Exceptional origin activation revealed by comparative analysis in two laboratory yeast strains

We mapped ssDNA formation in WT and rad53K227A cells in both A364a and W303 background. We also performed whole genome sequencing of the A364a strain. Briefly, cell cultures were grown to an OD600 of 0.25~0.3 in YPD medium, followed by G1 arrest with 200 nM α-factor. Pronase (0.02 mg/mL) was used to synchronously release cells into S phase in the presence of 200 mM hydroxyurea (HU). G1 control and S phase samples were collected prior to cell cycle release and after 1 h treatment of HU, respectively. Three-hundred ml of cells from each sample were collected and spheroplasted in agarose plugs for ssDNA labeling. Differentially labeled G1 (Cy5-dUTP) and S phase (Cy3-dUTP) DNA were co-hybridized onto Agilent Yeast Whole Genome ChIP-to-chip 4 × 44K (G4493A) microarrays and the data were extracted by the Agilent Feature Extraction Software (v9.5.1). The relative quantity of ssDNA at a given genomic locus was calculated as the ratio of the fluorescent signal from the S phase sample to that of the G1 control, followed by Loess-smoothing over a 6-kb window at a step size of 250 bp.