THE NUCLEAR-ENVELOPE PREVENTS REINITIATION OF REPLICATION BY REGULATING THE BINDING OF MCM3 TO CHROMATIN IN XENOPUS EGG EXTRACTS

Background: A complex of MCM proteins is implicated in ensuring that D NA replicates only once in each cell cycle, by 'replication licensing' . The nuclear membrane is also implicated in replication licensing, bu t the relationship between the MCM proteins and the nuclear membrane i s unclear. Here, we investigate the relationship between XMCM3 (a comp onent of the Xenopus MCM complex), nuclear envelope permeability and t he initiation of DNA replication once per cell cycle. Results: Our res ults show that the nuclear envelope does not prevent the entry of XMCM 3 into the nucleus, but that it does prevent the binding of XMCM3 to c hromatin. We have also identified another component of the Xenopus MCM complex as a homologue of the Schizosaccharomyces pombe protein Cdc21 . XMCM3 does not preferentially co-localize with sites of DNA replicat ion. Instead, it is almost uniformly distributed on chromatin and is s uddenly lost during replication, XMCM3 crosses intact nuclear membrane s of G2-phase HeLa cells but cannot then bind to chromatin. Permeabili zation of the nuclear envelope allows the binding of XMCM3 to G2-phase chromatin. We have therefore resolved replication licensing into two stages. The first requires the entry of a cytosolic 'loading factor' t hat is excluded by the nuclear membrane; subsequently, MCM3 can bind t o chromatin in the presence or absence of a nuclear membrane, but only if the loading factor has gained access in the absence of the membran e. Conclusions: The Xenopus MCM complex contains homologues of yeast M CM2, MCM3, MCM5 and Cdc21 proteins. XMCM3 is displaced from chromatin during replication. The nuclear envelope allows entry of XMCM3 into th e nucleus, but regulates its binding to chromatin; binding requires a loading factor which cannot cross the nuclear envelope. Based on these results, we present a two-stage model for replication licensing.