Figure 4: Depletion of RepID decreases the frequency of replication-initiation events.

(a) Cells were sequentially labelled with IdU followed by CldU. Top panel, a typical field with replication signals (IdU detected in green and CldU detected in red). Second panel, the same field with all fibres labelled with an antibody detecting single-strand DNA (ssDNA; grey). Third and fourth panels, an example of CldU-IdU (third) ssDNA (fourth) fibre tracks from RepID WT MEFs. Fifth and sixth panels, an example of CldU-IdU (fifth) ssDNA (sixth) fibre tracks from RepID −/− MEFs. Illustrations of replication fork patterns are shown below the ssDNA track. The lengths of fibres label associated with ldU and CIdU incorporation and inter-origin distances were measured (see Methods), and rates of replication fork progression were calculated based on these values. Ori, origin; ssDNA, DNA detected by anti-single strand antibody. (b,c) Measurements of the distribution of distances between replication origins in DNA fibres from WT MEFs and RepID −/− MEFs. (d,e) Measurements of the distribution of replication fork progression rates for WT and RepID −/−MEFs. The differences between measurements from fibres obtained from wild-type and RepID-deficient MEFs were significant at P<0.05 (P=0.0218 for inter-origin distance and P=0.0061 for replication fork speed as calculated using the Mann–Whitney test). Normality test by Kolmogorov–Smirnov test showed that the distributions of data for b–e are not normal (P<0.01).