Orc1 function regulates replication fork progression. (A) WT and orc1-161 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) Immunoblot analysis of phosphorylated Rad53 (Rad53-P); both rows are from the same blot and exposure. Molecular weight markers were not run on this gel; for the migration of size markers relative to the bands detected by this antibody, see Fig. 2 F. Black line indicates that intervening lanes have been spliced out. (D) 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). (E) 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. Data shown are from a single representative experiment out of two replicates, except data in D, and were calculated from both replicates.