The hat1Δ mutation reduces viability and enhances the cell-cycle defect of orc-ts cells. (A) Percentage of cells that form viable microcolonies when asynchronous cultures of orc5-1 hat1 (BSY538), orc5-1 (BSY535), hat1 (BSY528) and wild-type strain (JRY2334) were shifted to the nonpermissive temperature for 0–5 h. Viability was measured as the fraction of microcolonies that formed after the incubation at 36°C within 1–2 days at permissive temperature. (B) Percentage of orc5-1 hat1, orc5-1, hat1, and wild-type cells that form viable microcolonies when synchronized cultures were shifted to the nonpermissive temperature. Cells were arrested in G1 (α-factor) or in S-phase (hydroxyurea) at 23°C and then maintained at restrictive temperature (36°C) for 0 to 3 h in G1 phase, in S-phase or from G1 to S-phase arrest. Averages of two independent experiments are shown. (C) FACS analysis of wild-type, hat1Δ (BSY539) orc2-1 (BSY568) and orc2-1 hat1Δ (BSY569) cells at semi-permissive temperature (26°C). Cells were arrested in α-factor (5 μg/ml) and release was performed at 26°C for 0, 10, 20, 30, 40, 50, 60, 90, 120, 150, and 190 min. (D) Cell-cycle progression of orc5-1 hat1, orc5-1 and wild-type strain at restrictive temperature for orc5-1. G1-arrested cells were held at 36°C (restrictive temperature) for 1 h and then released into fresh (36°C) medium. Samples for FACS were taken at times indicated. For a detailed list of strains, see Additional file 7.