Phosphorylation of MCM4 by Cdc7 Kinase Facilitates Its Interaction with Cdc45 on the Chromatin

Abstract

Cdc7 kinase, conserved from yeasts to human, plays important roles in DNA replication. However, the mechanisms by which it stimulates initiation of DNA replication remain largely unclear. We have analyzed phosphorylation of MCM subunits during cell cycle by examining mobility shift on SDS-PAGE. MCM4 on the chromatin undergoes specific phosphorylation during S phase. Cdc7 phosphorylates MCM4 in the MCM complexes as well as the MCM4 N-terminal polypeptide. Experiments with phospho-amino acid-specific antibodies indicate that the S phase-specific mobility shift is due to the phosphorylation at specific N-terminal (S/T)(S/T)P residues of the MCM4 protein. These specific phosphorylation events are not observed in mouse ES cells deficient in Cdc7 or are reduced in the cells treated with siRNA specific to Cdc7, suggesting that they are mediated by Cdc7 kinase. The N-terminal phosphorylation of MCM4 stimulates association of Cdc45 with the chromatin, suggesting that it may be an important phosphorylation event by Cdc7 for activation of replication origins. Deletion of the N-terminal non-conserved 150 amino acids of MCM4 results in growth inhibition, and addition of amino acids carrying putative Cdc7 target sequences partially restores the growth. Furthermore, combination of MCM4 N-terminal deletion with alanine substitution and deletion of the N-terminal segments of MCM2 and MCM6, respectively, which contain clusters of serine/threonine and are also likely targets of Cdc7, led to an apparent nonviable phenotype. These results are consistent with the notion that the N-terminal phosphorylation of MCM2, MCM4, and MCM6 may play functionally redundant but essential roles in initiation of DNA replication.

Establishment of expression-state boundaries by Rif1 and Taz1 in fission yeast

“…3D), which fits the fact that Mcm4 is not the sole factor mediating the effects of Rif1 on origin firing. Part of the regulation might occur on other MCM subunits or on the initiation factor Sld3 (43,44). The silencing defects in mcm4ΔNSD mutants extend the mutational analyses presented above to reinforce the idea that replication control is necessary to heterochromatic silencing at the edge of the silent domain.…”

Cdc7 kinase mediates Claspin phosphorylation in DNA replication checkpoint

“…Cells ES, HeLa and U2OS cells were maintained as described previously (Kim et al, 2002;Masai et al, 2006). siRNA Cells were transfected twice (24 h interval) with siRNA duplexes by using the Oligofectamine (Invitrogen, Carlsbad, CA, USA), unless stated otherwise.…”

“…siRNA Cells were transfected twice (24 h interval) with siRNA duplexes by using the Oligofectamine (Invitrogen, Carlsbad, CA, USA), unless stated otherwise. Cdc7 (Cdc7-1, Cdc7-D and Cdc7-nc) and Luciferase siRNAs have been described previously (Montagnoli et al, 2004;Yoshizawa-Sugata et al, 2005;Masai et al, 2006). Claspin siRNA has been described (Yoshizawa-Sugata and Masai, 2007).…”

Multi-step Loading of Human Minichromosome Maintenance Proteins in Live Human Cells

“…MCM4 was chosen because it was shown to interact with the GINS complex (42), and its phosphorylation by Cdc7 facilitates the interaction with Cdc45 on chromatin (56). MCM2, as well as MCM3, carry nuclear localization sequences (NLSs) sequestering the other MCM subunits to the cell nucleus (57).…”