THE ROTE 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 re plication in vitro. Extracts supplemented with the protein phosphatase inhibitor microcystin-LR displayed various inhibitory effects at diff erent concentrations of the toxin. In the presence of cycloheximide, a dditions of microcystin did not induce histone H1-kinase activity. Nev ertheless, increasing concentrations of microcystin did sequentially p revent DNA replication, nuclear lamina assembly and nuclear envelope a ssembly, DNA replication was prevented when microcystin was added at 2 50 nM. Furthermore, this effect could be reversed after the addition o f the catalytic sub-unit of protein phosphatase 2A to inhibited extrac ts. At a concentration of 250 nM microcystin, nuclear membrane assembl y, nuclear lamina assembly and nuclear transport all occurred in egg e xtracts. In addition single-stranded M13 DNA replication was also perm itted. However, it appeared that replicase assembly was not completed, since nuclei assembled in microcystin-treated extracts displayed an u nusual distribution of proliferating cell nuclear antigen (PCNA). Alth ough PCNA was located at sites that resembled pre-replication foci, th is nuclear protein was readily solubilised when nuclei were isolated a nd extracted sequentially with Triton, nucleases and salts. Despite th is, nuclei containing pre-assembled replication forks could synthesise DNA when transferred into microcystin-treated extracts.