Fig. 6: Completion of replication of subtelomeric regions and difficult-to-replicate sequences requires the drop in Cdk activity that occurs during mitotic exit.

a Under-representation of subtelomeric regions in chromosome V for metaphase (MET3pr-CDC20, in green) and telophase (dbf2-2, in red) arrests. Shadows correspond to standard deviation across biological replicates (6 for MET3pr-CDC20, 5 for dbf2-2). b Distribution of sequences under-represented in metaphase across the whole genome, with values greater than a threshold of 20.5% (see Methods) shaded in green (1.2 Mb, about 10% of the genome). c Under-representation values for all 200 bp windows throughout the genome, with significantly underrepresented genomic regions colored green. d Late-replicating regions show higher under-representation in both non-subtelomeric and subtelomeric regions (N.S. p > 0.05; ***p < 0.0001, Wilcoxon rank test). All 200-bp windows of measured under-replication split into bins based on their replication timing (data from⁵¹). Colour-code corresponds to the proximity to telomeres (1:100 kb). ~0.1% of 200 bp regions have under-representation less than −20%; for visualization we plot them at -20%. e Regions with high frequency of G-quadruplexes, transposable elements and fragile sites exhibit higher under-replication in metaphase (***p < 0.0001, two-sided Wilcoxon rank test). f Transposable elements and fragile sites are uniformly distributed across the genome and show the same replication timing as a bulk genome, whereas G4-rich regions are mainly located in subtelomeric regions and replicated later than the majority of the genome. Each transposable element and fragile site correspond to a single functional element, and G4-regions correspond to a single 200 bp window with elevated fraction of G4-sequences. g Genome sequencing was performed in metaphase-arrested (Cdc20-depleted) cells before and after inhibition of Cdk using the ATP analogue-sensitive mutant cdc28-as1 (see Methods). h Inactivation of Cdk function allows chromatin bridge resolution in MEN-deficient (cdc15-as + 1-NA-PP1) cells. Arrowheads mark chromatin bridges and asterisks bridge resolution. Time 0 corresponds to the start of imaging (temperature shift). n = number of cells is indicated. Cells were pooled from three independent experiments. ****p < 0.0001, two-sided Fisher’s exact test. Scale bar: 1 µm.