Simultaneously alleviating controls on Mcm4, Sld3, and Dbf4 allows efficient late origin firing in HU despite an elevated checkpoint response. (A,B) Yeast cells were synchronized in G1 phase and released into YPD containing 0.2 M HU and 0.5 mM EdU for 90 min. (A) Replication profiles of Chromosome IV for the wild type (WT), single, double, and triple mutants with mcm4Δ74-174, sld3 38A, and/or dbf4 19A alleles as indicated on the top left of each profile. Gray bars under each profile indicate annotated replication origins in OriDB v2.1.0 (Siow et al. 2012). Orange bars indicate the position of the centromere. Red arrows point out some late origins that are inactive in the wild-type cells but fire in the mutants, in particular the triple mutant. (B) Distribution of fork progression from activated origins. Top panels show the peak width–height plots of all the recorded origins across the entire genome for each strain in A. Box plot shows the fork progression, excluding peaks, with heights <10% of the maximal height (also shown as black dashed line in individual width–height plots). Box and whiskers indicate 25–75 and 10–90 percentiles, respectively. (C) Cells of the indicated yeast strains were synchronized in G1, released into 0.2 M HU, and collected at the indicated time points. Protein samples were prepared using TCA extraction and analyzed by immunoblot. (D) Analysis of chromatin-bound proteins. Cells synchronized in G1 were released into 0.2 M HU and collected at the indicated time points. Chromatin-bound proteins were extracted from cells and analyzed by immunoblot.
