Figure 6

Detection of IR-induced DSBs by 3′-end-labelling of surface-spread horsetail nuclei.

(A) Maximum projection images showing spread nuclei stained for DSBs by 3′-end-labelling (Cy3-dCTP, red). Horsetail nuclei are tagged with Rec8-GFP dots (green); DSB signals (red); DNA is stained with DAPI (blue). Positive control: DNAse I-treated cells (DNAse) show an overall reddish labelling. Scale bar: 2 μm. (B) Graph showing the average frequency of irradiation-induced DSB foci in spread horsetail cells identified by punctate Rec8-GFP signals. Background labelling in non-irradiated controls was 0.46 foci/cell. There is a dose-dependent linear increase (R² = 0.9953) of IR-induced DSBs at 1.3 DSBs per 10 Gy. (C) Average frequency of DSB foci in H₂O₂-treated horsetail cells. Background labelling in non-exposed controls was 0.11 foci/cell. There is a dose-dependent linear increase of H₂O₂-induced DSBs with 2 DSBs at 100 mM. The average values (av.) are from 2 independent experiments as shown (e1, e2 = experiment 1 and 2). (D) Nuclear spreading of meiocytes leads to much wider chromatin distribution with S. cerevisiae pachytene cells (Zip1-GFP-tagged; see²⁵) than with S. pombe Rec8-GFP expressing horsetail meiocytes, suggesting a different chromatin organization/state in S. pombe nuclei (DNA, blue). Bar: 2 μm. (E) Graph showing average nuclear volume of fission and budding meiocyte nuclei (±SD) as revealed by image analysis of Rec8-GFP (S. pombe) and Zip1-GFP (S. cerevisiae) expressing live cells. While pachytene nuclei and extended horsetail-stage nuclei have a similar volume, the volume is significantly smaller in spo11∆ budding yeast nuclei (68% of wt; p<0.001) that do not form an SC. The rounded S. pombe horsetail-stage nuclei are even smaller in volume (46% of extended horsetail nuclei, or 51% of wild-type pachytene); ^ap < 0.001 relative to pachytene and ext. horsetail nuclei, ^bsignificantly smaller than pachytene (p < 0.0001), spo11∆ (p = 0.01) and ext. horsetail nuclei (p < 0.0001). Below: representative images of a S. cerevisiae pachytene (pachy) and a polycomplex (PC, white dot, used as stage marker) expressing spo11∆ SK1 meiocyte nucleus. S. pombe: prophase I nuclei expressing Rec8-GFP in rounded (h.tail cpt; the SPB passes the trailing edge on its way to the other cell tip) and extended horsetail-stage nuclei (h.tail ext.; SPB at the cell tip). Gray scale images, bar: 2 μm.