The structural basis of replicative helicase loading onto DNA

我们细胞中的DNA和其他生命形式的DNA给出了关于生命是如何形成和运作的精确指令。为了生长、更新和繁殖，生物细胞必须首先复制它们的DNA，这样每个子细胞才能从母细胞获得完整的基因构成。只有在复制时，细胞才会分裂。DNA复制是一个精心设计的过程，由大型蛋白质机器--“细胞工作人员”--沿着多个检查点协同组装，以确保复制的准确性。由于DNA分子本身是由两条缠绕的链组成的双螺旋结构，因此必须将其解开，才能为复制机器提供遗传信息。解离是由紧密结合的蛋白质复合体进行的，形成一种名为DNA解旋酶的酶，但其活性有时会受到错误调节，导致细胞应激或衰老，导致人类疾病或植物生长缺陷。在正常细胞中，环状的、成熟前的解旋酶在激活之前首先被包裹在DNA周围。我们最近发现了对成熟前解旋酶与帮助将其加载到DNA周围的蛋白质相互作用的详细见解，并发现了一种名为CDT1的蛋白质中的一段伸展，它可能起到打开和关闭环的钳子的作用。打开过程在稍后的展开过程中特别重要，因此必须进行探索，以扩展我们对正常过程与异常过程的理解。为了研究在体外DNA负载过程中的解旋酶，我们创造了几个解旋酶变体，它们在特定的点“冻结”解旋酶的负载过程，并使我们能够使用高分辨率冷冻电子显微镜和复杂的计算方法来详细研究结果。从目前的研究中获得的对准确的解旋酶加载到DNA上的机械洞察力将产生对最终解开DNA和DNA复制的几个基本早期步骤的概述。虽然这对于基本了解我们的细胞如何工作特别重要，但它对癌症等人类疾病有着重要的影响，但也将更直接地支持对传染病抑制剂的研究。

The DNA in our cells and that of other lifeforms gives the precise instructions on how life is shaped and works. To grow, renew and reproduce, biological cells must first duplicate their DNA, so that each daughter cell can receive the full genetic make-up from the mother cell. Only upon duplication, cell division occurs. DNA duplication is a carefully choreographed process, which is carried out by the "workers of the cell", large protein machineries, co-operatively assembled along many checkpoints to ensure faithful copying. Since the molecule DNA itself is shaped as a double helix, composed of two wound-up strands, it has to be unwound to grant access to the genetic information for the copy machinery. Unwinding is carried out by a tightly integrated complex of proteins, forming the enzyme called DNA helicase, but its activity can be occasionally misregulated, causing cellular stress or ageing, resulting in human disease or growth defects in plants. In the normal cell, the ring-shaped, pre-mature helicase is first being wrapped around DNA before its activation. We have recently uncovered detailed insights into the interactions of the pre-mature helicase with the protein that helps loading it around DNA and identified a stretch in a protein called Cdt1 that might act as a clamp to open and close the ring. The opening process is particular important later in the unwinding process and therefore has to be explored to extend our understanding of the normal versus an abnormal process. To study the helicase during the process of loading around DNA in vitro, we created several helicase variants that "freeze" the helicase loading process at specific points and enable us to study the outcome in detail using high-resolution cryo-electron microscopy and sophisticated computational methods. The mechanistic insight into accurate helicase loading onto DNA gained from the presented study will generate an overview of several essential early steps towards eventual unwinding of the DNA and DNA duplication. While this is particularly important for a basic understanding of how our cell works, it has important implications for human diseases such as cancer but also will more immediately support research into inhibitors of infectious diseases.