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Error bars, SD **< 0

Error bars, SD **< 0.01 (Students test). min after G1 release (Fig. 1column). In contrast, S-phase entry was significantly delayed with SDS treatment (Fig. 1column). However, cell cycle progression was completely normal when cells were treated with SDS after the origins have been fired, 20 min after G1 release (Fig. 1column). This finding indicates that S-phase entry is inhibited in response to TVB-3664 plasma membrane damage. To test which DNA replication step is affected by SDS treatment, we used a mutant to arrest the cell cycle. Cdc7 binds to Dbf4 to make the DDK complex, which then phosphorylates and activates Mcm4 to initiate DNA replication after origin licensing (39, 40). is a DDK temperature-sensitive mutant that arrests the cell cycle during G1-S phase, after pre-RC formation but with an inactive helicase not yet able to unwind DNA (41). When cells were treated with SDS upon block and release, the cell cycle proceeded normally (Fig. 1cells were arrested in G1-phase by -factor at 23 C and then released at 37 C, the restrictive temperature, to block the cells after pre-RC formation but with an inactive helicase. Cells were then released into YPD media at 23 C and collected every 20 min to monitor the cell cycle progression by FACS analysis in the presence or absence of SDS 0.0075% added at time 0 (green arrow). The diagrams below show at what point of the cycle that the cells were in when SDS was added. S-cyclin/CDK Activity Is Inhibited in Response to Plasma Membrane Damage. Next, we examined TVB-3664 cell cycle regulators to test if they play a role in G1 arrest induced by membrane damage. We investigated Sic1 protein expression, the Rabbit Polyclonal to STK10 S-phase CDK inhibitor, during cell cycle arrest induced by plasma membrane stress. Sic1 is rapidly degraded at the onset of the G1/S transition in untreated cells and is expressed again 60 min later during the next G1 phase (Fig. 2test. Open in a separate window Fig. S1. Clb5 is stabilized after SDS treatment. (cells were synchronized during G1 phase by -factor. The cell cycle block was released and SDS was added to the media. Samples were collected every TVB-3664 20 min for protein extractions. Each time point was subjected to Western blot analysis. Pgk1 was used as a loading control. Budding index is shown as percent of budded cells. (cells were used to monitor the Sld2 phosphorylation status upon G1 block and release as described in rescues the S-phase delay caused by SDS and observed that the cell cycle arrest was sustained (Fig. S2and Fig. S3deletion cells are sensitive to various stress stimuli, including TVB-3664 plasma membrane stress (Fig. 4deletion cells, indicating that Cdc6 degradation in response to plasma membrane stress requires Mck1 (Fig. 3or cells were grown to log-phase. Samples were collected every 15 min in the presence or absence of 0.0075% SDS. Protein was extracted from each time point for Western blotting to detect Cdc6-prA or Pgk1 as a loading control. The same experiment was repeated three times and the signal was quantified to show the average with SD. (cells were incubated in galactose-containing media first. The cell cycle was arrested in G1 by -factor or in mitosis by nocodazole. The cell cycle block was 86% in G1 and 83% in mitosis. Samples were collected TVB-3664 every 15 min in the presence or absence of 0.0075% SDS. Protein was extracted from.