Cdc7 functions upstream of Rad53 in fork regulation. (A) WT, cdc7-1 mcm5-bob1, and cdc7-1 mcm5-bob1 pph3Δ cells were synchronized with α-factor for 3 h at 23°C, shifted to 32°C for 1 h, and released from α-factor at 32°C with 0.033% MMS. (B) DNA content analysis by FACScan. (C) Heat maps of BrdU incorporation levels at origins are arranged according to each origin’s published replication timing from early to late (left to right). (D) Aliquots were pulsed with BrdU for the indicated intervals and analyzed by BrdU-IP-chip. Results for segments of chromosomes III and VI are plotted, with origin locations indicated above. (E) WT and cdc7-1 mcm5-bob1 cells expressing Rfa1-Myc18 were treated as in A and analyzed by ChIP-chip 35 min after release; plots are color coded as in D. (F) Immunoblot analysis of unphosphorylated (single asterisks) and phosphorylated Rad53 (double asterisk); molecular mass markers were visualized with Ponceau S. Black line indicates that intervening lanes have been spliced out. Data shown are from a single representative experiment out of two replicates, except data in C, and were calculated from both replicates.