Limits...
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.

Show MeSH

Related in: MedlinePlus

Recombinosome Model: Rad51 and Dmc1 each form short filaments on both spatially separated ends of a DSB.(Top) Short helical Rad51 and Dmc1 nucleoprotein homofilaments (green and red, respectively) form on adjacent segments of a single DSB-associated ssDNA tract. Each of these filaments is on the order of 100 nt or 33 protomers long and more than one Rad51-Dmc1 structure can form on a single tract of ssDNA. This single end of the DSB manifests itself cytologically as a side-by-side Rad51-Dmc1 co-focus (represented by the offset transparent red and green circles). (Left) Both Rad51 and Dmc1 similarly occupy the second end of the DSB. Prior to strand exchange, the two ends of the DSB are separated by 400 nm or less, resulting in focus pairing. The two ends of the DSB are both tethered to the axial element (purple box) by chromatin arms of variable length. Sister chromatids (pink circles) are often split in a DSB-independent manner. (Right) This architecture is maintained after strand exchange tethers the homolog at the predetermined distance of 400 nm or less.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4697796&req=5

pgen.1005653.g006: Recombinosome Model: Rad51 and Dmc1 each form short filaments on both spatially separated ends of a DSB.(Top) Short helical Rad51 and Dmc1 nucleoprotein homofilaments (green and red, respectively) form on adjacent segments of a single DSB-associated ssDNA tract. Each of these filaments is on the order of 100 nt or 33 protomers long and more than one Rad51-Dmc1 structure can form on a single tract of ssDNA. This single end of the DSB manifests itself cytologically as a side-by-side Rad51-Dmc1 co-focus (represented by the offset transparent red and green circles). (Left) Both Rad51 and Dmc1 similarly occupy the second end of the DSB. Prior to strand exchange, the two ends of the DSB are separated by 400 nm or less, resulting in focus pairing. The two ends of the DSB are both tethered to the axial element (purple box) by chromatin arms of variable length. Sister chromatids (pink circles) are often split in a DSB-independent manner. (Right) This architecture is maintained after strand exchange tethers the homolog at the predetermined distance of 400 nm or less.

Mentions: Assuming that Rad51 and Dmc1 sr foci represent the DNA bound helical filaments that promote strand exchange in vitro, the sr foci we observed by dSTORM suggest that the Rad51 and Dmc1 filaments that promote recombination in vivo are extremely short and that more than one Rad51 and/or Dmc1 filament can form on the same ssDNA tract. Images of Rad51 and Dmc1 sr foci are only about 115 nm long. Since the diameter of Rad51 and Dmc1 filaments are known to be 10 nm [43], but sr foci are 70 nm wide, the images of sr foci observed with super-resolution microscopy following indirect immunostaining are likely about 60 nm larger in each dimension than the underlying protein complex. This difference can be accounted for by considering the size of the primary and secondary antibodies decorating the structure. Thus, assuming that RecA homolog structures represent the canonical nucleoprotein filaments[43], they are about 55 nm long. Given that Rad51 and Dmc1 filaments contain about 2 nt per nm[41,43,47], this corresponds to roughly 100 nt, 33 protomers of Rad51 (or Dmc1), and 5 turns of the helical nucleoprotein filament. This length estimate suggests that an individual Rad51 or Dmc1 filament typically occupies less than 15% of a typical 800 nt ssDNA tract [8,48]. An alternative, but in our view less likely, interpretation, is that RecA homologs coat a more substantial fraction of each ssDNA tract in a previously unknown compact configuration. Additionally, a significant fraction of the closely spaced (<200 nm apart) Rad51 sr foci likely represent loading of distinct filaments on the same tract of ssDNA as evidenced by detection of nuclei with more than 4 Rad51 sr foci in VDE cut site heterozygote nuclei and the accumulation of clustered Rad51 sr foci at late time points in strand exchange mutants. Although 100 nt filaments are quite small relative to those that have been studied in many biochemical experiments[49], both ensemble and single molecule experiments have shown that only 8 nt is sufficient for recognition of homology by RecA-like strand exchange proteins [50,51]. Thus, the size of the structures we observe is more than sufficient to promote efficient recombination. The finding that Rad51 and Dmc1 structures are small relative to the average length of ssDNA tracts is in agreement with previous observations indicating that Rad51 foci display offset colocalization with foci formed by the recombination proteins RPA and Rad52. These observations suggest that RPA and Rad52 can simultaneously occupy ssDNA segments adjacent to regions bound by Rad51 and Dmc1[52]. Furthermore, the lack of an inverse relationship between Rad51 and Dmc1 staining intensity in a single co-focus supports the hypothesis that ssDNA tracts are not completely bound by RecA homologs. All of these observations suggest that the protein composition and organization of a tract of ssDNA associated with a meiotic DSB is highly heterogeneous (Fig 6, top).


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)

Recombinosome Model: Rad51 and Dmc1 each form short filaments on both spatially separated ends of a DSB.(Top) Short helical Rad51 and Dmc1 nucleoprotein homofilaments (green and red, respectively) form on adjacent segments of a single DSB-associated ssDNA tract. Each of these filaments is on the order of 100 nt or 33 protomers long and more than one Rad51-Dmc1 structure can form on a single tract of ssDNA. This single end of the DSB manifests itself cytologically as a side-by-side Rad51-Dmc1 co-focus (represented by the offset transparent red and green circles). (Left) Both Rad51 and Dmc1 similarly occupy the second end of the DSB. Prior to strand exchange, the two ends of the DSB are separated by 400 nm or less, resulting in focus pairing. The two ends of the DSB are both tethered to the axial element (purple box) by chromatin arms of variable length. Sister chromatids (pink circles) are often split in a DSB-independent manner. (Right) This architecture is maintained after strand exchange tethers the homolog at the predetermined distance of 400 nm or less.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005653.g006: Recombinosome Model: Rad51 and Dmc1 each form short filaments on both spatially separated ends of a DSB.(Top) Short helical Rad51 and Dmc1 nucleoprotein homofilaments (green and red, respectively) form on adjacent segments of a single DSB-associated ssDNA tract. Each of these filaments is on the order of 100 nt or 33 protomers long and more than one Rad51-Dmc1 structure can form on a single tract of ssDNA. This single end of the DSB manifests itself cytologically as a side-by-side Rad51-Dmc1 co-focus (represented by the offset transparent red and green circles). (Left) Both Rad51 and Dmc1 similarly occupy the second end of the DSB. Prior to strand exchange, the two ends of the DSB are separated by 400 nm or less, resulting in focus pairing. The two ends of the DSB are both tethered to the axial element (purple box) by chromatin arms of variable length. Sister chromatids (pink circles) are often split in a DSB-independent manner. (Right) This architecture is maintained after strand exchange tethers the homolog at the predetermined distance of 400 nm or less.
Mentions: Assuming that Rad51 and Dmc1 sr foci represent the DNA bound helical filaments that promote strand exchange in vitro, the sr foci we observed by dSTORM suggest that the Rad51 and Dmc1 filaments that promote recombination in vivo are extremely short and that more than one Rad51 and/or Dmc1 filament can form on the same ssDNA tract. Images of Rad51 and Dmc1 sr foci are only about 115 nm long. Since the diameter of Rad51 and Dmc1 filaments are known to be 10 nm [43], but sr foci are 70 nm wide, the images of sr foci observed with super-resolution microscopy following indirect immunostaining are likely about 60 nm larger in each dimension than the underlying protein complex. This difference can be accounted for by considering the size of the primary and secondary antibodies decorating the structure. Thus, assuming that RecA homolog structures represent the canonical nucleoprotein filaments[43], they are about 55 nm long. Given that Rad51 and Dmc1 filaments contain about 2 nt per nm[41,43,47], this corresponds to roughly 100 nt, 33 protomers of Rad51 (or Dmc1), and 5 turns of the helical nucleoprotein filament. This length estimate suggests that an individual Rad51 or Dmc1 filament typically occupies less than 15% of a typical 800 nt ssDNA tract [8,48]. An alternative, but in our view less likely, interpretation, is that RecA homologs coat a more substantial fraction of each ssDNA tract in a previously unknown compact configuration. Additionally, a significant fraction of the closely spaced (<200 nm apart) Rad51 sr foci likely represent loading of distinct filaments on the same tract of ssDNA as evidenced by detection of nuclei with more than 4 Rad51 sr foci in VDE cut site heterozygote nuclei and the accumulation of clustered Rad51 sr foci at late time points in strand exchange mutants. Although 100 nt filaments are quite small relative to those that have been studied in many biochemical experiments[49], both ensemble and single molecule experiments have shown that only 8 nt is sufficient for recognition of homology by RecA-like strand exchange proteins [50,51]. Thus, the size of the structures we observe is more than sufficient to promote efficient recombination. The finding that Rad51 and Dmc1 structures are small relative to the average length of ssDNA tracts is in agreement with previous observations indicating that Rad51 foci display offset colocalization with foci formed by the recombination proteins RPA and Rad52. These observations suggest that RPA and Rad52 can simultaneously occupy ssDNA segments adjacent to regions bound by Rad51 and Dmc1[52]. Furthermore, the lack of an inverse relationship between Rad51 and Dmc1 staining intensity in a single co-focus supports the hypothesis that ssDNA tracts are not completely bound by RecA homologs. All of these observations suggest that the protein composition and organization of a tract of ssDNA associated with a meiotic DSB is highly heterogeneous (Fig 6, top).

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