RESUMO
Claspin transmits replication stress signal from ATR to Chk1 effector kinase as a mediator. It also plays a role in efficient replication fork progression during normal growth. Here we have generated conditional knockout of Claspin and show that Claspin knockout mice are dead by E12.5 and Claspin knockout mouse embryonic fibroblast (MEF) cells show defect in S phase. Using the mutant cell lines, we report the crucial roles of the acidic patch (AP) near the C terminus of Claspin in initiation of DNA replication. Cdc7 kinase binds to AP and this binding is required for phosphorylation of Mcm. AP is involved also in intramolecular interaction with a N-terminal segment, masking the DNA-binding domain and a newly identified PIP motif, and Cdc7-mediated phosphorylation reduces the intramolecular interaction. Our results suggest a new role of Claspin in initiation of DNA replication during normal S phase through the recruitment of Cdc7 that facilitates phosphorylation of Mcm proteins.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular/genética , Proliferação de Células/fisiologia , Quinase 1 do Ponto de Checagem/metabolismo , Proteínas de Ligação a DNA/genética , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Purification of recombinant proteins of a large size often poses problems of instability or low expression in bacterial or insect cells. Here, we established a method for a high-level expression of large-sized recombinant proteins in mammalian cells and subsequent purification of the full-length proteins. We applied this method to express human Claspin and Tim-Tipin complex, which play important roles in replication checkpoint responses as fork-stabilizing factors, and successfully purified them in functional forms in amount sufficient for enzymatic characterization. Purified Claspin behaves as a monomer and binds preferentially to fork-like DNA. Over-expression of tagged Claspin in mammalian cells facilitated the detection of its interacting factors. Claspin interacts with many factors involved in checkpoint regulation and replication fork machinery, including ATR, ATM, Chk1, Tim, MCM4, MCM10, Cdc45, DNA polymerases α, δ, ε and Cdc7 kinase. We will discuss the potential implication of these findings in architecture of replication fork. We will also discuss the advantage of this system for purification and characterization of those proteins that are large and have been difficult to deal with.