A screen for genes with extended S phase. (A–D) Distribution of DNA content (“FACS profiles”) in asynchronous populations of wild-type cells (BY4741) (A) and cells identified in the screen (B–D). All FACS profiles were reproduced at least eight times. The color codes of the titles and axes in B–D denote known functional classifications of the genes. (OBO) Off-by-one (Pan et al. 2006). (E) Generation time and duration of each cell-cycle phase for the different mutants. Ordering is S phase duration. See Supplemental Table S2 for the raw data and summary of the genes and phenotypes. (F) FACS profiles of double mutant combinations as indicated.

Genetic characterization of S-phase mutants. (A) FACS profiles of the indicated cells treated with different dosage of HU. The large G₂ fraction of HU-treated rnr1 cells may be overestimated because of some difficulties in discriminating doublets (data not shown). (B) Synthetic interactions between genes associated with dNTP synthesis. FACS profiles are shown for mutants deleted of all gene-pair combinations. (C) A complete epistatic miniarray profile (E-Map) for the genes identified in our screen. Genetic interaction scores were computed and used to cluster the matrix (see Methods). (SL) Synthetic lethal. (Gray boxes) No data. Two cases that resulted in particularly long S phases are at left: dpb3 rad27 and mrc1 tda3. Note the strong aggravating interaction in mid-to-late S phase not evident in either of the single mutants.

Profiling the genomic DNA replication timing program. (A) Two million SYBR-Green stained cells are sorted separately from G₁ and S phases, differentially labeled with fluorescent Cy dyes, and competitively hybridized to an Agilent genomic tiling array of ∼290-bp resolution. The data from four biological replicates are averaged, and the averaged data are smoothed to generate the DNA replication timing profile. (B) The replication profile of chromosome IV generated in two experiments, each consisting of four biological repetitions. The profiles are scaled to 0–100, with 0 being the beginning of S phase and 100 the end. The black vertical lines correspond to positions reported as replication origin (“verified” origins, identified by a combination of experimental and sequence analysis; Nieduszynski et al. 2006). (C) Autocorrelation as a quality control: Shown are the average correlations between the DNA content in chromosomal positions separated by the indicated distances. The correlations are calculated from the raw (unsmoothed) data, are averaged over all chromosomal positions, and are normalized to a maximum of 1. The black region represents 100 separate calculations of autocorrelations on randomized data. (D) Comparison of origin activation times in wild-type cells, as identified in our experiment, with those characterized previously using a similar strain background (S288c [Green et al. 2006]; our strain background is BY4741, a derivative of S288c). See Supplemental Figure S2 for comparison with other data sets. (E) A matrix showing the genome-wide correlations between different data set, including the two repeats from our study. The different experiments used different strain backgrounds, as indicated (S288c and A364a [Green et al. 2006]; W303 [Yabuki et al. 2002]; RM14-3a [Raghuraman et al. 2001]).

Replication timing profiles of the mutants identified in our screen. (A,B) Replication profiles of chromosome IV (A) and VII (B) for wild-type, mrc1, and either clb5 (A) or ura7 (B). Axes, legends, and vertical lines are as in Fig. 3B, with the exception that the y-axis is scaled according to the length of S phase in each of the mutants. Other mutants and chromosomes are shown in Supplemental Figure S3. (C) Clustered correlation matrix of the entire data set. The dendrogram distance to mrc1 is not to scale (broken lines). Functional classifications are based on the literature, the FACS results, and analysis of the microarray data described in the main text. Correlations were calculated between the smoothed averages of four biological repeats, as described above (Fig. 3). See Supplemental Figure S4 for correlation between individual repeats. (D) Autocorrelations (calculated as in Fig. 3C) of the different mutants: Strains are ordered by increasing values of autocorrelation at 50 kb. Autocorrelation shown here was calculated using the smoothed profile. See Supplemental Figure S4 for autocorrelations for the raw data.

Scaling of the replication profiles. (A) Number of replication origins in each mutant. The broken vertical line represents the number of origins active in the wild-type strain. Total active origins are further divided into origins that appear in both the wild type and mutant (maintained, “Maint.”) and those that appear only in the mutant (activated, “Act.”). This later group is further divided into telomeric origins (“Act. tel.”; this includes the rDNA borders) and nontelomeric origins. Green bars represent origins that appear in the wild type but not in the mutants (suppressed, “Supp.”), again divided into these found in telomeric (“Supp. tel.”) vs. nontelomeric regions. (B) Scaling in all mutants apart from the mrc1 deletion strain: For each mutant, the scatter plot shows the activation timing of each maintained origin in the mutant (y-axis) as a function of its activation time in the wild type (x-axis). The wild-type data are scaled to 0–100, and the mutant data are scaled according to the relative length of S phase. Red broken lines represent identical timing between the wild type and mutant. The green line is a second-order polynomial fit to the data. The black broken line represents a perfect linear scaling. Typically, origin activation times are delayed more than by a linear factor. All mutants are shown in Supplemental Figure S5. (C) All polynomial fits (as in B) are shown together for comparison. The broken gray line represents no delay in origin activation. The differences between mrc1 and rrm3 and sic1 are more pronounced than apparent from this comparative figure when taking into account the significantly longer S phase of mrc1 cells (see B).

Model of replication timing control. Perturbations of normal replication fork progression (or origin activation), brought about by disruption of replication factors, cause a proportional delay in the activation timing of the different origins, maintaining their relative order of firing. While the structure of the replication timing program is overall maintained, certain origin are rendered inactive because of passive replication. Conversely, in mrc1-deleted cells with a similar disruption of replication, origins are not delayed and the overall structure of the replication program is altered. When combined with the slower progression of the replication fork, this enables the firing of dormant origins, which in wild type are passively replicated. The replication profiles (solid lines) correspond to: green, wild type (unperturbed; normal replication); black, cells subject to a replication perturbation (mutation) with intact MRC1 (scaled replication program); red, cells subject to a replication perturbation with defective MRC1 (no scaling). Broken lines correspond to different classes of replication origins: green, origins active in all situations; black, origins rendered inactive because of scaling when MRC1 is intact and replication is perturbed; red, normally inactive origins that become activated when replication is perturbed and MRC1 is nonfunctional.