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. 2019 Mar 1;33(5-6):276–281. doi: 10.1101/gad.321836.118

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© 2019 Daniloski et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 2. — rDNA resolution requires CAP-D3/condensin II. (A) Schematic diagram of condensins I and II. Horizontal lines indicate CAP-D2 and D3. TNKS-binding sites at positions 5 and 519 are indicted in CAP-D3. (B–D) Depletion of SMC2 leads to rDNA stretching. Following a 48-h transfection with GFP or SMC2 siRNA, HeLa cells were analyzed by immunoblot (B) or isolated by mitotic shake-off and analyzed by FISH using rDNA (red) and 13/21 cen (green) probes (C) and quantified (D). Mitotic cells with three or more stretched rDNA lines were scored as having stretched rDNA. Average of two independent experiments (n = 63–70 cells each) ± SEM. (**) P ≤ 0.01, Student's unpaired t-test. (E–G) CAP-D3, not D2, is required for rDNA resolution in anaphase. HeLa cell lines stably expressing CAP-D3 or CAP-D2 shRNAs were analyzed by immunoblot (E) or isolated by mitotic shake-off following a 4.5-h incubation in nocodazole and 45-min release into anaphase and analyzed by FISH using rDNA (red) and 13/21 cen (green) probes (F) and quantified (G). Anaphase cells (separated centromeres) with ≥3 stretched rDNA lines were scored as having stretched rDNA. Average of two independent experiments ± SEM. n = 56–112 cells each. (*) P ≤ 0.05, Student's unpaired t-test. (H) CAP-D3, but not D2, coimmunoprecipitates with TNKS1. Immunoblot analysis of HeLa cells immunoprecipitated with control or TNKS1 antibodies and probed with the indicated antibodies. (I–K) The TNKS-binding site in CAP-D3 at amino acid 519 is required for resolution of cohesion. CAP-D3 shRNA#2 cells were transfected with GFP-Vec, GFP-CAP-D3 wild type, or GFP-CAP-D3 single G10A or G524, or double GAA mutant plasmids for 18 h and analyzed by immunoblot (I) or isolated by mitotic shake-off following a 4.5 h incubation in nocodazole and 45-min release into anaphase (J) and analyzed by FISH using rDNA (red) probes and quantified (K). Anaphase cells (separated centromeres) with three or more stretched rDNA lines were scored as having stretched rDNA. Average of two independent experiments ± SEM. n = 56–112 cells each. (**) P ≤ 0.01, Student's unpaired t-test. (L) CAP-D3 GAA mutant does not bind tankyrase 1 but retains its association with SMC2. Immunoblot analysis of U2OS cells immunoprecipitated with anti-GFP antibody following transfection with GFP-Vec, GFP-CAP-D3 wild type, or GFP-CAP-D3 GAA and probed with the indicated antibodies. (C,F,J) DNA was stained with DAPI (blue). Bar, 2 μm.