Ddc2ATRIP promotes Mec1ATR activation at RPA-ssDNA tracts
Fig 4
Effect of ddc2-S4 mutation on S-phase checkpoint signaling.
(A) Wild-type (KSC4233), ddc2-S4 (KSC4234) and ddc2Δ (KSC4235) cells expressing Mrc1-HA were arrested with α-factor at G1 and released into medium containing 0.05% MMS. Cells were collected at the indicated time and analyzed as in Fig 1A. Cell cycle progression from G1 to S phase was monitored by DNA flow cytometry. (B) Wild-type (KSC1178), ddc1Δ dna2-AA (KSC4219), ddc2-S4 (KSC3153) and ddc2Δ (KSC1234) cells carrying the YCpT-Rad53-HA plasmid were synchronized with α-factor at G1 and released into medium containing 0.05% MMS or 100 mM HU. Cells were collected at the indicated time (45 min for MMS and 60 min for HU) and analyzed as in Fig 4A. (C) Effect of ddc1Δ dna2-AA or ddc2-S4 on sensitivities to MMS and HU. Wild-type (KSC1178), ddc1Δ dna2-AA (KSC4219), ddc2-S4 (KSC3153) and ddc2Δ (KSC1234) cells were serially diluted and spotted on plates medium containing MMS or HU. (D) Wild-type (KSC1178), ddc1Δ dna2-AA (KSC4219), ddc1Δ dna2-AA mec1Δ (KSC4238) or mec1Δ (KSC1186) cells were synchronized with α-factor at G1 and released into medium containing 0.05% MMS. Cells were collected at the indicated time and subjected to immunoblotting analysis with anti-Rad53 or anti-tubulin antibody. Cell cycle progression from G1 to S phase was monitored by DNA flow cytometry.