Fig 1.
Genome-wide ssDNA mapping in two laboratory yeast strains.
(A) Schematic experimental procedures of ssDNA mapping by microarray. (B) Reproducibility of ssDNA ratios (S/G1) from two independent experiments. Pearson correlation co-efficient values are shown. (C) Normalized ssDNA profiles of ChrIII (ssDNA ratios of S/G1 plotted against chromosome coordinates in kb) for RAD53 and rad53-K227A cells in the A364a (left) and the W303 (right) background from one of the two replicate experiments (Exp 1).
Fig 2.
Comparative analysis of origins of replication in the A364a and W303 background.
(A) Consensus origins identified by both biological replicate experiments in the A364a (left) and W303 (right) background, respectively, were divided into three classes of origins, “Rad53-unchecked”, “Rad53-checked”, and “Rad53-dependent”, based on the comparison between RAD53 and rad53 cells. See text for detail. (B) Heatmaps of the normalized ssDNA ratios (compressed to a scale of 1 to 2) from the 419 unique origins from (A) in each of the four strains. (C) Quantification of average AUC values for the uncompressed ssDNA signals at the 419 origins in each strain from two experiments. (D) Western blots of Rad53 levels in cells at 0 or 60 min post release from G1 arrest into S phase in the presence of 200 mM HU, respectively. A cellular protein Vma1 was used as loading control. Overall protein levels were also controlled by Ponceau staining. The Rad53 level was quantified as the ratio of Rad53/Vma1. Each ratio was then normalized to the ratio from A364a_RAD53 cells, which was set to 1. Data from three independent measurements were averaged and plotted. Error bars stand for standard deviation.
Fig 3.
ChIP-qPCR analysis of initiation factor binding to origins.
(A) Schematic diagram (drawn to scale) of the chromosomal loci used for qPCR analysis. The prefix “ARS” was dropped for simplicity. Note the ARS306-dist locus that is located 20 kb upstream of ARS306 and 15 kb downstream of ARS305 was used as a negative control region to normalize signals from all other loci. (B) Cdc45-ChIP was performed in cells collected at 0, 20, 40, and 60 min post release of G1-arrested cells into S phase in the presence of 200 mM HU. "A", A304a; "W", W303. Relative enrichment of the ChIP DNA signals was calculated as the ratio of %Input at a given locus over %Input at the control locus. %Input was calculated as the percentage of ChIP-DNA in input DNA, averaged from at least three replicate experiments. The error bars represent standard deviations. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple test. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. (C) Relative enrichment of Rad53-ChIP DNA at the same loci as in (B). The analysis was done identically as in (B) except only cells collected at 0, and 60 min post G1 arrest/release were analyzed.
Fig 4.
Validation of Rad53-checked/unchecked origin firing status using 2-D agarose gel electrophoresis.
(A) Comparison of origin usage in the two strains leads to further classification of active origins into 15 categories. The number of origins in each category is listed below the diagram. The numbers with asterisks are the adjusted number after manual inspection. See text for detail. (B-E) DNA fragments containing select origins for analysis (ARS305, ARS1006, ARS207.1, and ARS813) were produced by restriction digestion with the indicated restriction enzymes. DNA probes are shown as black bars. Origin position is as indicated on the ssDNA profiles from two biological replicate experiments. “Bubble arcs” indicative of origin activation are indicated as red arrowheads. The origin classes were indicated using the key described in (A).
Fig 5.
Examples of strain-specific origins with polymorphisms.
The corresponding origin status is color coded same as in Fig 4. (A) Two origins with polymorphisms in A364a or W303 background resulting in a shift of initiation sites from the canonical origins. The SNPs (red font) are indicated in the ARS regions based on OriDB curation on the right. The near-matches of ACS elements are underlined in black or, in the case of overlapping elements, in magenta. The sequences shared by overlapping elements are underlined by wavy magenta lines. (B) Six origins that show binary pattern of activation in A364a but not W303 or vice versa, without shifting initiation sites.
Fig 6.
(A) Numbers of Rad53-dependent origins parsed into the shown categories. (B & C) 2-D gel validation of ARS(13:269) and ARS(16:560) performed similarly as described in Fig 4. (D) Relative gene expression levels (S/G1) at genomic loci of Rad53-dependent origins that overlap with ORFs. The relative locations of the origins and location of ORC binding sites are derived from OriDB. The ORF that is nearest to the ORC binding site is bolded in each locus. The primers (P#) used for RT-qPCR are shown as a light grey bar, not drawn to scale.