Summary

Aspects of the regulation of DNA replication and mitosis have been studied using a cell-free extract of Xenopus eggs. The extract is characterized by repeated cycles of DNA replication and mitosis, which are accompanied by periodic synthesis and degradation of cyclins as well as fluctuations in the level of Histone HI kinase activity. DNA replication in this system is dependent upon the formation of a nucleus. However, while nuclear structures are clearly required for initiation, a complete nuclear membrane does not appear to be necessary. Indirect immunofluorescence and D IC microscopy indicate that nuclear reformation from chromosomes occurs asynchronously around individual chromatids. Lamin polymerization, biotin-11-dUTP incorporation and associ ation of polymerases with chromatin occur before membrane formation is complete. S phase nuclei are typified by the co-distribution of both anti-DNA polymerase a and anti-PCNA antibodies as discrete spots of fluorescence which align the chromatin. However, as DNA replication is terminated, PCNA fluorescence fades and DNA polymerase a dissociates from the chromatin and is redistributed throughout the nucleoplasm. By inhibiting DNA replication with aphidicolin, both DNA polymerase a and PCNA remain associated with the chromatin throughout prolonged incubation. Under these conditions mitosis is delayed by up to 70min, although both the general rate of protein synthesis and more importantly the rate of cyclin synthesis and histone kinase activation are unaffected. Upon nuclear envelope breakdown and lamin dispersal, cyclins degrade; however, no chromosomes are formed, and both PCNA and DNA polymerase a remain associated with the chromatin. Also, histone kinase activity is maintained at elevated levels