Phosphorylation of MCM4 by Cdc7 kinase facilitates its interaction with Cdc45 on the chromatin.
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.
Masai
Hisao
H
Genome Dynamics Project, Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan. hmasai@rinshoken.or.jp
Taniyama
Chika
C
Ogino
Keiko
K
Matsui
Etsuko
E
Kakusho
Naoko
N
Matsumoto
Seiji
S
Kim
Jung-Min
JM
Ishii
Ai
A
Tanaka
Taku
T
Kobayashi
Toshiko
T
Tamai
Katsuyuki
K
Ohtani
Kiyoshi
K
Arai
Ken-Ichi
K
D000595:Amino Acid Sequence; D000818:Animals; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000502:physiology*; D002843:Chromatin / Q000737:chemistry; D004265:DNA Helicases / Q000737:chemistry / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D051379:Mice; D064148:Minichromosome Maintenance Complex Component 4; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000502:physiology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000502:physiology*; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
C113571:CDC45 protein, human; C491876:Cdc45 protein, mouse; D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C068619:CDC7 protein, human; C489403:Cdc7 protein, mouse; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C099243:MCM4 protein, human; C516793:Mcm4 protein, mouse; D064148:Minichromosome Maintenance Complex Component 4