Dynamics of ssDNA formation in WT cells and rad53 cells. (A) Flow cytometric analysis of an asynchronous cell population (Asy) and of cells undergoing S phase in the presence of 200 mM HU; the times indicated are those following the release from an alpha factor block. (B) Amount of ssDNA (% of genomic total) in WT (black bars) and rad53 (grey bars) cells at the indicated times post release from an alpha factor block as determined by slot blot analysis (see Fig. 1C). (C)&(D) Time course of ssDNA formation on chromosome III in WT cells (purple in C&D) and rad53 cells (orange in D). The relative ratios of ssDNA from S phase samples to that from the G1 control sample are plotted against chromosomal coordinates. Raw but normalized data instead of smoothed data are used here. For smoothed versions please see Supplementary Information, Fig. S1&2. Note the 4-fold difference in the scale on Y axes for WT and rad53 cells. The locations of known origins are marked by vertical lines and annotated above the plots. The centromere is marked as a filled grey circle on the X-axis. Examples of migrating ssDNA peaks in WT cells are indicated by grey arrowheads. (Note, the peak between 80 and 85 kb in WT cells is the consequence of hybridization to a single ORF, YCL021W-A, does not correspond to any known origin, ARS, or Pro-ARS sequence, and does not move laterally with time. For these reasons, we consider this peak to be a possible artifact.) The replication profile for chromosome III (T_rep--the time of half maximal replication5--vs. chromosomal coordinate) is shown at the bottom of the WT panels.

New origin identification from the ssDNA profiles of rad53 cells. (A)&(D) ssDNA profiles for rad53 cells at 1, 2 and 3 hour (yellow, orange, and red respectively) post release from alpha factor arrest into S phase in the presence of 200 mM HU for chromosome III (A) and X (D). Green diamonds, locations of Pro-ARSs identified by Wyrick et al.13 Blue circles, locations of clustered origins identified from ssDNA profiles of rad53 cells. Red circles, locations of peaks identified in only one of the three rad53 timed samples. (B) Map and (C) two-dimensional agarose gel analysis of the predicted origin ORI314.5. Genomic DNA isolated from RM14-3a cells5 in a normal S phase (30 minutes post release from an alpha factor block and a subsequent cell cycle block with cdc7-1 mutation) was digested with XbaI (coordinates as indicated) and probed with a fragment (orange bar) corresponding to the 5' end of TAF2 shown as the long black arrow. The resulting autoradiogram is shown. (E) Map and (F) two-dimensional gel analysis of the predicted origin ORI1007.5. Genomic DNA obtained as described above was digested with NcoI and probed with a fragment corresponding to MRS3 (orange bar). (G) Correlations between T_rep in minutes (solid diamonds, dashed line) or efficiency (open circles, solid line) for origins on ChrVI16 and the extent of ssDNA formation in rad53 cells at 1 hour post release into HU. The correlation coefficient values are shown.

Comparison between the distributions of inter-origin distances in S. cerevisiae (cross-hatched bars) and S. pombe (solid black bars). The bin size for the histogram was set at 10 kb.

Outline of experimental procedures. (A) Synchronization and yeast cell sample collections. (B) Labeling of DNA for microarray hybridization. (C) Slot blotting and hybridization for quantification of ssDNA. The total amount of ssDNA in the genome for each sample was calculated and used for normalization of microarray data. (D) Data analysis. The relative amount of ssDNA was calculated as a ratio of the signal from the S phase sample to that from the control G1 sample. The ssDNA profile was constructed by plotting the ratio of ssDNA as a function of the chromosome coordinate.

Elevated levels of ssDNA at telomeric regions. (A) Overlay of ssDNA profiles of chromosome VI for WT cells (light purple, purple, and dark blue for 1, 2, and 3 hours post release, respectively) and rad53 cells (yellow, orange, and red for 1, 2, and 3 hours post release, respectively). The Y-axis for the rad53 cells (right) is offset slightly from the Y-axis for the WT cells (left) to facilitate comparisons of coincident peaks. “T”, regions defined as telomeric (see text). (B) Bar graph of the ratio of telomeric ssDNA to internal (non-telomeric) ssDNA in WT (gray bars), rad53 (black bars) and rad53 exo1 (crosshatched bars) cells at different times following release from G1 arrest in media containing HU. The error bars show the standard deviations of telomere to internal ssDNA ratios among the 16 chromosomes.

Single-stranded DNA profiles of S. pombe chromosomes (higher resolution profiles are shown in Fig. S3). Chromosomal coordinates were downloaded from the from the Sanger Center (sequence version AL137130.1) and from the National Center for Biotechnology Information. Chromosome I is shown in two halves as “Ia” and “Ib”. The ratio of ssDNA (S/G1) was plotted as a function of chromosomal coordinates for wild type (purple, Y1 axis) and Δcds1 (orange, Y2 axis) cells. The approximate position of each centromere is marked by a grey circle. Purple circles, locations of significant ssDNA peaks identified from wild type cells. Orange circles, locations of significant ssDNA peaks identified from Δcds1 cells. Standard deviation for baseline variation was determined similarly as described for S. cerevisiae (see Supplementary Information, Identification of significant ssDNA peaks). A given local maximum is considered a significant peak if the peak height differential is above 2 standard deviations from either of its flanking local minima. The positions of known origins that have been identified as significant ssDNA peaks are marked by vertical grey lines19 and the origin names are listed above the graphs. The two known origins that were not detected by a significant ssDNA peak, pcr1 and pARS727, are each marked by a box.