Sensing of Replication Stress and Mec1 Activation Act through Two Independent Pathways Involving the 9-1-1 Complex and DNA Polymerase ε
Figure 3
Low levels of Rad53 activity are sufficient to prevent replication fork breakdown.
(A) HU recovery assay: K699 (WT), YFP20 (dpb11-1), YAN21/8d (ddc1Δ), YFP62/1d (ddc1Δdpb11-1) and YMIC5A3 (mec1-1) were synchronized in G1 with α-factor and released into fresh medium supplemented with 200 mM HU. 90 min later cells were transferred to fresh YPD + nocodazole and allowed to resume DNA replication. Progression into S phase was monitored by FACS analysis. (B) The indicated strains were synchronized in G1 with α-factor and released into 100 mM HU + nocodazole. 3 and 5 hours later cells were harvested and total DNA was analyzed by Pulse Field Gel Electrophoresis (PFGE). (C) The strains in panel A were synchronized in G1 and released in YPD supplemented with 200 mM HU. 90 min later 10-fold serial dilution were prepared and spotted onto YPD plates. The same was done with the G1-synchronised cultures as control.