A Synthetic Interaction between CDC20 and RAD4 in Saccharomyces cerevisiae upon UV Irradiation
Figure 1
UV resistance of Δrad4 mutants was diminished by the introduction of temperature-sensitive cdc20-1 allele. (a) The optical density A 600 of overnight cultures was normalized to 1.0, and a 1 : 10 serial dilution was prepared. Onto YPD, 5 μL of each dilution was spotted and plates were exposed to UV radiation at energy levels corresponding to 10 and 25 J/m2. Plates were then incubated at 22°C for 3 days. The growth of Δrad4 harboring cdc20-1 was greatly reduced relative to the Δrad4 single mutant or the wild-type strain (Y2454). (b) Diluted cultures of yeast were plated on YPD and then exposed to UV at energy levels of 10 and 25 J/m2, or none at all. Following 3 days of incubation at 22°C, colonies were counted and percent survival was calculated as the total number of colonies on the treated plates divided by the number of colonies on the untreated plates. The Δrad4/cdc20-1 mutant exhibits a tenfold decrease in survivability relative to the Δrad4 strain at both energy levels tested. (c) Plasmids were constructed in which either CDC20 or cdc20-1 was placed under the control of the gal1 promoter. These plasmids, in addition to the empty vector, were transformed into yeast strains containing Δrad4 and Δrad4/cdc20-1. Survivability was determined as described in (c), with cells plated on SCD lacking uracil rather than YPD. Overexpression of CDC20 (pCDC20) resulted in increased survivability of the Δrad4/cdc20-1 strain, while overexpression of cdc20-1 (pcdc20-1) did not. (d) Yeast cells were prepared as in (a) and spotted onto YPD media containing 5 μg/mL phleomycin, 12 μg/mL benomyl, 0.02% MMS, 200 mM hydroxyurea, and 1 μg/mL 4-NQO. The Δrad4/cdc20-1 strain exhibited decreased growth on 4-NQO relative to Δrad4 but was otherwise similar in growth patterns to the cdc20-1 mutant.
