Why does transcription present a major barrier to genome duplication?

为什么转录是基因组复制的主要障碍？

• 批准号: BB/I003142/1
• 负责人: Nigel Savery
• 金额: $ 44.58万
• 依托单位: University of Bristol
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2011
• 资助国家: 英国
• 起止时间: 2011 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FI003142%2F1
• 关键词: Why; does; transcription; present; major

When a cell divides, all of its DNA must be copied so that each of the two new cells contains a complete set of genes. The copying and passing on of genetic information is a central feature of life, and is performed in a similar manner by organisms from bacteria to humans. DNA replication must be accurate because mistakes can corrupt the genetic message with potentially fatal consequences. The DNA replication machinery must also be able to overcome any obstacles that it encounters, because a cell cannot divide until its genome has been fully duplicated. Potential obstacles to DNA replication are common, because DNA within cells is coated with proteins that package, repair or read the genetic material. In this project we aim to determine how the DNA replication machinery deals with a common and potent obstacle: collisions with the molecular machinery that 'reads' the information contained within genes. The central components of this machinery are enzymes called RNA polymerases. These enzymes unwind the double-stranded DNA molecule at the beginning of a gene so that they can use one of the two strands as a template for the construction of a temporary copy, called mRNA. In order to copy a complete gene the RNA polymerase must move along the DNA, unwinding it as it goes. During this process RNA polymerase binds tightly to the DNA to stop it falling off before it reaches the end. The copying of genes by RNA polymerase is regulated: the cell needs more copies of some genes than others, and so RNA polymerases are found rarely on some genes and in nose-to-tail traffic jams on others. Like RNA polymerase, the enzymes that replicate DNA move along, and unwind, the DNA double helix. As the DNA replication machinery has to copy the entire genome it will collide frequently with RNA polymerases that are copying individual genes. Because RNA polymerases bind tightly to DNA, the DNA replication machinery often has difficulty moving past them, and in some cases needs help from other proteins in order to overcome the obstacle. In our preliminary experiments we have identified two proteins that help the DNA replication machinery to push its way through obstacles. We have also shown that one of the main reasons that these proteins are needed by cells is to help overcome the 'roadblock' effect caused by RNA polymerases. The experiments that we now wish to undertake will enable us to discover what determines whether a particular RNA polymerase blocks DNA replication or is easily bypassed (for example, does the length of an RNA polymerase 'traffic jam' determine how easily the replication machinery can get through?). They will also enable us to define the various helper-systems that the cell uses to overcome such obstacles, and understand which systems help at which types of obstacle. There are many reasons why it is interesting and important to undertake this study. These experiments are exciting because they aim to understand the interface between two of the most fundamental and important processes in the cell: the copying and expression of the genetic material. These processes are well conserved in all organisms, and our findings will have implications beyond the experimentally tractable model system in which we will work. The understanding that we will gain will also have practical implications. It is now possible for scientists to construct organisms in which large sections of the genome, or even the entire genome, are artificially designed and constructed. A sound understanding of the interplay between genome replication and gene expression will be important if these designed organisms are to function correctly. Furthermore, by understanding how cells help the DNA replication machinery to overcome obstacles we can identify opportunities to disrupt those processes: drugs that target the helper-systems and prevent complete DNA replication may have applications in anti-bacterial or anti-tumour chemotherapy.

当一个细胞分裂时，它的所有DNA都必须被复制，这样两个新细胞中的每一个都包含一套完整的基因。遗传信息的复制和传递是生命的核心特征，从细菌到人类的生物体都以类似的方式进行。DNA复制必须是准确的，因为错误可能会破坏遗传信息，造成潜在的致命后果。DNA复制机制还必须能够克服它遇到的任何障碍，因为细胞在其基因组完全复制之前不能分裂。DNA复制的潜在障碍是常见的，因为细胞内的DNA被蛋白质包裹，修复或读取遗传物质。在这个项目中，我们的目标是确定DNA复制机制如何处理一个共同的和强大的障碍：与“读取”基因中包含的信息的分子机制的碰撞。这种机器的核心成分是称为RNA聚合酶的酶。这些酶在基因的起始处解开双链DNA分子，以便它们可以使用两条链中的一条作为模板来构建临时拷贝，称为mRNA。为了复制一个完整的基因，RNA聚合酶必须沿着DNA运动，并在运动过程中将DNA解旋。在这个过程中，RNA聚合酶与DNA紧密结合，以阻止它在到达末端之前脱落。RNA聚合酶的基因复制是受调控的：细胞需要的某些基因的拷贝数比其他基因多，因此RNA聚合酶很少出现在某些基因上，而在另一些基因上则是首尾相接。与RNA聚合酶一样，复制DNA的酶也会沿着DNA双螺旋运动并展开。由于DNA复制机制必须复制整个基因组，它将经常与复制单个基因的RNA聚合酶发生碰撞。由于RNA聚合酶与DNA紧密结合，DNA复制机制通常难以通过它们，在某些情况下需要其他蛋白质的帮助才能克服障碍。在我们的初步实验中，我们已经确定了两种蛋白质，它们可以帮助DNA复制机器克服障碍。我们还表明，细胞需要这些蛋白质的主要原因之一是帮助克服由RNA聚合酶引起的“路障”效应。我们现在希望进行的实验将使我们能够发现是什么决定了一种特定的RNA聚合酶是否会阻止DNA复制或容易被绕过（例如，RNA聚合酶“交通堵塞”的长度是否决定了复制机器可以多容易地通过？）。它们还将使我们能够定义细胞用来克服这些障碍的各种辅助系统，并了解哪些系统有助于克服哪些类型的障碍。有很多原因可以解释为什么进行这项研究是有趣和重要的。这些实验是令人兴奋的，因为它们旨在了解细胞中两个最基本和最重要的过程之间的界面：遗传物质的复制和表达。这些过程在所有生物体中都是保守的，我们的发现将产生超出我们将工作的实验易处理的模型系统的影响。我们将获得的谅解也将产生实际影响。现在科学家可以构建生物体，其中大部分基因组，甚至整个基因组都是人工设计和构建的。如果这些设计的生物体要正确地发挥作用，那么对基因组复制和基因表达之间的相互作用的充分理解将是重要的。此外，通过了解细胞如何帮助DNA复制机制克服障碍，我们可以确定破坏这些过程的机会：靶向辅助系统并阻止完整DNA复制的药物可能在抗菌或抗肿瘤化疗中有应用。

项目成果

Multipartite control of the DNA translocase, Mfd.

• DOI: 10.1093/nar/gks775
• 发表时间: 2012-11-01
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Smith AJ;Pernstich C;Savery NJ
• 通讯作者: Savery NJ

Inhibiting translation elongation can aid genome duplication in Escherichia coli.

• DOI: 10.1093/nar/gkw1254
• 发表时间: 2017-03-17
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Myka KK;Hawkins M;Syeda AH;Gupta MK;Meharg C;Dillingham MS;Savery NJ;Lloyd RG;McGlynn P
• 通讯作者: McGlynn P

The conserved C-terminus of the PcrA/UvrD helicase interacts directly with RNA polymerase.

• DOI: 10.1371/journal.pone.0078141
• 发表时间: 2013
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Gwynn EJ;Smith AJ;Guy CP;Savery NJ;McGlynn P;Dillingham MS
• 通讯作者: Dillingham MS

The structure and function of an RNA polymerase interaction domain in the PcrA/UvrD helicase.

• DOI: 10.1093/nar/gkx074
• 发表时间: 2017-04-20
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Sanders K;Lin CL;Smith AJ;Cronin N;Fisher G;Eftychidis V;McGlynn P;Savery NJ;Wigley DB;Dillingham MS
• 通讯作者: Dillingham MS