Fig. 4: Replication origins that initiate replication early during normal mitotic growth are over-activated during re-replication.

a HCT116 cells were treated with 250 nM of MLN4924 for 30 h and EdU click-it to label S phase cells. Control cells showed typical S phase cell foci patterns (ES: early S phase; MS: mid-S phase; LS: late S phase). MLN4924 treated cells lost these typical S phase replication foci patterns, showing ES-like foci patterns (see Supplementary Fig. 7a for more details). Three biological repeats yielded similar results. b Total genomic DNA of HCT116 cells was purified from G1 cells, asynchronous normal mitotic growing cells and asynchronous re-replicating cells induced by MLN4924 as shown in Supplementary Fig. 7b and sequenced at >30× coverage. IGV screenshot of replication timing of chromosome 2 for normal replication and re-replication (same y-axis scale) showing increased copy number at the early replication genomic regions and decreased copy number at the late replication genomic regions in re-replicating cells. c Genomic regions were divided into four equal groups according to their replication timing and amplification scores of these segments were calculated in control and re-replicating nascent DNA. 5051, 8624, 6746 and 3076 origins from one representative experiment for early, mid-early, mid-late and late, respectively, were analyzed. Box plots are showing the median, 25^th and 75^th percentiles as box boundaries and the 5^th and 95^th percentile ranges. Wilcoxon test was used for the statistical significance analysis. p < 2.2e-16 by using the default two-sided test. 3 independent experiments showed similar results. d Top panel: Density plots comparing reads per peak (reflecting the frequency of initiation per each origin) in groups of replication origins from cells undergoing normal replication (control) and re-replication (MLN4924 for 45 h). Origins were classified into six replication timing groups, ranging from very early to very late, using the replication timing data illustrated in panel b. Data points from origins that exhibited similar frequencies of initiation during normal replication and re-replication are expected to localize on the diagonal dotted line. Data points above the diagonal dotted line represent origins that initiated more frequently during re-replication than during normal S-phase, and vice versa. Bottom panel: origins in the density plot were further divided into 10 fractions ranging from origins that showed the highest ratio of initiation frequency during re-replication vs. during normal replication (MLN 45 h, left side of each bar graph) to origins that showed the highest ratio of initiation frequency during normal replication vs. during re-replication (Control 45 h, right side of each bar graph). The number of peaks in each of the 10 fractions showed the gradually shift from more origins initiated in MLN4924 treated sample than in control at early replicating genomic regions to fewer origins initiated in MLNN4924 treated sample than in control at late replication genomic regions.