The structural basis of replicative helicase loading onto DNA

DNA 上复制解旋酶的结构基础

• 批准号: BB/S001387/1
• 负责人: Christian Speck
• 金额: $ 57.83万
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FS001387%2F1
• 关键词: structural; basis; replicative; helicase; loading

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.

我们和其他生命形式细胞中的DNA给出了生命如何形成和运作的精确指示。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遗传组成。只有在复制时，细胞分裂才会发生。DNA复制是一个精心设计的过程，它是由“细胞的工人”，大型蛋白质机器，合作组装沿着许多检查点，以确保忠实复制。由于DNA分子本身的形状是由两条缠绕的链组成的双螺旋，它必须被解开才能为复制机器提供遗传信息。解旋是由一种紧密整合的蛋白质复合物进行的，形成了一种称为DNA解旋酶的酶，但它的活性偶尔会被错误调节，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、未成熟的解旋酶在其激活之前首先包裹在DNA周围。我们最近发现了关于早熟解旋酶与蛋白质相互作用的详细见解，该蛋白质有助于将其装载在DNA周围，并确定了一种名为Cdt1的蛋白质中的一段，该蛋白质可能充当打开和关闭环的夹子。开放的过程是特别重要的，后来在解旋过程中，因此必须探讨，以扩大我们的理解正常与异常的过程。为了研究在体外加载DNA的过程中的解旋酶，我们创建了几种解旋酶变体，这些变体在特定点“冻结”解旋酶加载过程，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详细研究结果。从本研究中获得的精确解旋酶加载到DNA上的机械见解将产生对DNA和DNA复制最终解旋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具有重要意义，而且还将更直接地支持对传染病抑制剂的研究。

项目成果

Structural mechanism of helicase loading onto replication origin DNA by ORC-Cdc6

• DOI: 10.1073/pnas.2006231117
• 发表时间: 2020-07-28
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Yuan, Zuanning;Schneider, Sarah;Speck, Christian
• 通讯作者: Speck, Christian

An integrated workflow for crosslinking mass spectrometry

• DOI: 10.15252/msb.20198994
• 发表时间: 2019-09-01
• 期刊: MOLECULAR SYSTEMS BIOLOGY
• 影响因子: 9.9
• 作者: Mendes, Marta L.;Fischer, Lutz;Rappsilber, Juri
• 通讯作者: Rappsilber, Juri

The structure of ORC-Cdc6 on an origin DNA reveals the mechanism of ORC activation by the replication initiator Cdc6.

• DOI: 10.1038/s41467-021-24199-1
• 发表时间: 2021-06-23
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Feng X;Noguchi Y;Barbon M;Stillman B;Speck C;Li H
• 通讯作者: Li H

Evolution of DNA replication origin specification and gene silencing mechanisms.

• DOI: 10.1038/s41467-020-18964-x
• 发表时间: 2020-10-14
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Hu Y;Tareen A;Sheu YJ;Ireland WT;Speck C;Li H;Joshua-Tor L;Kinney JB;Stillman B
• 通讯作者: Stillman B