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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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Elongated Dmc1 structures and higher order clustered Rad51 structures accumulate in strand exchange mutants at late times.(A-D) 8 hr mnd1 nucleus stained for Dmc1. (A) dSTORM and (B) widefield micrographs each with 1 μm scale bars. (C) Magnified region from (A) indicated by arrowhead, highlighting elongated Dmc1 structure. Scale bar is 100 nm wide. (D) Surface plot of (C). (E-H) 3.5 hr dmc1 nucleus stained for Rad51, highlighting a common pair (low order cluster) of sr foci. (I-L) 8 hr mnd1 nucleus and (M-P) 8 hr dmc1 nucleus stained for Rad51, highlighting higher order clusters of Rad51 sr foci. (Q-T) dSTORM micrographs of elongated Rad51 structures observed in spread nuclei of mitotic srs2 mutants overexpressing Rad51. Scale bar is 1 μm.
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pgen.1005653.g005: Elongated Dmc1 structures and higher order clustered Rad51 structures accumulate in strand exchange mutants at late times.(A-D) 8 hr mnd1 nucleus stained for Dmc1. (A) dSTORM and (B) widefield micrographs each with 1 μm scale bars. (C) Magnified region from (A) indicated by arrowhead, highlighting elongated Dmc1 structure. Scale bar is 100 nm wide. (D) Surface plot of (C). (E-H) 3.5 hr dmc1 nucleus stained for Rad51, highlighting a common pair (low order cluster) of sr foci. (I-L) 8 hr mnd1 nucleus and (M-P) 8 hr dmc1 nucleus stained for Rad51, highlighting higher order clusters of Rad51 sr foci. (Q-T) dSTORM micrographs of elongated Rad51 structures observed in spread nuclei of mitotic srs2 mutants overexpressing Rad51. Scale bar is 1 μm.

Mentions: We next asked if Rad51 and Dmc1 sr foci were altered when strand exchange is blocked. In wild type cells, Dmc1 foci are present and strand exchange occurs between 3 and 6 hours[8,13,16]; foci disappear and strand exchange is complete before 8 hours. If strand exchange is blocked in an mnd1 or a dmc1 mutant, DSB-associated ssDNA tracts become much longer than normal by 8 hours[8,13,34]. dSTORM microscopy of 3.5 hour mnd1 nuclei revealed a punctate Dmc1staining pattern very similar to that seen in wild type (Fig 4H and 4P). However, the Dmc1 staining patterns seen in 8-hour mnd1 nuclei were dramatically different; they contained numerous elongated structures with contour lengths often reaching 250 nm (Fig 5A–5D). This result indicates that elongation of Dmc1-containing structures is limited by Mnd1 function, likely because completion of Hop2-Mnd1-dependent strand exchange is associated with Dmc1 disassembly, as has been argued for Rad51 based on biochemical observations[45]. Importantly, no corresponding elongated Dmc1 structures have been observed in strand exchange-proficient cells (for example, Fig 4F and 4P). Like Dmc1 staining, little or no difference in the Rad51 staining was seen in either mnd1 or dmc1 mutants, as compared to wild type, at 3.5 hours (Figs 4G and 4P and 5E–5H). However, at 8 hours, mnd1 nuclei frequently displayed clusters of about 3–7 poorly resolved Rad51 sr foci (Fig 5I–5L). These clusters lacked obvious elongated structure, in dramatic contrast to the fibrous staining patterns seen for Dmc1 using duplicate slides from the same cultures and time points (compare Fig 5A to 5I). Similar clustering of Rad51 sr foci was seen at 8 hours in dmc1 cells (Fig 5M–5P), suggesting Dmc1 does not influence formation of Rad51 clusters when strand exchange is blocked. The Rad51 staining results are in agreement with previous low resolution studies showing Rad51 focus staining intensity increases with time in dmc1 and mnd1 mutants[16,33].


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)

Elongated Dmc1 structures and higher order clustered Rad51 structures accumulate in strand exchange mutants at late times.(A-D) 8 hr mnd1 nucleus stained for Dmc1. (A) dSTORM and (B) widefield micrographs each with 1 μm scale bars. (C) Magnified region from (A) indicated by arrowhead, highlighting elongated Dmc1 structure. Scale bar is 100 nm wide. (D) Surface plot of (C). (E-H) 3.5 hr dmc1 nucleus stained for Rad51, highlighting a common pair (low order cluster) of sr foci. (I-L) 8 hr mnd1 nucleus and (M-P) 8 hr dmc1 nucleus stained for Rad51, highlighting higher order clusters of Rad51 sr foci. (Q-T) dSTORM micrographs of elongated Rad51 structures observed in spread nuclei of mitotic srs2 mutants overexpressing Rad51. Scale bar is 1 μm.
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pgen.1005653.g005: Elongated Dmc1 structures and higher order clustered Rad51 structures accumulate in strand exchange mutants at late times.(A-D) 8 hr mnd1 nucleus stained for Dmc1. (A) dSTORM and (B) widefield micrographs each with 1 μm scale bars. (C) Magnified region from (A) indicated by arrowhead, highlighting elongated Dmc1 structure. Scale bar is 100 nm wide. (D) Surface plot of (C). (E-H) 3.5 hr dmc1 nucleus stained for Rad51, highlighting a common pair (low order cluster) of sr foci. (I-L) 8 hr mnd1 nucleus and (M-P) 8 hr dmc1 nucleus stained for Rad51, highlighting higher order clusters of Rad51 sr foci. (Q-T) dSTORM micrographs of elongated Rad51 structures observed in spread nuclei of mitotic srs2 mutants overexpressing Rad51. Scale bar is 1 μm.
Mentions: We next asked if Rad51 and Dmc1 sr foci were altered when strand exchange is blocked. In wild type cells, Dmc1 foci are present and strand exchange occurs between 3 and 6 hours[8,13,16]; foci disappear and strand exchange is complete before 8 hours. If strand exchange is blocked in an mnd1 or a dmc1 mutant, DSB-associated ssDNA tracts become much longer than normal by 8 hours[8,13,34]. dSTORM microscopy of 3.5 hour mnd1 nuclei revealed a punctate Dmc1staining pattern very similar to that seen in wild type (Fig 4H and 4P). However, the Dmc1 staining patterns seen in 8-hour mnd1 nuclei were dramatically different; they contained numerous elongated structures with contour lengths often reaching 250 nm (Fig 5A–5D). This result indicates that elongation of Dmc1-containing structures is limited by Mnd1 function, likely because completion of Hop2-Mnd1-dependent strand exchange is associated with Dmc1 disassembly, as has been argued for Rad51 based on biochemical observations[45]. Importantly, no corresponding elongated Dmc1 structures have been observed in strand exchange-proficient cells (for example, Fig 4F and 4P). Like Dmc1 staining, little or no difference in the Rad51 staining was seen in either mnd1 or dmc1 mutants, as compared to wild type, at 3.5 hours (Figs 4G and 4P and 5E–5H). However, at 8 hours, mnd1 nuclei frequently displayed clusters of about 3–7 poorly resolved Rad51 sr foci (Fig 5I–5L). These clusters lacked obvious elongated structure, in dramatic contrast to the fibrous staining patterns seen for Dmc1 using duplicate slides from the same cultures and time points (compare Fig 5A to 5I). Similar clustering of Rad51 sr foci was seen at 8 hours in dmc1 cells (Fig 5M–5P), suggesting Dmc1 does not influence formation of Rad51 clusters when strand exchange is blocked. The Rad51 staining results are in agreement with previous low resolution studies showing Rad51 focus staining intensity increases with time in dmc1 and mnd1 mutants[16,33].

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