Identification of MCM4 as a target of the DNA replication block checkpoint system.
Inhibition of the progression of DNA replication prevents further initiation of DNA replication and allows cells to maintain arrested replication forks, but the proteins that are targets of the replication checkpoint system remain to be identified. We report here that human MCM4, a subunit of the putative DNA replicative helicase, is extensively phosphorylated in HeLa cells when they are incubated in the presence of inhibitors of DNA synthesis or are exposed to UV irradiation. The data presented here indicate that the consecutive actions of ATR-CHK1 and CDK2 kinases are involved in this phosphorylation in the presence of hydroxyurea. The phosphorylation sites in MCM4 were identified using specific anti-phosphoantibodies. Based on results that showed that the DNA helicase activity of the MCM4-6-7 complex is negatively regulated by CDK2 phosphorylation, we suggest that the phosphorylation of MCM4 in the checkpoint control inhibits DNA replication, which includes blockage of DNA fork progression, through inactivation of the MCM complex.
Ishimi
Yukio
Y
Biomolecular and Technology Department, Mitsubishi Kagaku Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194-8511, Japan. yukio@libra.ls.m-kagaku.co.jp
Komamura-Kohno
Yuki
Y
Kwon
Hyun-Ju
HJ
Yamada
Kouichi
K
Nakanishi
Makoto
M
D018797:Cell Cycle Proteins / Q000378:metabolism*; D004261:DNA Replication / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D064148:Minichromosome Maintenance Complex Component 4; D064151:Minichromosome Maintenance Complex Component 6; D064168:Minichromosome Maintenance Complex Component 7; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D014466:Ultraviolet Rays
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C099243:MCM4 protein, human; C103258:MCM6 protein, human; C101624:MCM7 protein, human; D064148:Minichromosome Maintenance Complex Component 4; D064151:Minichromosome Maintenance Complex Component 6; D064168:Minichromosome Maintenance Complex Component 7