Cell Cycle- and Chromatin Binding State-dependent Phosphorylation of Human MCM Heterohexameric Complexes

Abstract

The mammalian MCM protein family, presently with six members, exists in the nuclei in two forms, chromatin-bound and unbound. The former dissociates from chromatin with progression through the S phase. Recently, we have established a procedure to isolate chromatin-bound and unbound complexes containing all six human MCM (hMCM) proteins by immunoprecipitation. In the present study, we applied this procedure to HeLa cells synchronized in each of the G 1 , S, and G 2 /M phases and could detect hMCM heterohexameric complexes in all three. In addition, depending on the cell cycle and the state of chromatin association, hMCM2 and 4 in the complexes were found to variously change their phosphorylation states. Concentrating attention on G 2 /M phase hyperphosphorylation, we found hMCM2 and 4 in the complexes to be good substrates for cdc2/cyclin B in vitro. Furthermore, when cdc2 kinase was inactivated in temperature-sensitive mutant murine FT210 cells, the G 2 /M hyperphosphorylation of the murine MCM2 and MCM4 and release of the MCMs from chromatin in the G 2 phase were severely impaired. Taken together, the data suggest that the six mammalian MCM proteins function and undergo cell cycle-dependent regulation as heterohexameric complexes and that phosphorylation of the complexes by cdc2 kinase may be one of mechanisms negatively regulating the MCM complex-chromatin association.The MCM protein family, presently with six members, was originally identified from its involvement in the initiation of DNA replication at autonomously replicating sequences in budding yeast (1-7) and later found to be conserved through eukaryotes (8 -16). Although definite functions of the MCM proteins remain largely unknown, they have been implicated in the regulatory machinery allowing DNA to replicate only once during the S phase (reviewed in Refs. 17 and 18).In mammalian cells, MCM proteins are present in the nuclei in two different forms, one extractable by nonionic detergents and the other tightly associated with chromatin, which is resistant to such extraction. The level of mammalian MCM does not greatly vary during the cell cycle, but the chromatin-bound form gradually becomes dissociated with progression through the S phase (19 -23). It is now assumed that the bound form is associated with prereplicative chromatin and released at the time of replication; the soluble form existing abundantly in G 2 nuclei is considered inactive and no longer capable of binding to chromatin. At least in budding yeast, the chromatin regions to which MCM7 binds during the G 1 phase contain the replication origins (24). However, details of the mode of MCM-chromatin binding remain unclear. In budding yeast and the Xenopus egg extract system, it has been shown that MCM-chromatin binding is regulated through multiple mechanisms, while MCM binds to chromatin depending on CDC6 and the origin recognition complex (24 -27), where the binding is negatively regulated by both S phase and mitotic CDKs (24, 28 -30). However, there is so far no direct evidence as to wheth...

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