A molecular understanding of how MCM2-7 becomes loaded onto DNA to maintain genomic stability

我们的细胞含有DNA，代表着“生命的遗传蓝图”。DNA由两条互补的链组成，它们的中心包含遗传信息。在细胞分裂之前，DNA必须被复制，这样每个子细胞才能从母细胞获得完整的遗传补充。DNA复制是一个精心编排的过程，许多蛋白质协同作用，在复制起点组装大型DNA复制机器。DNA复制的核心是一种酶——DNA解旋酶——它将两条DNA链分开，使复制机器能够获得位于DNA中心的信息。最初，解旋酶必须装载到DNA上，然后才能变得活跃并参与DNA复制。然而，解旋酶装载反应在衰老、细胞应激或癌症中经常被错误调节，导致人类疾病或植物减产。幸运的是，细胞已经进化出一种高效的机制，可以将解旋酶装载到DNA上。虽然这一过程在简单细菌中被很好地理解，但我们最近的工作表明，在真核生物中，这一过程是完全不同的。我们现在已经发现了解旋酶本身对装载过程至关重要的证据，这在细菌中还没有被发现到如此程度。现在我们想要揭示解旋酶是如何在DNA上组装的以及它是如何装载到DNA上的，从而产生一个环绕DNA的环状复合体。我们已经创建了许多解旋酶变体，它们在特定点“冻结”解旋酶装载反应。此外，我们开发了几个独特的工具来分析与解旋酶装载相关的详细结构和功能变化。因此，我们建议在这个项目中使用这些解旋酶变体来揭示这种分子机器如何能够以精确和渐进的方式加载到DNA上，并定义在酵母细胞中引入解旋酶变体的生物体后果。我们的工作将产生一个机制框架，显示解旋酶装载实际上是如何工作的。这将支持相关领域研究人员的工作，但也可能导致开发具有抗真菌活性的解旋酶负载抑制剂。

Our cells contains DNA, representing a "genetic blueprint of life". DNA is composed of two complementary strands, which contain the genetic information in their centre. It is imperative that prior to cell division the DNA becomes duplicated, so that each daughter cell receives a full genetic complement from the mother cell. DNA duplication is a careful choreographed process, where numerous proteins synergize to assemble large DNA replication machinery at replication origins. Central for DNA duplication is an enzyme - the DNA helicase - that separates the two strands of DNA, to give copying machine access to the information that lies in the centre of the DNA. Initially the helicase must be loaded onto DNA, before it can become active and participate in DNA duplication. However, the helicase loading reaction is frequently misregulated during aging, cellular stress or in cancer, resulting in human disease or reduced crop yields in plants. Fortunately, cells have evolved a highly efficient mechanisms that allows the loading of the helicase onto DNA. While this process in simple bacteria is well understood, our recent work has revealed that in eukaryotes this process is quite different. We have now uncovered evidence that the helicase itself is crucial for the loading process, which has not been seen to such an extent in bacteria. Now we want to uncover exactly how the helicase assembles on DNA and how it is loaded onto DNA, producing a ring shaped complex that encircles DNA. We have created a number of helicase variants that "freeze" the helicase loading reaction at specific points. Additionally, we have developed several unique tools to analyse the detailed structural and functional changes associated with helicase loading. Therefore we propose for this programme to use these helicase variants to uncover how this molecular machine is able to be loaded in an accurate and processive way onto DNA and to define the organismal consequences of introducing the helicase variants in yeast cells.Our work will generate a mechanistic framework, showing how helicase loading actually works. This will support the work of researchers in related fields, but could also lead to the development of helicase loading inhibitors with anti-fungal activity.