A functional analysis of p53 during early development of Xenopus laevis.

p53 is a nuclear protein that acts like a tumor suppressor and is involved in regulation of cellular growth. In Xenopus, the p53 protein is highly expressed during oogenesis and is strictly cytoplasmic in the oocyte. We have analysed its participation in DNA replication and transcription during early development, using the egg and oocyte as model-systems. The injection of sperm nuclei into Xenopus eggs is followed by DNA replication and mitotic events. We show that the endogenous p53 enters the nuclei and moves through a series of discrete sub-nuclear loci whose distribution is S-phase specific. A specific peripheral nuclear localization of p53 is observed before entry into S-phase, followed by an internal localization which is strictly dependent on ongoing DNA synthesis. At no stage in the cell cycle, however, did we observe any co-localization with RPA or PCNA, which were used as initiation or elongation markers for DNA replication. We also show that injection into the nucleus of the oocyte of small amounts of either Xenopus or human p53 - less than 10% of the cytoplasmic storage - is sufficient to block RNA polymerase II-dependent transcription from a coinjected TATA-box-containing reporter plasmid. Transcription is rescued by microinjection of the TATA-box binding protein (TBP), suggesting that nuclear exclusion of p53 during oogenesis may be necessary for transcription of maternal genes. These characteristics are discussed in relation to the regulation of nuclear activities during early embryogenesis.

Evidence for different MCM subcomplexes with differential binding to chromatin in Xenopus.

MCM proteins are molecular components of the DNA replication licensing system in Xenopus. These proteins comprise a conserved family made up of six distinct members which have been found to associate in large protein complexes. We have used a combination of biochemical and cytological methods to study the association of soluble and chromatin-bound Xenopus MCM proteins during the cell cycle. In interphase, soluble MCM proteins are found organized in a core salt-resistant subcomplex that includes MCM subunits which are known to have high affinity for histones. The interphasic complex is modified at mitosis and the subunit composition of the resulting mitotic subcomplexes is distinct, indicating that the stability of the MCM complex is under cell cycle control. Moreover, we provide evidence that the binding of MCM proteins to chromatin may occur in sequential steps involving the loading of distinct MCM subunits. Comparative analysis of the chromatin distribution of MCM2, 3, and 4 shows that the binding of MCM4 is distinct from that of MCM2 and 3. Altogether, these data suggest that licensing of chromatin by MCMs occurs in an ordered fashion involving discrete subcomplexes.