Structure and Mutagenesis Studies of the C-terminal Region of Licensing Factor Cdt1 Enable the Identification of Key Residues for Binding to Replicative Helicase Mcm Proteins
Structure and Mutagenesis Studies of the C-terminal Region of Licensing Factor Cdt1 Enable the Identification of Key Residues for Binding to Replicative Helicase Mcm Proteins
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DOI：
10.1074/jbc.m109.075333
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发表时间：
2010-05-21
影响因子：
4.8
通讯作者：
Shirakawa, Masahiro
中科院分区：
文献类型：
--
作者：
Jee, JunGoo;Mizuno, Takeshi;Shirakawa, Masahiro
在真核生物中，在细胞分裂开始之前，DNA 复制在单个细胞周期中启动一次，以维持基因组的稳定性。这一事件受到一系列蛋白质的严格控制。 Cdt1 是许可因子之一，与 Cdc6 一起参与将复制 DNA 解旋酶 Mcm2-7 蛋白招募到复制前复合物中。在 Cdt1 中，C 末端区域充当 Mcm2-7 蛋白的结合位点，尽管这些相互作用的细节仍然很大程度上未知。在这里，我们报告了该区域的结构以及与 Mcm 蛋白结合的关键残基。我们通过 NMR 确定了小鼠 Cdt1 的 C 端片段（残基 450-557）的溶液结构。该结构由翼状螺旋结构域组成，与 Cdc6 的 C 末端结构域和 Cdt1 的中心片段显示出意想不到的相似性，从而暗示了功能和进化关系。基于结构的诱变和体外结合测定使我们能够查明与 Mcm 蛋白相互作用的区域。此外，通过进行体外结合和出芽酵母活力实验，我们发现位于结构区N端方向的类似45个残基对于识别Mcm蛋白同样至关重要。我们的数据表明翼状螺旋结构域可能作为通用模块在 DNA 复制许可过程中与复制解旋酶相互作用。
In eukaryotes, DNA replication is fired once in a single cell cycle before cell division starts to maintain stability of the genome. This event is tightly controlled by a series of proteins. Cdt1 is one of the licensing factors and is involved in recruiting replicative DNA helicase Mcm2-7 proteins into the pre-replicative complex together with Cdc6. In Cdt1, the C-terminal region serves as a binding site for Mcm2-7 proteins, although the details of these interactions remain largely unknown. Here, we report the structure of the region and the key residues for binding to Mcm proteins. We determined the solution structure of the C-terminal fragment, residues 450-557, of mouse Cdt1 by NMR. The structure consists of a winged-helix domain and shows unexpected similarity to those of the C-terminal domain of Cdc6 and the central fragment of Cdt1, thereby implying functional and evolutionary relationships. Structure-based mutagenesis and an in vitro binding assay enabled us to pinpoint the region that interacts with Mcm proteins. Moreover, by performing in vitro binding and budding yeast viability experiments, we showed that similar to 45 residues located in the N-terminal direction of the structural region are equally crucial for recognizing Mcm proteins. Our data suggest the possibility that winged-helix domain plays a role as a common module to interact with replicative helicase in the DNA replication-licensing process.