An inactive geminin mutant that binds cdt1.
An inactive geminin mutant that binds cdt1.
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DOI：
10.3390/genes6020252
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发表时间：
2015-05-15
期刊：
影响因子：
3.5
通讯作者：
McGarry TJ
中科院分区：
文献类型：
--
作者：
Suchyta M;Miotto B;McGarry TJ
DNA复制的起始受到严格调控，以确保基因组在每个细胞周期仅复制一次。在脊椎动物细胞中，不稳定的调节蛋白Geminin通过抑制必需的复制因子Cdt 1来阻止第二轮DNA复制。Cdt 1在DNA复制起点招募微型染色体维持复合物（MCM 2 -7），复制解旋酶，进入复制前复合物（pre-RC）。Geminin通过Cdt 1抑制MCM 2 -7负载的机制还不完全清楚。传统的模型是，Geminin空间阻碍Cdt 1和MCM 2 -7之间的直接物理相互作用。在这里，我们描述了一个非活性的错义突变体的Geminin，GemininAWA，它结合Cdt 1与正常的亲和力，但完全是无活性的复制抑制剂，即使在巨大的过量添加。事实上，GemininAWA可以与GemininWT竞争结合Cdt 1，并阻止其抑制DNA复制。GemininAWA在体内不抑制MCM 2 -7加载到DNA上，并且在GemininAWA存在下，核DNA在单个S期内大量过度复制。我们的结论是，Geminin不抑制MCM加载通过简单的空间干扰Cdt 1-MCM 2 -7相互作用，而是通过非空间机制，可能通过抑制组蛋白乙酰转移酶HBO 1。
The initiation of DNA replication is tightly regulated in order to ensure that the genome duplicates only once per cell cycle. In vertebrate cells, the unstable regulatory protein Geminin prevents a second round of DNA replication by inhibiting the essential replication factor Cdt1. Cdt1 recruits mini-chromosome maintenance complex (MCM2-7), the replication helicase, into the pre-replication complex (pre-RC) at origins of DNA replication. The mechanism by which Geminin inhibits MCM2-7 loading by Cdt1 is incompletely understood. The conventional model is that Geminin sterically hinders a direct physical interaction between Cdt1 and MCM2-7. Here, we describe an inactive missense mutant of Geminin, GemininAWA, which binds to Cdt1 with normal affinity yet is completely inactive as a replication inhibitor even when added in vast excess. In fact, GemininAWA can compete with GemininWT for binding to Cdt1 and prevent it from inhibiting DNA replication. GemininAWA does not inhibit the loading of MCM2-7 onto DNA in vivo, and in the presence of GemininAWA, nuclear DNA is massively over-replicated within a single S phase. We conclude that Geminin does not inhibit MCM loading by simple steric interference with a Cdt1-MCM2-7 interaction but instead works by a non-steric mechanism, possibly by inhibiting the histone acetyltransferase HBO1.
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