Functional in vitro- and in vivo-characterisation of the ring-shaped, replicative helicase MCM2-7 to identify the role of subunit interfaces in ring-opening, which in turn control loading and release of the helicase from double-stranded DNA.

染色体DNA的精确复制对于保存细胞的遗传互补是必不可少的。最近，Speck博士的研究小组已经重建了DNA复制的第一步，使用纯化的蛋白质将复制的MCM 2 -7解旋酶装载到dsDNA上。这个重要过程的重建，称为复制前复合物（前RC）的形成，现在能够详细表征这个反应。将环状MCM 2 -7装载到DNA上需要起始识别复合物（ORC）、Cdc 6和Cdt 1。在该反应期间，六聚体MCM 2 -7环必须打开以允许DNA进入其中央通道。有人提出，Mcm 2和Mcm 5之间的弱界面可能参与加载过程，产生DNA进入的门。在拟议的项目中，我正在使用重建的预RC组装系统和体内方法来测试这一假设。我已经将FK 506和雷帕霉素结合蛋白（FKBP）或FKBP-雷帕霉素结合（FRB）结构域融合到MCM 2 -7的单个亚基上，以产生雷帕霉素诱导的蛋白质相互作用。最初，测试了这些修饰的蛋白质是否在酵母中耐受。然后，使用复溶的pre-RC体外试验，我们已经表明在Mcm 2/5界面处的门的阻断阻碍了MCM 2 -7的加载。相比之下，在不存在雷帕霉素的情况下进行的相同反应，即具有打开的门，允许有效的加载。此外，到目前为止测试的所有其他接口都不受雷帕霉素的影响。在未来，这些结构将在体内进行测试。这些研究将重点关注它们抑制前RC形成以及在DNA复制完成后释放MCM 2 -7的能力。这项工作有可能确定特定的Mcm亚基接口作为调节DNA加载和卸载。由于复合物MCM 2 -7在癌症中经常过表达，因此该界面可能是体内调节MCM活性的有趣靶点。

The precise duplication of chromosomal DNA is essential for preserving the genetic complement of the cell. Recently, the group of Dr. Speck has reconstituted the first step in DNA replication, loading of the replicative MCM2-7 helicase onto dsDNA, using purified proteins. Reconstitution of this important process, termed pre-replication complex (pre-RC) formation, enables now the detailed characterisation of this reaction. Loading of the ring-shaped MCM2-7 onto DNA requires the origin recognition complex (ORC), Cdc6, and Cdt1. During this reaction the hexameric MCM2-7 ring must be opened to allow entry of the DNA into its central channel. It has been proposed that a weak interface between Mcm2 and Mcm5 could be involved in the loading process, generating a gate for DNA entry. In the proposed project I am testing this hypothesis using the reconstituted pre-RC assembly system and an in vivo approach. I have already fused FK506- and rapamycin-binding protein (FKBP) or FKBP-rapamycin binding (FRB) domains to the individual subunits of MCM2-7 to generate a rapamycin-inducible protein interaction. Initially, it was tested whether those modified proteins are tolerated in yeast. Then, using the reconstituted pre-RC in vitro assay we have shown that the blocking of the gate at the Mcm2/5 interface hinders loading of MCM2-7. In contrast, the same reaction performed in the absence of rapamycin, i.e. with an open gate, allows efficient loading. Also, all other interfaces tested so far remained unaffected by rapamycin. In the future these constructs will be tested in vivo. These studies will focus on their ability to inhibit pre-RC formation as well as the release of MCM2-7 after completion of DNA replication. This work has the potential to identify specific Mcm subunit interfaces as regulators of DNA loading and unloading. Since the complex MCM2-7 is frequently overexpressed in cancer this interface could be an interesting target to regulate MCM activity in vivo.