A) Schematic of ori2004 and ori2060 (figure not drawn to scale). B) FACS analysis indicates that S occurs between 50 and 80 minutes after release from cdc25-22 arrest. C) Orp1 binding to ori2004 was assayed in a synchronous culture after release from cdc25-22 arrest. Each point represents the result of qPCR using primers over region III. Orp1 binding to ori2004 is periodic during the cell cycle, reaching a maximum at M/G1. D) Association of an integrated, fully functional Orp4-HA fusion with ori2004 as determined by ChIP. The same primers as in (C) were used. Orp4 shows similar timing and periodicity of binding at ori2004 as Orp1. E) Comparison of Orp1 and Mcm4 binding during the cell cycle. Each Mcm4 point represents the occupancy over region II of ori2004. F) The peak of Cdc45 association with ori2004 coincides with the start of replication, after maximal Mcm4 binding. IPs were performed from the same extracts, and each point represents the occupancy over region II of ori2004.

A) Time course of Orp1 binding at wild type ori2004 and three mutant alleles: ori2004ΔI, ori2004ΔIII, and ori2004ΔIΔIII (see Figure 1A). For wild type and ori2004ΔI, each point represents the occupancy of Orp1 over region III; ori2004ΔIII occupancy was quantified using primers over region I, while ori2004ΔIΔIII was assayed using primers over region II. Orp1 binding is reduced in the single ori2004 mutants, absent in the double mutant, and specifically delayed in ori2004ΔIII. B, C) Comparison of Mcm4 and Cdc45 binding in wild type and ori2004 deletions. Primers over region II were used to determine occupancy for both Mcm4 (B) and Cdc45 (C). Mcm4 occupancy is reduced in the ori2004 mutants, and ori2004ΔIII shows a small delay in Mcm4 binding. Cdc45 binding is delayed in ori2004ΔIII and ori2004ΔIΔIII.

A) Left: FACS analysis of cells synchronized using cdc25-22 and released into MBC shows only a small susbset of cells entering S after 70 minutes. Right: ChIP of Orp1 from MBC-treated cells. Orp1 accumulates at ori2004, ori2060, and ars727 and remains bound during MBC treatment. B) Representative regions from array analyses showing reduction in the usage of early-firing efficient origins in cells treated with MBC and released into HU. MBC-treated and HU control cells are represented by black and gray lines, respectively. Asterisks mark reproducible decreases in three experiments. C) A subset of inefficient regions show increased BrdU incorporation after MBC treatment. Asterisks mark reproducible increases. D) Relative origin usage in MBC-treated samples and HU controls. BrdU IPs were assayed by qPCR. Bars represent the ratio of signal in MBC-treated to HU control samples, and a region that does not replicate in HU-treated cells was used for normalization. Values are averaged from three experiments, and error bars show the standard deviation. (i) The five efficient origins tested display reduced signals in the MBC samples. (ii) Eight low-efficiency regions, labeled by chromosome, show reproducible increases in replication after MBC treatment. (D) Origin efficiencies following MBC treatment. Replication efficiency was determined using qPCR of genomic DNA from both MBC-treated and HU-control cells. Values are averaged from three experiments.

A) BrdU signal is increased at efficient and inefficient origins in HU-treated synchronous cultures of strains overexpressing cdc45, hsk1, or dfp1. Bars represent the ratio of signal in overexpressing vs. wildtype cells, and a region that does not replicate in HU was used for normalization. Values are averaged from at least three experiments. B) Relative origin usage in cells constitutively overproducing Cdc45, Hsk1, or Dfp1 after MBC treatment. qPCR of BrdU IPs showed increased signal in overexpressing strains at both efficient and inefficient origins.

A, B) Time course of Orp1 and Mcm4 binding at ori2060 and ori2004. For ori2060, each point represents the occupancy between the two AT-hook regions. Orp1 binding at ori2060 peaks around 40 minutes, while Mcm4 binding is maximal 50-60 minutes post-release from cdc25-22 arrest. ORC binding and pre-RC formation at ori2060 occur approximately 10 minutes later than at ori2004. C) Comparison of Cdc45 binding at ori2060 and ori2004. Maximal Cdc45 association at ori2060 is delayed compared to ori2004. D-F) Time course of Orp1 (D), Mcm4 (E), and Cdc45 (F) at ori2004, ori2060, and ars727 by ChIP shows a delay in ORC, pre-RC and pre-IC formation at late-firing and inefficient or inactive origins.

A) cdc25-22 synchronized cells were released into HU at 25°C for time course analysis. ChIP of Mcm4 at ori2004, ori2060, and ars727 show that pre-RC formation is generally not affected by HU treatment. B) HU treatment significantly reduces the maximal levels of Cdc45 binding at late-firing inefficient origins.

A-C) Time course of Orp1, Mcm4, and Cdc45 binding by ChIP shows that all three steps are delayed at ori1140 compared with ori2004. D) ChIP of Orp1 from MBC-treated cells. Orp1 accumulates at ori2004 and ori1140 and remains bound at the origin throughout the MBC treatment. E) The timing of Mcm4 and Cdc45 binding at ori1140 after MBC treatment and growth in HU is advanced and coincides with these events at ori2004.