Figure 1.

Transient exposure of hst3Δ hst4Δ cells to MMS or HU causes loss of viability and prevents the completion of DNA replication. (A) hst3∆ hst4∆ cells are sensitive to transient exposure to MMS and HU during S phase. Cells were arrested in G₁ and released into the cell cycle in the presence of increasing concentrations of MMS (left panel) or HU (right panel) at 25°. Appropriate dilutions of cells were plated on YPD during G₁ arrest and after 90 min of MMS exposure. Viability was defined as the ratio of colonies that arose after MMS or HU treatment to colonies formed by G₁-synchronized cells (see Materials and Methods). (B and C) Transient exposure to MMS or HU delays the completion of DNA replication in hst3∆ hst4∆ mutants. Cells were synchronized in G₁ with α-factor and released toward S phase in a medium containing 0.03% MMS or 200 mM HU for 90 min. Genotoxic agents then were washed away and inactivated using 2.5% sodium thiosulfate in the case of MMS, and cells were released into fresh medium lacking genotoxins. Samples were processed for cell-cycle analysis by FACS at the indicated time points. Asyn, asynchronous cells. (D and E) hst3∆ hst4∆ mutants cannot complete chromosome duplication after transient exposure to MMS. Cells were arrested in G₁ and released into the cell cycle in the presence of 0.03% MMS or 200 mM HU for 1.5 hr. They were washed with YPD (containing 2.5% sodium thiosulfate in the case of MMS) and resuspended in fresh medium lacking genotoxins. Samples were taken at the indicated time points and processed for pulse-field gel electrophoresis.