The role of protein phosphorylation in the assembly of a replication competent nucleus: investigations in Xenopus egg extracts using the cyanobacterial toxin microcystin-LR.

Cell-free extracts of Xenopus eggs support nuclear assembly and DNA replication in vitro. Extracts supplemented with the protein phosphatase inhibitor microcystin-LR displayed various inhibitory effects at different concentrations of the toxin. In the presence of cycloheximide, additions of microcystin did not induce histone H1-kinase activity. Nevertheless, increasing concentrations of microcystin did sequentially prevent DNA replication, nuclear lamina assembly and nuclear envelope assembly. DNA replication was prevented when microcystin was added at 250 nM. Furthermore, this effect could be reversed after the addition of the catalytic sub-unit of protein phosphatase 2A to inhibited extracts. At a concentration of 250 nM microcystin, nuclear membrane assembly, nuclear lamina assembly and nuclear transport all occurred in egg extracts. In addition single-stranded M13 DNA replication was also permitted. However, it appeared that replicase assembly was not completed, since nuclei assembled in microcystin-treated extracts displayed an unusual distribution of proliferating cell nuclear antigen (PCNA). Although PCNA was located at sites that resembled pre-replication foci, this nuclear protein was readily solubilised when nuclei were isolated and extracted sequentially with Triton, nucleases and salts. Despite this, nuclei containing pre-assembled replication forks could synthesise DNA when transferred into microcystin-treated extracts.