Figure 5.

Swi1 and Swi3 are essential to maintain rid mutant viability. (A) Mutants defective in DNA replication initiation require swi1 and swi3 to maintain cell viability. Fivefold serial dilutions of the double mutant strains cdc30Δswi1, cdc20Δswi1, and orp2Δswi, cdc30Δswi3, cdc20Δswi3, orp2Δswi3, the rid single mutant strains, and wild-type were plated on YEA plates and incubated at their respective critical temperature. In the absence of Swi1 or Swi3, rid mutant viability was dramatically decreased compared with the single mutants or wild-type cells. (B) Neither Swi1 nor Swi3 is required for Chk1 activation in rid mutants. Phosphorylation of tagged Chk1-protein was detected by Western immunoblotting using anti-HA antibodies. On activation, Chk1 shows a characteristic mobility shift as indicated by the arrow. The double mutants of cdc20-M10Δswi1 and cdc20-M10Δswi3 were shifted to the critical temperature of 31°C for 3 h and analyzed for Chk1 activation. Chk1 activation was not abolished in rid mutants deleted for swi1 or swi3 at 31°C, indicating they are not required for Chk1 activation in rid mutants (lanes 3–6). (C) cdc20Δmrc1Δchk1 cells have a lower critical temperature than cdc20Δchk1 or cdc20Δmrc1. The strain cdc20Δmrc1 was crossed to the Δchk1 strain and triple mutants were isolated and assayed for cell viability at different temperatures. The critical temperature for cdc20Δmrc1Δchk1 was lower than the critical temperature for either of the two double mutants.