSTORM and widefield micrographs, 100 nm in top right insets. (I-K) Distributions of Rad51→Rad51 sr foci nearest neighbor measurements for (I) wild type, (J)mnd1, and (K)zip1 mutants. Micrographs from cultures 3.5 hours after meiotic induction. (L-O) Each Rad51 focus in a pair of Rad51 foci imaged under widefield can contain more than one Rad51 sr focus. (L-N) Micrographs of paired Rad51 foci observed at widefield resolution (left) and with dSTORM (right) containing 1 and 1 (L); 1 and 2 (M); and 2 and 2 (N) Rad51 sr foci. Scale bar is 400 nm. (O) Distribution of the frequencies of Rad51 sr foci in one focus (blue) or both foci (red) in paired Rad51 foci at low resolution. 48 widefield pairs of Rad51 foci were scored. (P) Characterization of the dimensions of Rad51 and Dmc1 sr foci. An ellipse was fit to each sr focus. Lmajor is the length of the ellipse’s long axis; aspect ratio equals Lmajor/Lminor. Scoring of 432, 212, and 304 sr foci from 5 wild type, 5 mnd1, and 5 zip1 nuclei for Rad51 scoring; 359 and 592 sr foci from 3 wild type and 6 mnd1 nuclei for Dmc1 scoring. Errors are S.D.
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pgen.1005653.g004: Rad51 and Dmc1 sr foci are extremely small and clustered at sub-diffraction distances.(A-D) dSTORM micrographs of Rad51 filaments assembled on a linear 2.7 kbp dsDNA in vitro in the presence of the meiotic protein Hed1. Corresponding widefield micrographs are inset at top right and the scale bars are 1 μm wide (E-H) Example dSTORM and widefield (bottom right insets) micrographs of wild type nuclei stained for (E) Rad51 and (F) for Dmc1; mnd1 nuclei stained for (G) Rad51 and (H) Dmc1. Arrowheads indicate examples of 100 nm paired Rad51 sr foci, magnified in the upper right insets. Scale bar is 1 μm for dSTORM and widefield micrographs, 100 nm in top right insets. (I-K) Distributions of Rad51→Rad51 sr foci nearest neighbor measurements for (I) wild type, (J)mnd1, and (K)zip1 mutants. Micrographs from cultures 3.5 hours after meiotic induction. (L-O) Each Rad51 focus in a pair of Rad51 foci imaged under widefield can contain more than one Rad51 sr focus. (L-N) Micrographs of paired Rad51 foci observed at widefield resolution (left) and with dSTORM (right) containing 1 and 1 (L); 1 and 2 (M); and 2 and 2 (N) Rad51 sr foci. Scale bar is 400 nm. (O) Distribution of the frequencies of Rad51 sr foci in one focus (blue) or both foci (red) in paired Rad51 foci at low resolution. 48 widefield pairs of Rad51 foci were scored. (P) Characterization of the dimensions of Rad51 and Dmc1 sr foci. An ellipse was fit to each sr focus. Lmajor is the length of the ellipse’s long axis; aspect ratio equals Lmajor/Lminor. Scoring of 432, 212, and 304 sr foci from 5 wild type, 5 mnd1, and 5 zip1 nuclei for Rad51 scoring; 359 and 592 sr foci from 3 wild type and 6 mnd1 nuclei for Dmc1 scoring. Errors are S.D.
Mentions: To characterize recombination complexes in more detail we used direct stochastic optical reconstruction microscopy (dSTORM), a method with higher resolution than standard widefield microscopy[39,40]. We validated our experimental system by imaging long Rad51 filaments prepared by the same method as that used previously for analysis by electron microscopy[41–43]. The Rad51 filaments were assembled on dsDNA in vitro, deposited on a coverslip, immunostained, and imaged. As expected, reconstructed dSTORM micrographs clearly show long Rad51 filaments often exceeding one micron in length (Fig 4A–4D). Following indirect immunostaining, the structures observed are 70 nm wide, 60 nm wider than the underlying protein filament as a consequence of both antibody decoration and the finite resolution of the imaging method. Given this apparent filament width, only filaments longer than about 70 nm have readily identifiable long axes in dSTORM reconstructions. Such elongated filaments were readily identified. After validating our dSTORM imaging procedure, we utilized the methodology to interrogate the molecular structures underlying widefield Rad51 and Dmc1 foci in vivo.

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