Functional Centromeres Determine the Activation Time of Pericentric Origins of DNA Replication in Saccharomyces cerevisiae
(A) Cartoon depiction of experimental setup. Cells were grown in medium containing heavy carbon (13C) and nitrogen (15N) isotopes. Upon genome saturation with the heavy isotopes, cells were arrested by the addition of alpha factor and released synchronously in medium containing light carbon (12C) and nitrogen (14N) isotopes. The cells were then collected over the next 140 minutes and their DNA was extracted, digested with EcoRI, and separated via ultra centrifugation in cesium chloride gradients such that unreplicated DNA resides lower in the gradient than newly replicated DNA. DNA samples were then collected and analyzed through drip fractionation. (B) S phase progression of WT (left) and rearranged (right) cells as measured by flow cytometry. Cells from both strains entered S-phase by 40 minutes and achieved 2C DNA content by 140 minutes as indicated by the peak shift from 1C to 2C DNA content. (C) Replication kinetic curves for met2 or MET2, ARS1410, and ARS1426 in WT (top panel) and rearranged (bottom panel) cells. The kinetic curves for ARS306 and R11 are shown as dashed and dotted lines, respectively. Trep is the time of half-maximal replication for each locus (see Materials and Methods). (D) Replication indices for met2 or MET2 (green), ARS1410 (blue), and ARS1426 (magenta) in WT (solid diamonds) and rearranged (empty diamonds) strains. ARS306 (black dashed arrow) and R11 (black dotted arrow) were used as early and late timing standards, respectively. In the WT strain, MET2, ARS1410, and ARS1426 had replication indices of 0.87, 0.77, and 0.16 respectively. In the rearranged strain, met2, ARS1410, and ARS1426 had replication indices of 0.24, 0.23, and 0.79, respectively. Direction of the black arrows indicates the direction of the shift in replication index for each locus between WT and rearranged strains.