Fig. 3: Re-replication is driven by the same set of origins utilized during normal mitotic growth.

a Schematic representation of the method used to map replication origins in re-replicating DNA. Cells were treated with MLN4924 for 24 h, including exposure to BrdU for 16 h. Genomic DNA was fragmented and re-replicated DNA (MLN HH DNA from cells incubated with BrdU for less than an entire doubling period) was isolated by BrdU-CsCl gradient. Normal replicated DNA (Control HL DNA and MLN HL DNA, only one DNA strand labeled with BrdU) was also collected to map normal replication origins as a control. Following the CsCl gradient, newly replicated nascent DNA was isolated from both normal- and re-replicated DNA using sucrose gradient and lambda exonuclease. DNA fragments that are resistant to λ-endonuclease digestion (RNA primed, yellow squares) are nascent strands⁴⁵. b IGV screenshot showing representative nascent DNA outputs in genomic control, during normal mitotic replication (Control-HL and MLN-HL), and during re-replication (MLN4924-HH). c Density plots (top panel) comparing replication origin usage in 2 normal mitotic replication samples (Control-HL and MLN4924-HL, left) and in one normal and one re-replication samples (MLN4924-HL and MLN4924-HH, middle). The location of each data point is proportional to the number of reads per origins. Origins that initiate replication with similar frequency during normal mitotic growth and in re-replicating cells are located on the diagonal dotted line; for the middle panel, origins above the diagonal dotted line initiate more frequently in re-replicating cells and vice versa. The right panel used as positive control for dormant origin activation showing augmented origin activation in aphidicolin treated cells (0.8 µM of Aphidicolin for 24 h). Reads per origin data of the above density plots were further divided into 10 fractions ranging from origins that showed the highest ratio of initiation frequency of y-axis sample (for example MLN4924 HH of the middle panel) vs. x-axis sample (for example MLN4924 HL of the middle panel) to origins that showed the highest ratio of initiation frequency of x-axis sample vs. y-axis sample). The number of peaks in each of the 10 fractions is shown as bar graphs (bottom panel) to show the cumulative distribution of small peaks with 250–400 reads (green) and large peaks >400 reads (blue). The vertical dash line in the middle equal to the diagonal dash line in the above density plot.