Figure 1. Effect of Ubc9dn on origin firing in early S phase.

(a) Sperm nuclei (2,000 nuclei per μl) were replicated in Xenopus egg extracts supplemented (+) or not (−) (Ctrl, control) with 350 ng μl⁻¹ Ubc9dn. At each time-point after addition of sperm nuclei, DNA replication in samples with Ubc9dn was defined relative to that in Ctrl samples (set at 100%). DNA replication was measured by standard [³³P]-labelled dCTP incorporation; mean±s.d. % Of DNA replicated in nine independent experiments based on four different egg extracts that replicated 90–100% of the input DNA. (b) Time-course of DNA replication in S-phase Xenopus egg extracts with or without Ubc9dn. The graph represents the percentage of input DNA that is replicated at each indicated time-point. (c) Quantification of the number of initiation events (number of BrdU tracks per Mb) on combed DNA fibres from the 30-min time-point with or without Ubc9dn of the replication reaction shown in b. (d) Distribution of initiation events on combed DNA fibres at the 30-min time-point. BrdU tracks are represented in green (bar, 50 kb). This representation was generated by using the IDeFIx software. (e) IOD, length of BrdU tracks and of DNA gaps between tracks measured for each condition (with or without Ubc9dn) at the 30-min time-point. Boxes and whiskers indicate the 5–95 percentiles. The vertical line within the boxes represents the median (in kb). Data not included between the whiskers are plotted as outliers (dots). Statistical significance was determined using the unpaired t-test (**P<0.001 and ***P<0.0001).