|Number of samples:||10|
|Release date:||Jan 30 2006|
|Last update date:||Jan 18 2013|
|Dataset link||Single stranded DNA formation during S phase in the presence of hydroxyurea in S. cerevisiae and S. pombe|
In order to investigate the dynamics of ssDNA formation on a genomic scale, we harvested cells at discrete times after releasing them from late G1 phase arrest with alpha factor into a synchronous S phase in the presence of 200 mM HU. Chromosomal DNA isolated from these S phase samples and an alpha factor arrested G1 control sample were differentially labeled with Cy-conjugated deoxyribonucleotides by random priming and synthesis without denaturation of the DNA, followed by co-hybridization to a microarray. Because the labeling was done without denaturation of the template DNA, single-stranded regions of the genome should preferentially act as templates for dye incorporation. Comparison of experimental (S phase) and control (G1 phase) samples from the microarray hybridization revealed regions of the genome that became single-stranded in S phase. We also assessed the total percentage of ssDNA in the samples by blotting native (undenatured) genomic DNA and fully denatured genomic DNA, followed by hybridization with a genomic DNA probe. The calculated total percentages of ssDNA in the samples were then used to normalize the raw ratio of ssDNA (S/G1) (raw data) to generate the normalized ratio of ssDNA (S/G1) (raw normalized data). The normalized relative ratio of ssDNA was then smoothed over a 4 kb window (smoothed data) via Fourier transformation. For origin mapping in S. pombe, we used S. pombe wild type and deltacds1 (cds1 encodes for the homolog of Rad53) cells in a comparative analysis. DNA isolated from cells that were exposed to HU (“early S phase” sample) and cells that were starved for nitrogen source (G1 phase control sample) were differentially labeled with Cy-conjugated dUTPs without denaturation of the template DNA to enrich for labeling of ssDNA region in the genome. These DNAs were then co-hybridized to a microarray. The relative amount of ssDNA was quantitated as the ratio of fluorescent signal from the “early S phase” sample to that from the control sample. The raw ratio of ssDNA (early S/G1) was then normalized by the total percentage of ssDNA in the samples similarly as for S. cerevisiae data. The raw normalized ratio was then smoothed over a 12 kb window via Fourier transformation.