Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Mol Cell. 2010 Nov 12;40(3):353-63. doi: 10.1016/j.molcel.2010.10.017.
Activation of the eukaryotic replicative DNA helicase, the Mcm2-7 complex, requires phosphorylation by Cdc7/Dbf4 (Dbf4-dependent kinase or DDK), which, in turn, depends on prior phosphorylation of Mcm2-7 by an unknown kinase (or kinases). We identified DDK phosphorylation sites on Mcm4 and Mcm6 and found that phosphorylation of either subunit suffices for cell proliferation. Importantly, prior phosphorylation of either S/T-P or S/T-Q motifs on these subunits is required for DDK phosphorylation of Mcm2-7 and for normal S phase passage. Phosphomimetic mutations of DDK target sites bypass both DDK function and mutation of the priming phosphorylation sites. Mrc1 facilitates Mec1 phosphorylation of the S/T-Q motifs of chromatin-bound Mcm2-7 during S phase to activate replication. Genetic interactions between priming site mutations and MRC1 or TOF1 deletion support a role for these modifications in replication fork stability. These findings identify regulatory mechanisms that modulate origin firing and replication fork assembly during cell cycle progression.
真核复制 DNA 解旋酶 Mcm2-7 复合物的激活需要 Cdc7/Dbf4（Dbf4 依赖性激酶或 DDK）的磷酸化，而 DDK 的磷酸化又依赖于 Mcm2-7 被未知激酶（或激酶）的先磷酸化。我们确定了 Mcm4 和 Mcm6 上的 DDK 磷酸化位点，并发现这两个亚基中的任何一个亚基的磷酸化都足以促进细胞增殖。重要的是，这些亚基上的 S/T-P 或 S/T-Q 模体的先磷酸化对于 Mcm2-7 的 DDK 磷酸化以及正常的 S 期通过是必需的。DDK 靶位的磷酸模拟突变可以绕过 DDK 功能和引发磷酸化位点的突变。Mrc1 在 S 期促进 Mec1 对染色质结合的 Mcm2-7 的 S/T-Q 模体的磷酸化，以激活复制。引发位点突变与 MRC1 或 TOF1 缺失之间的遗传相互作用支持这些修饰在复制叉稳定性中的作用。这些发现确定了调节机制，这些机制在细胞周期进展过程中调节起始点火和复制叉组装。
