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Pushing proteins off DNA - how do helicases unwind protein-coated DNA?

将蛋白质从 DNA 上推开 - 解旋酶如何解开蛋白质包被的 DNA？

基本信息

批准号：
BB/P000746/1
负责人：
Mark Leake
金额：
$ 52.05万
依托单位：
University of York
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2017
资助国家：
英国
起止时间：
2017 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FP000746%2F1
关键词：
Pushing proteins off DNA how

项目摘要

DNA encodes all the genetic information needed to act as a blueprint for life. This blueprint, though, needs to be converted ultimately into the building blocks of a cell. However, the information that DNA stores is buried deep within the structure of this molecule and can only be accessed by stripping apart the two strands that constitute DNA, so-called unwinding. This critical function is performed by enzymes called helicases that are tiny nanomotors that ratchet along DNA, separating the two DNA strands as they go. All organisms have a variety of different helicases and defects in just one type of helicase can result in catastrophe for a cell, potentially causing lethality or debilitating mutations within genes.The central importance of helicases in maintaining the viability of all cells and in passing the genetic blueprint from one generation to the next has been known for decades. Recently we have also come to appreciate that helicases face a particular problem when attempting to unwind DNA. DNA is coated in a wide variety of different proteins inside cells. These proteins play important roles in packaging DNA, reading the genetic blueprint and coordinating the movement of chromosomes inside cells. Unfortunately we now know that these bound proteins also present problems to helicases since any proteins bound to the DNA must also be pushed off to allow separation of the two DNA strands by a helicase. However, we know very little about how helicases displace proteins bound to the nucleic acid.We have been studying a helicase called Rep that plays an important role in copying of the genetic blueprint by displacing proteins that are bound to DNA. Our preliminary work has discovered that removal of part of this helicase activates DNA unwinding but at the same time inhibits displacement of proteins from the DNA. This discovery is important because it shows that displacement of proteins from DNA must involve something more than the helicase merely ratcheting along the DNA. This helicase has therefore evolved specific features to help push proteins off DNA although currently we do not understand what these features are. We aim to investigate how this helicase displaces proteins from DNA by using a combination of different molecular tools to investigate the properties of Rep and versions of this helicase that have increased or decreased abilities to push proteins off DNA. This work will cast light on how this important class of enzyme deals with the vast array of proteins that coat DNA. This problem is one that all organisms must face and so our findings will help us to understand how DNA is maintained effectively inside cells and, just as importantly, how things might go wrong. Mistakes made by helicases can result in very harmful rearrangements within the genetic code, contributing to genetic disease, and so our proposed work will shed light on potential sources of corruption of the genetic code. Conversely, this work may also reveal new ways of deliberately inhibiting helicases. Such inhibitors have potential uses as antiviral, antibacterial and anticancer compounds since helicases are so important for survival.

DNA编码了所有需要作为生命蓝图的遗传信息。然而，这个蓝图最终需要转化为细胞的构建模块。然而，DNA储存的信息被深埋在这个分子的结构中，只能通过剥离构成DNA的两条链来获取，即所谓的解旋。这种关键功能是由称为解旋酶的酶执行的，解旋酶是一种微小的纳米马达，可以沿着DNA旋转，分离两条DNA链。所有生物体都有各种不同的解旋酶，仅一种类型的解旋酶的缺陷就可能导致细胞的灾难，可能导致致命或基因内的衰弱突变。几十年来，人们已经知道解旋酶在维持所有细胞的活力和将遗传蓝图从一代传递到下一代方面的核心重要性。最近，我们也开始认识到，解旋酶在试图解开DNA时面临着一个特殊的问题。DNA在细胞内被各种不同的蛋白质包裹。这些蛋白质在包装DNA、阅读遗传蓝图和协调细胞内染色体的运动方面起着重要作用。不幸的是，我们现在知道，这些结合的蛋白质也给解旋酶带来了问题，因为任何与DNA结合的蛋白质也必须被推开，以允许解旋酶分离两条DNA链。然而，我们对解旋酶如何取代与核酸结合的蛋白质知之甚少，我们一直在研究一种名为Rep的解旋酶，它通过取代与DNA结合的蛋白质，在遗传蓝图的复制中起着重要作用。我们的初步工作已经发现，去除部分解旋酶会激活DNA解旋，但同时会抑制DNA中蛋白质的置换。这一发现很重要，因为它表明，蛋白质从DNA上的置换必须涉及比解旋酶仅仅沿着DNA棘轮运动更多的东西。因此，这种解旋酶进化出了特定的功能，以帮助将蛋白质从DNA上推下来，尽管目前我们还不知道这些功能是什么。我们的目标是研究如何通过使用不同的分子工具的组合来研究Rep和这种解旋酶的版本，增加或减少了将蛋白质从DNA上推下来的能力的特性，从而研究这种解旋酶如何从DNA上取代蛋白质。这项工作将揭示这类重要的酶如何处理DNA外壳的大量蛋白质。这个问题是所有生物体都必须面对的问题，因此我们的发现将帮助我们了解DNA如何在细胞内有效地维持，以及同样重要的是，事情可能会出错。解旋酶的错误可能导致遗传密码中非常有害的重排，导致遗传疾病，因此我们提出的工作将揭示遗传密码腐败的潜在来源。相反，这项工作也可能揭示故意抑制解旋酶的新方法。由于解旋酶对生存非常重要，因此此类抑制剂具有作为抗病毒、抗菌和抗癌化合物的潜在用途。