Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235, USA.
J Biol Chem. 2011 Apr 22;286(16):14157-67. doi: 10.1074/jbc.M111.218305. Epub 2011 Mar 1.
Sld3 is essential for the initiation of DNA replication, but Sld3 does not travel with a replication fork. GINS binds to Cdc45 and Mcm2-7 to form the replication fork helicase in eukaryotes. We purified Sld3, Cdc45, GINS, and Mcm2-7 and studied their interaction and assembly into complexes. Sld3 binds tightly to Cdc45 in the presence or absence of cyclin-dependent kinase activity. Furthermore, Sld3 binds tightly to the Mcm2-7 complex, and a ternary complex forms among Cdc45, Mcm2-7, and Sld3, with a 1:1:1 stoichiometry (CMS complex). GINS binds directly to Mcm2-7, and GINS competes with Sld3 for Mcm2-7 binding. GINS also binds directly to Cdc45, and GINS competes with Sld3 for Cdc45 binding. Cdc45, Mcm2-7, and GINS form a ternary complex with a stoichiometry of 1:1:1 (CMG complex). Size exclusion data reveal that when Sld3, Cdc45, Mcm2-7, and GINS are added together, the result is a mixture of CMS and CMG complexes. The data suggest that GINS and Sld3 compete with one another for Mcm2-7 and Cdc45 binding. Our results are consistent with a model wherein GINS trades places with Sld3 at a replication origin, contributing to the activation of the replication fork helicase.
Sld3 对于 DNA 复制的起始是必不可少的，但 Sld3 不会随复制叉移动。GINS 与 Cdc45 和 Mcm2-7 结合，在真核生物中形成复制叉解旋酶。我们纯化了 Sld3、Cdc45、GINS 和 Mcm2-7，并研究了它们之间的相互作用和组装成复合物的情况。在有或没有细胞周期蛋白依赖性激酶活性的情况下，Sld3 与 Cdc45 紧密结合。此外，Sld3 与 Mcm2-7 复合物紧密结合，形成 Cdc45、Mcm2-7 和 Sld3 之间的三元复合物，具有 1:1:1 的化学计量比（CMS 复合物）。GINS 直接与 Mcm2-7 结合，并且 GINS 与 Sld3 竞争 Mcm2-7 结合。GINS 还直接与 Cdc45 结合，并且 GINS 与 Sld3 竞争 Cdc45 结合。Cdc45、Mcm2-7 和 GINS 形成具有 1:1:1 化学计量比的三元复合物（CMG 复合物）。尺寸排阻数据显示，当 Sld3、Cdc45、Mcm2-7 和 GINS 一起添加时，结果是 CMS 和 CMG 复合物的混合物。数据表明，GINS 和 Sld3 相互竞争与 Mcm2-7 和 Cdc45 的结合。我们的结果与一种模型一致，即在复制起点处，GINS 与 Sld3 交换位置，有助于复制叉解旋酶的激活。
