Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA, USA.
EMBO J. 2011 Nov 1;30(24):4885-96. doi: 10.1038/emboj.2011.394.
Eukaryotic origins of replication are selected by loading a head-to-head double hexamer of the Mcm2-7 replicative helicase around origin DNA. Cdt1 plays an essential but transient role during this event; however, its mechanism of action is unknown. Through analysis of Cdt1 mutations, we demonstrate that Cdt1 performs multiple functions during helicase loading. The C-terminus of Cdt1 binds Mcm2-7, and this interaction is required for efficient origin recruitment of both proteins. We show that origin recognition complex (ORC) and Cdc6 recruit multiple Cdt1 molecules to the origin during helicase loading, and disruption of this multi-Cdt1 intermediate prevents helicase loading. Although dispensable for loading Mcm2-7 double hexamers that are topologically linked to DNA, the essential N-terminal domain of Cdt1 is required to load Mcm2-7 complexes that are competent for association with the Cdc45 and GINS helicase-activating proteins and replication initiation. Our data support a model in which origin-bound ORC and Cdc6 recruit two Cdt1 molecules to initiate double-hexamer formation prior to helicase loading and demonstrate that Cdt1 influences the replication competence of loaded Mcm2-7 helicases.
真核生物的复制起点是通过将 Mcm2-7 复制解旋酶的对头双六聚体加载到原点 DNA 上来选择的。Cdt1 在这个过程中起着至关重要但短暂的作用；然而，其作用机制尚不清楚。通过对 Cdt1 突变的分析，我们证明 Cdt1 在解旋酶加载过程中具有多种功能。Cdt1 的 C 末端与 Mcm2-7 结合，这种相互作用对于两种蛋白质的有效原点募集是必需的。我们表明，在解旋酶加载过程中，起始识别复合物（ORC）和 Cdc6 将多个 Cdt1 分子募集到原点，并且破坏这种多 Cdt1 中间物会阻止解旋酶加载。尽管对于加载拓扑连接到 DNA 的 Mcm2-7 双六聚体是可有可无的，但 Cdt1 的必需 N 端结构域对于加载与 Cdc45 和 GINS 解旋酶激活蛋白以及复制起始相容的 Mcm2-7 复合物是必需的。我们的数据支持这样一种模型，即原点结合的 ORC 和 Cdc6 招募两个 Cdt1 分子，在解旋酶加载之前启动双六聚体形成，并证明 Cdt1 影响加载的 Mcm2-7 解旋酶的复制能力。
