G2 checkpoint targets late replicating DNA.

In the multinucleate cells induced in Allium cepa L. meristems, the nuclei surrounded by the largest cytoplasm environment complete replication earlier (advanced nuclei), but have a longer G2, than the others (delayed nuclei). Thus, all nuclei break down the nuclear envelope and start metaphase simultaneously. The present report shows that this synchronization relies on a checkpoint mechanism. When completion of replication was prevented in the delayed nuclei (due to in vivo 5-aminouracil feeding initiated when the advanced nuclei were already in G2), the metaphase was also further delayed in the advanced ones. In turn, some of the delayed nuclei overrode the G2 checkpoint (adaptation) and entered into mitosis with broken chromatids (Del Campo et al., 1997). Anoxic UVA (313 nm) irradiation apparently prevents the binding of regulatory proteins to Br-DNA. The present report shows that late replicating sequences are the targets of the checkpoint signal produced by the still replicating nuclei. This signal delays metaphase in the advanced nuclei, whose DNA is already fully replicated. Thus, when the already replicated sequences of late replicating DNA was modified in the advanced nuclei by bromosubstitution followed by anoxic UVA irradiation, they entered into mitosis without any delay, ignoring the inhibitory signals produced by the still replicating nuclei.