Figure 4. Depleting topoisomerase I causes lethal DNA damage in G2-M.
(A) Depleting Top1 in rnh1∆ rnh201∆ cells shows similar onset of Rad52-GFP at the S/G2-M border. Cultures of rnh1∆ rnh201∆ TOP1-AID cells were arrested in G1 using alpha factor, treated with auxin for 2 hr, then released into media containing nocodazole and auxin. Samples were taken at 15 min intervals and 300 cells per time point were scored for Rad52-GFP foci. (B) Depleting Top1 in rnh1∆ rnh201∆ cells after completion of S-phase causes accumulation of Rad52-GFP foci. Cultures of rnh1∆ rnh201∆ TOP1-AID cells were released from alpha factor into nocodazole. Once cells had completed S-phase, auxin was added. Samples were taken at 30 min intervals and 300 cells per time point were scored for Rad52-GFP foci. (C) Cultures of rnh1∆ rnh201∆ TOP1-AID cells were allowed to divide asynchronously, arrested in S-phase using hydroxyurea, or arrested in Mid-M phase using nocodazole. Once cells were arrested, auxin was added for four hours. Left – Cells were washed and plated on YPD for recovery. Viability was measured by normalizing colony-forming units from auxin-treated cells to untreated cells. Bars represent mean +/- standard deviation (n = 4). Right – Cells were fixed and scored for Rad52-GFP foci. Bars represent mean +/- standard deviation (n = 3, 300 cells scored per replicate).
Figure 4—figure supplement 1. Details on rnh1∆ rnh201∆ TOP1-AID cells released from alpha factor into nocodazole.
