Quantitative Analysis of a Cell Cycle Checkpoint in Xenopus laevis Cell-Free Egg Extracts

Abstract

In somatic cells, checkpoint pathways trigger cell cycle arrest in response to unreplicated or damaged DNA by inhibiting the activity of cyclin-dependent kinases (Cdks). In the Xenopus laevis embryo, checkpoints are not operational until the midblastula transition (MBT). Studies in cell-free egg extracts indicate that a threshold concentration of nuclei, which approximates the MBT concentration, is required to elicit a checkpoint. The checkpoint response to unreplicated DNA in the extract prevents transition into mitosis by inhibiting Cdk1/cyclin B, causing an increase in the minimum amount of cyclin B necessary to enter mitosis, termed the cyclin threshold. Once the threshold of cyclin is maintained or exceeded, the system will proceed into mitosis after a lag time. We have investigated the relationship between nuclear concentration and cell cycle regulation in the extract. By precisely regulating the concentration of cyclin B and nuclear content in extract samples, we have found 1) the concentration of nuclei affects cyclin B thresholds and lag time of entry into mitosis, 2) elevated cyclin thresholds caused by DNA replication blocks are further increased by increasing the concentration of nuclei, and 3) double-stranded DNA breaks in the extract system do not affect cyclin thresholds or lag time of entry into mitosis within the range of nuclear concentrations that can be efficiently replicated. This data provides evidence of the importance of the nucleocytoplasmic ratio in normal cell cycle progression and its importance for checkpoint acquisition during early Xenopus laevis development.