The DNA damage checkpoint in embryonic cell cycles is dependent on the DNA-to-cytoplasmic ratio.

In Xenopus, cell cycle checkpoints monitoring DNA damage, DNA replication, and spindle assembly do not appear until after the midblastula transition (MBT; 4000 cells). We show that a DNA damage checkpoint can slow the cell cycle even in 2-cell embryos when the DNA content is increased. Slowing follows caffeine-sensitive activation of the checkpoint kinase, Chk1; degradation of the cell cycle phosphatase, Cdc25A; and inhibitory phosphorylation of Cdc25C and cyclin-dependent kinases (Cdks). Alterations in the DNA-to-cytoplasmic ratio elicit a dose-dependent DNA damage checkpoint, and the ratio required to activate Chk1 for the damage response is lower than that associated with "developmental" activation of Chk1 shortly after the MBT. Our results indicate that a maternal damage response, independent of zygotic transcription, is present even in very early embryos, and requires both double-stranded DNA ends and a threshold DNA-to-cytoplasmic ratio to significantly affect the cell cycle.

Mammalian Polo-like kinase 3 (Plk3) is a multifunctional protein involved in stress response pathways.

The Polo-like kinases (Plks) are a conserved family of kinases that contribute to cell cycle regulation, particularly in G2 and mitosis. In mammals, there are at least three members of the Plk family. Here we show that Plk3 is a stress response protein that becomes phosphorylated following DNA damage or mitotic spindle disruption. Phosphorylation enhances its kinase activity and is dependent upon ataxia telangiectasia-mutated (ATM) in the former case but not the latter. Plk3 associates with complexes of multiple sizes ranging from 150 to greater then 600 kDa. In its unphosphorylated form it elutes from a sizing column at about 400 kDa whereas it associates with complexes of 150 and 600 kDa when phosphorylated. Among the proteins with which it physically associates and utilizes, as substrates are Chk2 and P53. It phosphorylates Chk2 on a residue different from threonine 68 (Thr68), the principal target for ATM. While ATM is necessary for phosphorylation and activation of Chk2 in vivo, Plk3 seems to contribute to its full activation. In its phosphorylated form it also coelutes and forms a complex with unpolymerized tubulin. In aggregate, the data argue that Plk3 is a multifunctional protein that associates with multiple complexes and that contributes to response to stress incurred by DNA damage and mitotic spindle disruption, albeit via different pathways.