Fig. 8: The Tel1-Mre11 (TM) checkpoint prolongs checkpoint arrest when Arp2/3 is blocked before DSB induction.

A Experimental setup to measure the percentage of G₂/M arrested cells after DSB induction. CK-666 or IAA were added 20 min or 1 h before galactose to block Arp2/3 activity before DSB induction. To block Arp2/3 activity after DSB induction, CK-666 or IAA was added 2 h after galactose. Example images of G₁, small budded, and large-budded (G₂/M) cells. B Percentage of G₂/M arrested cells in FZ015 (WT) and FZ075 (Las17-AID) ± IAA after DSB induction. IAA was added before galactose as described in (A). Data was collected from 3 separate days (n = 3) with >100 cells counted at each timepoint per day. Mean ± SEM is shown. C Western blot of Las17-AID ± IAA probed with α-Myc to show Las17-AID degradation, α-Rad53 shows both an unphosphorylated and phosphorylated protein, and α-Pgk1 as a loading control. Source data are provided as a Source Data file. D Percentage of G₂/M arrested cells in FZ015 (WT) ± CK-666, FZ033 (myo3∆), and FZ034 (myo5∆) after DSB induction. CK-666 was added before galactose as described in (A). Data was collected from 3 separate days (n = 3) with >100 cells counted at each timepoint per day. Mean ± SEM is shown. Source data are provided as a Source Data file. E Western blot of myo5∆ probed with α-Rad53 shows both an unphosphorylated and phosphorylated protein, and α-Pgk1 as a loading control. Source data are provided as a Source Data file. F Percentage of G₂/M arrested cells in a FZ015 (WT), FZ019 (mre11∆) ± CK-666, and YSL56 (tel1∆) ± CK-666 strains, where CK-666 was added before galactose as described in (A). Data are shown from 3 independent experiments with error bars representing the standard error of the mean (SEM). Data was collected from 3 separate days (n = 3) with >100 cells counted at each timepoint per day. Mean ± SEM is shown. Source data are provided as a Source Data file. G Western blot of WT and mre11∆ strains after DSB induction, probed with α-Rad53 shows both an unphosphorylated and phosphorylated protein, and α-Pgk1 as a loading control. Source data are provided as a Source Data file. H Percentage of G₂/M arrested cells in FZ015 (WT) and FZ192 (Las17-AID mre11∆) ± IAA DSB induction. IAA was added before galactose as described in (A). Data was collected from 3 separate days (n = 3) with >100 cells counted at each timepoint per day. Mean ± SEM is shown. Source data are provided as a Source Data file. I Western blot of Las17-AID mre11∆ ± IAA probed with α-Myc to show Las17-AID degradation, α-Rad53 shows both an unphosphorylated and phosphorylated protein, and α-Pgk1 as a loading control. Source data are provided as a Source Data file. J Percentage of G₂/M arrested cells in FZ015 (WT) ± CK-666 and FZ075 (Las17-AID) ± IAA after DSB induction. CK-666 or IAA were added 2 h after galactose as described in (A). Data was collected from 3 separate days (n = 3) with >100 cells counted at each timepoint per day. Mean ± SEM is shown. Source data are provided as a Source Data file. K Western blot of Las17-AID ± IAA probed with α-Myc to show Las17-AID degradation, α-Rad53 shows both an unphosphorylated and phosphorylated protein, and α-Pgk1 as a loading control. Source data are provided as a Source Data file.