(A) Flow cytometry of the indicated strains grown overnight in YPraffinose, then arrested in G2/M with nocodazole. After addition of fresh nocodazole, 2% galactose was added for the indicated times to express the licensing mutants. Right, overlay between the 0 and 2 hr time points for the licensing mutant strain with (red) or without (black) the sld3-A/dbf4-A alleles. (B) Copy number analysis of Chromosome III from the experiment in (A), after 4 hr in galactose. As in (A), the strains overexpress wild type Cdc6 and the indicated pre-replicative complex mutants from the galactose inducible promoter. DNA sequencing read depth per 1 kb bin was normalised to the 0 hr time point for each strain. Baseline was set at two as the strains are arrested in G2. The y-axis shows the DNA copy number after these steps. (C) Schematic diagram of re-replication-induced gene amplification (RRIGA) assay for gene amplification events. Re-replication of the split LEU2 gene from origin ARS317 results in tandem head to tail gene duplications, leading to a functional LEU2 gene. (D) RRIGA assay in (C) was performed with the indicated strains. Strains were grown overnight in YPraffinose then arrested in G2/M with nocodazole (pre-induction, blue time point). After addition of fresh nocodazole, 2% galactose was added for 3 hr to express the licensing mutants (red time point). Cells were plated on YPD (viable cell count) and SC-leu plates (Leu+ count) and the % of Leu+ colonies out of the viable cell population was plotted. N = 3, error bars are SD. *The p value was calculated as 0.0481 using an unpaired t-test.