Department of Biology, University of Waterloo, Waterloo, Ontario N2L3G1, Canada.
J Biol Chem. 2013 May 24;288(21):14926-35. doi: 10.1074/jbc.M112.392910. Epub 2013 Apr 2.
The essential cell cycle target of the Dbf4/Cdc7 kinase (DDK) is the Mcm2-7 helicase complex. Although Mcm4 has been identified as the critical DDK phosphorylation target for DNA replication, it is not well understood which of the six Mcm2-7 subunits actually mediate(s) docking of this kinase complex. We systematically examined the interaction between each Mcm2-7 subunit with Dbf4 and Cdc7 through two-hybrid and co-immunoprecipitation analyses. Strikingly different binding patterns were observed, as Dbf4 interacted most strongly with Mcm2, whereas Cdc7 displayed association with both Mcm4 and Mcm5. We identified an N-terminal Mcm2 region required for interaction with Dbf4. Cells expressing either an Mcm2 mutant lacking this docking domain (Mcm2ΔDDD) or an Mcm4 mutant lacking a previously identified DDK docking domain (Mcm4ΔDDD) displayed modest DNA replication and growth defects. In contrast, combining these two mutations resulted in synthetic lethality, suggesting that Mcm2 and Mcm4 play overlapping roles in the association of DDK with MCM rings at replication origins. Consistent with this model, growth inhibition could be induced in Mcm4ΔDDD cells through Mcm2 overexpression as a means of titrating the Dbf4-MCM ring interaction. This growth inhibition was exacerbated by exposing the cells to either hydroxyurea or methyl methanesulfonate, lending support for a DDK role in stabilizing or restarting replication forks under S phase checkpoint conditions. Finally, constitutive overexpression of each individual MCM subunit was examined, and genotoxic sensitivity was found to be specific to Mcm2 or Mcm4 overexpression, further pointing to the importance of the DDK-MCM ring interaction.
Dbf4/Cdc7 激酶（DDK）的基本细胞周期靶标是 Mcm2-7 解旋酶复合物。虽然 Mcm4 已被确定为 DNA 复制中 DDK 磷酸化的关键靶标，但尚不清楚哪六个 Mcm2-7 亚基实际上介导了该激酶复合物的对接。我们通过双杂交和共免疫沉淀分析系统地检查了每个 Mcm2-7 亚基与 Dbf4 和 Cdc7 的相互作用。观察到明显不同的结合模式，因为 Dbf4 与 Mcm2 相互作用最强，而 Cdc7 则与 Mcm4 和 Mcm5 都有联系。我们鉴定了一个与 Dbf4 相互作用所需的 Mcm2 N 端区域。表达缺乏此对接域的 Mcm2 突变体（Mcm2ΔDDD）或缺乏先前鉴定的 DDK 对接域的 Mcm4 突变体（Mcm4ΔDDD）的细胞显示出适度的 DNA 复制和生长缺陷。相比之下，将这两种突变结合在一起会导致合成致死性，表明 Mcm2 和 Mcm4 在 DDK 与复制起点处的 MCM 环的结合中发挥重叠作用。与该模型一致，通过 Mcm2 过表达诱导 Mcm4ΔDDD 细胞的生长抑制，作为滴定 Dbf4-MCM 环相互作用的一种手段。通过将细胞暴露于羟基脲或甲基甲磺酸硫酸盐中，这种生长抑制作用加剧，为 DDK 在 S 期检查点条件下稳定或重新启动复制叉提供了支持。最后，检查了每个单独的 MCM 亚基的组成型过表达，并且发现遗传毒性敏感性特异性地针对 Mcm2 或 Mcm4 的过表达，这进一步表明 DDK-MCM 环相互作用的重要性。
