Figure 4.

Trichostatin-A-induced changes in replication dynamics of cells from cell line 422 are abrogated by exogenous precursors. (A) Relative frequency of initiation events at the genome-wide level as determined by the ratio of the number of initiation events relative to replication events (replication forks and initiations). Frequency of initiation was normalized to that of 0 h −CT. −CT and +CT, without and with cytidine and thymidine added in the medium. Values were normalized to that of 0 h. At least 100 DNA fibres were analysed for each time-point and culture condition. −CT and +CT conditions were compared by using a χ²-test. Densities of initiation are not significantly different between the two conditions at 0 h, whereas they are at 24 h (P=0.01). Similarly, densities of initiation are not significantly different between 0 h and 24 h in +CT conditions. (B) Localization of initiation events along the AMPD2 locus. At least 30 DNA fibres were analysed for each condition. Circles symbolize: orange, major origin; grey, minor origin; tan, average efficiency. Fisher's exact test indicated that initiation profiles are not significantly different between Ctl and TSA 6 h+CT, whereas they are different between Ctl and TSA 6 h (P=0.007). (C) Fork speed (left) and examples of combed DNA molecules labelled with IdU then with CldU (right). −CT and +CT conditions were compared using a Mann–Whitney–Wilcoxon test. Replication speeds are not significantly different between the two conditions at 0 h, whereas they are at 24 h (P<2e16). (D,E) CTP synthetase and TS messenger RNA levels. For each condition, levels were normalized to that of zero time point (arbitrary units). Errors bars represent standard error of the mean of two experiments. AMPD2, adenosine monophosphate deaminase 2; AU, arbitrary units; CldU, chlorodeoxyuridine; IdU, iododeoxyuridine; TS, thymidylate synthase.