Kinetics of replicon initiation during S phase of Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells were synchronized by a thymidine-hydroxyurea block. At different times after release from the block, cells were treated with trioxsalen and long-wavelength ultraviolet light to crosslink DNA in vivo and were labelled with [3H]thymidine for 30 min. This technique permits labelling of only the short nescent DNA fragments initiated between crosslinks. The amount of radioactivity incorporated in these fragments during the labelling period reflects the number of replicon initiation events and allows us to follow the replicon initiation pattern after removing the inhibitor. It was shown that the rate of initiation was high at the beginning of S phase and then steadily decreased.