The rid2-1 mutant displays a DNA replication initiation defect. (A) Experimental Strategy. See Text for a description of the experimental design for the hydroxyurea block and release experiment. (B) Control experiment showing the behavior of two different mutants known to be defective in DNA replication initiation (cdc20) and DNA replication elongation (cdc27). The cdc20 mutant completes cell division on schedule and doubles following release from HU. As expected for an initiation mutant, no increase in cell number is observed during the second cell cycle period. On the other hand, mutants defective in replication elongation (cdc27) show no significant increase in cell number throughout the course of the experiment. (C) Following release from the HU block wildtype cells show normal cell cycle kinetics. The first cell division or population doubling occurs at 4 hrs (33°C) and 6 hrs (25°C) that are longer than normal due to the time needed for recovery from HU. All subsequent cell cycles occur with normal kinetics for the indicated temperatures and are approximately 2 hrs at 33°C and 4 hrs at 25°C. The mutant rid2-1 grows slowly and requires at least 10 hours for the first population doubling which is not effected by shifting cells to the semi-permissive temperature of 33°C. However, during the second cell cycle period, rid2-1 cells at 33°C have a significant cell cycle delay as compared to cells that remain at 25°C, suggesting that DNA replication initiation is defective in these mutants.