The PP1 Docking Motifs in Rif1 Are Required to Establish the Replication Timing of Budding Yeast Telomeres and Fission Yeast DNA Replication Origins
(A) Analysis of the association of C-terminally Myc-tagged Pol2 with selected telomeres and origins in RIF1 wild-type, rif1-PP1, and rif1-Δ budding yeast cells after synchronous release from G1 arrest. ARS607 (blue) and ARS1412 (orange) were used as markers of early and late S phase, respectively. To account for differences in efficiencies in the immunoprecipitations among different experiments, each profile for each amplicon was normalized against its highest peak. The data represent the average of three independent experiments for each strain. The significance of the change in the position of the telomere peak for each rif1 mutant against the wild-type was assessed by applying a Wilcoxon test (one sided; p < 0.024).
(B) Analysis of the replication timing of ARS607 and the VI-R and XV-L telomeres, in reference to ARS1412. DNA amounts for cells after release from G1 arrest were quantified by quantitative PCR (qPCR). For each of the three loci analyzed, normalization was first carried out against the ARS1412 locus at the same time point, and subsequently against the G1 time point (0 min). A minimum of three experiments were averaged for the analysis. Two-tailed t tests were carried out for significance for each mutant against wild-type at the same time point (p < 0.05 is indicated by asterisks). See also .
(C) Replication efficiency of early and late origins in fission yeast, in wild-type, rif1-Δ, rif1-PP1, and sds21-Δ strains. Log-phase cultures were arrested in G2 at 36°C using the cdc25-22 temperature-sensitive allele and then released into 25 mM hydroxyurea for 140 min. Genomic DNA was prepared for the G2 (0 min time point) and late S phase (140 min time point) cells and quantified by qPCR. The ratio of the amount of genomic DNA in late S phase to that in G2 was calculated for each locus. The non-ori1 locus was used for normalization (). Two-tailed t test for each mutant against wild-type were performed from at least eight replicates. A p value < 0.05 was deemed significant and is indicated by an asterisk in the graph. SDs are indicated in all panels.