Condensin targeting to the nucleolus is impaired in cells arrested by DNA replication checkpoint. (A) Condensin enriches nucleoli in nocodazole-arrested cells. The strain expressing both Smc4-GFP (condensin subunit) and Sik1-mRFP (a nucleolar marker) (640-EY0987) was arrested with nocodazole for 3 h at 30°C. Arrows indicate the colocalization of condensin-enriched areas with nucleoli. Scale bar 5 μm. (B) Condensin fails to target to nucleoli in hydroxyurea-arrested cells. Conditions the same as in (A), except hydroxyurea (200mM) was used to arrest cells. (C) rDNA chromatin is not dispersed in hydroxyurea-arrested cells. The SIR4-42 strain expressing Sir2-GFP (622-YPH499-4) was arrested with hydroxyurea or nocodazole for 3 h at 23°C. In this strain Sir2p is exclusively bound to rDNA chromatin (47). The images were deconvolved and 3-D segmentation was conducted (see Results) using a DeltaVision workstation. Scale bar 5 μm.

Condensin binds chromatin in the absence of replication, but with altered pattern in rDNA. (A) Western blot analysis of the chromatin-bound fraction of condensin subunits at the replicated (Cdc6⁺) and unreplicated (Cdc6−) chromatin prepared as in (33). The cdc15-2 pGAL:CDC6 strain (5757) (27) was grown in the presence of galactose (Cdc6+) or dextrose (Cdc6−) and arrested with nocodazole. (B) The layout of condensin-binding qPCR ChIP probes in the rDNA repeat. Probes design and numbering are as in (46). (C) Condensin has an altered distribution pattern in unreplicated rDNA in mitosis. After the release from cdc15 arrest the pGAL:CDC6 (5757, Cdc6−) (27) and CDC6 (SLJ127, Cdc6+) strains were grown as described in Results in the presence of or dextrose (Cdc6−) and arrested with nocodazole. The qPCR ChIP results were averaged from three independent experiments. TUB2 PCR was a negative control.

rDNA condensation fails in mitotic cells without DNA replication. (A) FACS and FISH analysis of the cdc15-2 pGAL:CDC6 strain (5757) (27) released from the cdc15 arrest (t=0 min) and assayed at the next mitosis (t=150 min). Haploid cells arrested at the cdc15-2-specific point show 2C DNA content, probably due to delayed cytokinesis, and thus many cells reach apparent 4C DNA content in the next cycle. However, similarly arrested cdc15-2 cells, progressing through the first cell cycle with depleted Cdc6p, show their true 1C DNA content after 150 min. Arrows in the upper rDNA-specific FISH panel show replicated, segregated and condensed rDNA signals (green) in the anaphase Cdc6+ cell. Propidium iodide (red) stains the bulk of nuclear DNA. The lower panel arrow points to the unsegregated decondensed rDNA signal in the Cdc6-anaphase. Scale bar 5μm. (B) rDNA FISH analysis of cdc15-2 pGAL:CDC6 strain (5757) grown in the presence of galactose (Cdc6+) or dextrose (Cdc6−) and arrested with nocodazole. Colors are as in (A). Scale bar 5μm. (C) rDNA condensation fails in the absence of replication. Relative rDNA compaction was calculated on the basis of experiments identical to (B), as a ratio between the rDNA FISH signal area and propidium iodide – stained area, according to (5). pGAL:CDC6 – 5757 cells, CDC6 - SLJ127 cells.

Hypothetical parallel between the two-site condensin loading in yeast rDNA and sequential loading of condensins I and II in vertebrates. The transcriptionally active repeat is shown for simplicity, with origin firing downstream, based on (48). Shaded box – the 35S RNA gene, open box – the 5S rRNA gene, open circle – rARS (origin of replication), vertical line – the RFB site, dotted line – Pol I transcription. Blue triangles – condensin loading (or condensin-chromatin contacts) resulting from replication fork termination. Green triangles – condensin loading (or condensin-chromatin contacts) to 35S promoter junction enabled by transcription downregulation in mitosis. The kinetics of condensin I and condensin II loading is according to (34, 35); color coding denotes the match to hypothetically equivalent yeast condensin pool).