Replication times of origins activated in wild-type cells (DCY1671) assigned according to previously published timing data. Replication times were assigned to all activated origins identified in this study (Supplementary Table II) according to two earlier studies (Raghuraman et al, 2001; Yabuki et al, 2002). Replication times ranged from 10 to 60 min for Raghuraman et al and 16 to 34 min for Yabuki et al. The ‘cutoffs' for early-firing origins used by Raghuraman et al and Yabuki et al were 28 and 24 min, respectively. Assigned replication times are shown for origins activated in wild-type cells after 80 (A) or 120 min (B) in HU. (X% <28 min) indicates that X% of origins are activated within 28 min after G1 release.

Overlap of activated and compromised replication origins between wild-type cells and S-phase checkpoint mutants. Venn diagrams showing the overlap for activated early origins between wild-type, mec1-1 and rad53-1 (A), or CEOs (B) and activated late origins (C) between mec1-1 and rad53-1. The total number of origins activated/compromised in each strain is indicated. The definition of early or late is based on our array results and on a previous study (Raghuraman et al, 2001). All origins activated in our wild-type strain (DCY1671) at the 80 min time point (66 in total) were deemed early. However, 22 of these origins are considered late in the KK14-3a background (Raghuraman et al, 2001) and six of these origins are defined as late in the W303 background (Yabuki et al, 2002).

2D gel electrophoresis of replication intermediates reveals the accumulation of replication forks at CEOs in S-phase checkpoint mutants. 2D gel electrophoresis was carried out on replication intermediates isolated from wild-type, mec1-1 and rad53-1 cells arrested in 0.2 M HU for 80 min. The maps above the 2D gels indicate the locations of the restriction sites and probes used for each origin. The distance to neighboring origins is indicated as well. Replication patterns are shown for an early-firing origin, ARS305 (A), and CEOs, ARS315 (B) and ARS1305 (C). (D) A cartoon depicting the replication pattern of a restriction fragment containing a CEO. Accumulation of arrested forks is indicated by the spot on the fork arc. (E) A model illustrating fork arrest in CEO-flanking regions at points a and b. We hypothesize that forks collapse, inducing breakage at a or b, which results in the accumulation of fork structures as shown in panel D. RE indicates restriction enzyme cleavage sites. Please note that the width of the fork arcs at the CEOs is narrower in the mutants than wild-type cells.

Chromosome fragility adjacent to CEOs in the absence of HU. (A) A two-step LMPCR method was used to detect DSBs near replication origins in asynchronous wild-type, mec1-1 and rad53-1 cells in the presence and absence of Rad52. DNA was isolated and a linker was ligated to all double-stranded DNA ends. DNA was amplified first by a 25-cycle PCR reaction with one origin- and one linker-specific primer, followed by a nested 10-cycle PCR reaction using a second origin-specific and the same linker-specific primer. PCR fragments were fractionated on agarose gels. As a control, breaks were introduced at a specific restriction site ∼1 kb downstream of the origin, and the same LMPCR was performed. Results are shown for early origins ARS305, ARS121 (proARS1021) and ARS1 (proARS416), and for CEOs ARS310, ARS1305, ARS315 and ARS606. (B) LMPCR analysis of DSBs adjacent to the CEO ARS315 in asynchronous wild-type, mec1-1 and rad53-1 cells (in the presence and absence of Rad52), containing a deletion of ARS315. As a control, DSBs were detected at the CEO ARS310 in these strains. (C) LMPCR analysis of DSBs in asynchronous wild-type, mec1-1 and rad53-1 cells (in the presence and absence of Rad52), containing an extra copy of the CEO ARS315 integrated into the URA3 locus. LMPCR analysis of ARS310 was conducted in the same strains as a positive control.

Origin activation profiles for wild-type chromosomes III and XIII. Origin activation profiles (80 min in 0.2 M HU) for chromosomes III (A) and XIII (B) are shown. The distribution of replication origins on each chromosome is indicated by black bars in the cartoon at the top. The letter E indicates early-firing origins (Raghuraman et al, 2001). Each point on the graph represents the average of three experiments. The dotted connecting line does not represent data points. The significance level used is one standard deviation above background (corresponding to approximately 20% of total signal). All scored origins had P-values <0.05. Replication profiles for chromosomes III (C) and XIII (D) based on an earlier study (Yabuki et al, 2002) are also shown. The significance level that was used by Yabuki and co-workers is 10% of total signal. Activated origins in panels A–D are indicated by their respective numbers.

Origin activation profiles for chromosomes III and XIII in mec1-1 and rad53-1 strains. Chromosomes III (A) and XIII (B) are shown as cartoons at the top of the figure (see Figure 1 for details). Each point on the graph represents the average of three experiments. The dotted connecting line does not represent data points. Significance levels 1 and 2 correspond to one and one-half of a standard deviation above background, respectively. Late origins are activated in rad53-1 and mec1-1 mutants (P-values <0.05) and are marked by the letter L. Compromised early-firing origins (CEOs), which are activated in wild-type cells (Figure 1) but not efficiently replicated in the checkpoint mutants (copy number change <significance level 2 and P-value <0.05), are indicated by CEO and highlighted by a circle over the corresponding point on the graph.

Analysis of replication intermediates from wild-type and rad53-1 cells in the presence of HU. G1-arrested cells were released into 0.2 M HU for 0–120 min at 25°C (A–C) or at 30°C (D, E). Replication intermediates (RIs) isolated from these cells at the indicated time points were studied using alkaline agarose gel electrophoresis (Santocanale and Diffley, 1998). RIs are shown for the early-firing origin ARS305 (A, D), and CEOs ARS310 (B, E) and ARS1305 (C).

ChIP analysis shows accumulation of replication forks adjacent to a CEO in S-phase checkpoint mutants. Wild-type, mec1-1 and rad53-1 cells expressing Myc-tagged Mcm10 were synchronized in G1 phase and released into 0.2 M HU at 30°C. Samples were taken at the indicated time points. ChIP was performed using an anti-Myc antibody. PCR products were visualized on agarose gels. Results are shown for origin and origin-flanking regions at an early-firing origin, ARS305 (A), a CEO, ARS315, at its native locus (B), and ARS315 integrated into the URA3 locus (C). The distance to the origin is indicated and R11 was used as a non-origin control.