Fig. 1.

DNA replication and Uhrf1-dependent chromatin loading of Dnmt1 in Xenopus egg extracts. Schematic representation of Geminin and p27 functions in DNA replication. ORC: origin recognition complex, Pre-RC: pre-replication complex, MCM: minichromosome maintenance (upper panel). Meiotic metaphase II (MII) arrested cytostatic factor (CSF) extracts were incubated with demembraned Xenopus sperm (XSP) nuclei to assemble mitotic chromatin for 30 min. The extracts arrested in MII were released into interphase by addition of CaCl₂, leading to degradation of mitotic cyclins by the APC/C, exit from mitosis and initiation of DNA replication (middle panel). Loading of Dnmt1 and Uhrf1 is dependent on DNA replication. DMSO (Control), Geminin or GST-p27 were added to the activated CSF extracts for inhibition of DNA replication. Sperm chromatin fractions at the indicated times and mitotic chromatin (-Ca²⁺) were analyzed by immunoblotting (IB) H3 Ser10-P: phosphorylation of H3 at Ser 10, a marker of mitosis (lower panels).