Stephenson Robert, Hosler Marcus R, Gavande Navnath S, Ghosh Arun K, Weake Vikki M
From the Departments of Biochemistry and.
Chemistry and Medicinal Chemistry, and.
J Biol Chem. 2015 Jan 16;290(3):1332-47. doi: 10.1074/jbc.M114.597948. Epub 2014 Dec 1.
Cdc7 is a serine-threonine kinase that phosphorylates components of the pre-replication complex during DNA replication initiation. Cdc7 is highly conserved, and Cdc7 orthologs have been characterized in organisms ranging from yeast to humans. Cdc7 is activated specifically during late G1/S phase by binding to its regulatory subunit, Dbf4. Drosophila melanogaster contains a Dbf4 ortholog, Chiffon, which is essential for chorion amplification in Drosophila egg chambers. However, no Drosophila ortholog of Cdc7 has yet been characterized. Here, we report the functional and biochemical characterization of a Drosophila ortholog of Cdc7. Co-expression of Drosophila Cdc7 and Chiffon is able to complement a growth defect in yeast containing a temperature-sensitive Cdc7 mutant. Cdc7 and Chiffon physically interact and can be co-purified from insect cells. Cdc7 phosphorylates the known Cdc7 substrates Mcm2 and histone H3 in vitro, and Cdc7 kinase activity is stimulated by Chiffon and inhibited by the Cdc7-specific inhibitor XL413. Drosophila egg chamber follicle cells deficient for Cdc7 have a defect in two types of DNA replication, endoreplication and chorion gene amplification. However, follicle cells deficient for Chiffon have a defect in chorion gene amplification but still undergo endocycling. Our results show that Cdc7 interacts with Chiffon to form a functional Dbf4-dependent kinase complex and that Cdc7 is necessary for DNA replication in Drosophila egg chamber follicle cells. Additionally, we show that Chiffon is a member of an expanding subset of DNA replication initiation factors that are not strictly required for endoreplication in Drosophila.
Cdc7是一种丝氨酸 - 苏氨酸激酶，在DNA复制起始过程中磷酸化前复制复合体的组分。Cdc7高度保守，并且在从酵母到人类的各种生物体中都已鉴定出Cdc7直系同源物。Cdc7在G1/S期晚期通过与它的调节亚基Dbf4结合而被特异性激活。黑腹果蝇含有一种Dbf4直系同源物Chiffon，它对于果蝇卵室中的卵壳蛋白扩增至关重要。然而，尚未鉴定出果蝇的Cdc7直系同源物。在此，我们报道了果蝇Cdc7直系同源物的功能和生化特性。果蝇Cdc7和Chiffon的共表达能够弥补含有温度敏感型Cdc7突变体的酵母的生长缺陷。Cdc7和Chiffon在物理上相互作用，并且可以从昆虫细胞中共纯化。Cdc7在体外磷酸化已知的Cdc7底物Mcm2和组蛋白H3，并且Cdc7激酶活性受到Chiffon的刺激并被Cdc7特异性抑制剂XL413抑制。缺乏Cdc7的果蝇卵室卵泡细胞在两种类型的DNA复制中存在缺陷，即核内复制和卵壳蛋白基因扩增。然而，缺乏Chiffon的卵泡细胞在卵壳蛋白基因扩增方面存在缺陷，但仍能进行核内循环。我们的结果表明，Cdc7与Chiffon相互作用形成功能性的依赖Dbf4的激酶复合体，并且Cdc7对于果蝇卵室卵泡细胞中的DNA复制是必需的。此外，我们表明Chiffon是DNA复制起始因子不断扩大的子集中的一员，这些因子在果蝇的核内复制中并非严格必需。
