Department of Medicine, Feinberg Cardiovascular Research Institute, Feinberg School of Medicine, Northwestern University Chicago, IL 60610, USA.
Epigenetics and Cell Fate, Sorbonne Paris Cité, University Paris Diderot, UMR 7216 CNRS, Paris 75013, France.
Genes (Basel). 2015 May 15;6(2):252-66. doi: 10.3390/genes6020252.
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.
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中仅复制一次。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 Cdt1 来阻止第二轮 DNA 复制。Cdt1 将微型染色体维持复合物 (MCM2-7)，即复制解旋酶，募集到 DNA 复制起点的预复制复合物 (pre-RC) 中。Geminin 通过 Cdt1 抑制 MCM2-7 加载的机制尚未完全理解。传统模型认为 Geminin 阻碍了 Cdt1 和 MCM2-7 之间的直接物理相互作用。在这里，我们描述了 Geminin 的一种无活性错义突变体 GemininAWA，它与 Cdt1 正常结合亲和力结合，但即使添加大量过量，作为复制抑制剂也完全失活。事实上，GemininAWA 可以与 GemininWT 竞争结合 Cdt1 并阻止其抑制 DNA 复制。GemininAWA 不会抑制体内 MCM2-7 加载到 DNA 上，并且在 GemininAWA 的存在下，核 DNA 在单个 S 期内过度复制。我们得出结论，Geminin 不是通过与 Cdt1-MCM2-7 相互作用的简单空间干扰来抑制 MCM 加载，而是通过非空间机制起作用，可能通过抑制组蛋白乙酰转移酶 HBO1。
