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Figure 3


Fig. 3. Rad53 activity persists after HU exposure in cells lacking both CCR4 and DUN1. (A) Wild-type (BY4741), ccr4{Delta} (MT3768), dun1{Delta} (MT3769) and ccr4{Delta} dun1{Delta} (MT3772) strains were treated with 200 mM HU for 3 hours followed by assessment of Rad53 kinase activity by in situ assay (ISA), as manifest in Rad53 autophosphorylation in vitro (upper panel), and by the extent of phosphorylation-dependent Rad53 mobility shift, as detected by the DAB001 Rad53 antibody (lower panel). (B) Cell cycle progression after replication stress is severely delayed in a ccr4{Delta} dun1{Delta} strain. DNA content of the strains in A was determined by FACS analysis of either asynchronous mid-log phase cultures (ASN) or after S-phase synchronization in 200 mM HU for 3 hours (+HU) followed by washout of HU for the indicated times.





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