Role of Senataxins in resolving transcription-replication conflicts

Senataxins 在解决转录复制冲突中的作用

基本信息

  • 批准号:
    BB/W014793/1
  • 负责人:
  • 金额:
    $ 49.75万
  • 依托单位:
  • 依托单位国家:
    英国
  • 项目类别:
    Research Grant
  • 财政年份:
    2023
  • 资助国家:
    英国
  • 起止时间:
    2023 至 无数据
  • 项目状态:
    未结题

项目摘要

Our DNA sequence, encoded on 46 DNA molecules (chromosomes), contains our genetic instructions. To pass this information to the our cells' machinery, short regions (genes) are copied (transcribed) into RNA. This is like photocopying a page of an instruction manual. In contrast, when a cell duplicates into two cells, the 46 DNA molecules must be entirely duplicated (replicated) so that a complete genetic instruction manual is passed to both new cells. These processes, transcription and replication, involve different molecular machines that read the DNA sequence. What happens when these two machines try to read the same bit of the DNA (a transcription-replication conflict, or a T-R conflict)?Cells have ways of minimising the number of T-R conflicts, but they still occur in large numbers in healthy cells, and in even larger numbers in cancerous cells (where some of the ways to minimise T-R conflicts no longer function). Nonetheless, our cells contain mechanisms that resolve these T-R conflicts and prevent them causing problems. For example, a protein called Senataxin can displace the machine that is copying (transcribing) the DNA into RNA to allow the passage of the machine that is replicating the DNA molecule. This is important because loss of one photocopy of a page of information is trivial, but a mistake in duplication of the original manual would result in a daughter cell with incorrect instructions - an outcome known as 'genetic instability'.Here we propose to use two model organisms (both simple unicellular eukaryotes: S. cerevisiae and S. pombe) to explore how Senataxin functions to achieve resolution of T-R conflicts. It is known in S. cerevisiae that a proportion of Senataxin is associated with the replication machine. Our preliminary data using S. pombe shows that, at some genes that are transcribed (copied) at high frequency, loss of Senataxin results in the replication machine stopping. We observed this using a methodology we recently developed (Pu-seq) that allows us to track the movement of the replication machines in a population of millions of cells. It was interesting that a gene had to be copied at a high rate to show this effect on replication, but that not all genes that are copied at high rates show the effect. This means there must be other features of these regions of the DNA that dictate that Senataxins are required to resolve the T-R conflicts.One aim we have is to understand what feature of our 'instruction manual' make it necessary for Senataxins to resolve T-R conflicts. To achieve this we will apply a second method of tracking the DNA replication machine which provides very high resolution information on the movement of the replication machine on individual DNA molecules from single cells. When we have collected enough data, this will allow us to understand much more detail about what is causing these T-R conflicts and why they are resolved by Senataxin. For example, is transcription level really a predictor, or is it simply necessary to visualise the effect in population studies? Are there specific DNA sequences or sequence patterns associated with the sites of T-R conflicts? The data will also allow us to determine if this phenomenon is conserved in the two model organisms. If it is, it is likely to operate similarly in human cells.We will also address the mechanism by which Senataxins displace the transcription machine. Currently there are two likely models. One is that when the T-R conflict happens, the RNA being copied gets 'tangled up' with the DNA being replicated (known as an R-loop) and this triggers Senataxin into action. We have preliminary data that argues again this and we thus favour a second model; Senataxin on the replication fork recognises the RNA being copied and uses this to target and displace the transcription machine before any problems arise from a T-R conflict. We propose a range of genetic experiments that will help distinguish between these two models.
我们的 DNA 序列由 46 个 DNA 分子(染色体)编码,包含我们的遗传指令。为了将这些信息传递给我们的细胞机器,短区域(基因)被复制(转录)成 RNA。这就像复印一页说明手册。相比之下,当一个细胞复制成两个细胞时,46个DNA分子必须完全复制(复制),以便将完整的遗传指导手册传递给两个新细胞。这些过程(转录和复制)涉及读取 DNA 序列的不同分子机器。当这两台机器尝试读取相同的 DNA 位时会发生什么(转录复制冲突或 T-R 冲突)?细胞有办法最大限度地减少 T-R 冲突的数量,但它们在健康细胞中仍然大量发生,癌细胞中的数量甚至更多(其中一些最小化 T-R 冲突的方法不再起作用)。尽管如此,我们的细胞含有解决这些 T-R 冲突并防止它们引起问题的机制。例如,一种名为 Senataxin 的蛋白质可以取代将 DNA 复制(转录)为 RNA 的机器,从而允许复制 DNA 分子的机器通过。这很重要,因为丢失一页信息的复印件是微不足道的,但复制原始手册的错误会导致子细胞的指令不正确 - 这种结果被称为“遗传不稳定”。在这里,我们建议使用两个模型生物(都是简单的单细胞真核生物:酿酒酵母和粟酒裂殖酵母)来探索 Senataxin 如何发挥作用来解决 T-R 冲突。已知在酿酒酵母中,一部分Senataxin 与复制机器相关。我们使用粟酒裂殖酵母的初步数据表明,在某些高频转录(复制)的基因中,Senataxin 的丢失会导致复制机器停止。我们使用我们最近开发的方法(Pu-seq)观察到了这一点,该方法使我们能够跟踪复制机器在数百万个细胞群中的运动。有趣的是,基因必须以高速率复制才能显示出这种对复制的影响,但并非所有高速率复制的基因都显示出这种效果。这意味着 DNA 的这些区域必须有其他特征,决定需要 Senataxins 来解决 T-R 冲突。我们的一个目标是了解我们的“说明书”的哪些特征使得 Senataxins 必须解决 T-R 冲突。为了实现这一目标,我们将应用第二种跟踪 DNA 复制机的方法,该方法提供有关复制机在来自单细胞的单个 DNA 分子上的运动的非常高分辨率的信息。当我们收集到足够的数据时,这将使我们能够更详细地了解导致这些 T-R 冲突的原因以及为什么这些冲突可以通过 Senataxin 解决。例如,转录水平真的是一个预测因子,还是仅仅需要在群体研究中可视化效果?是否存在与 T-R 冲突位点相关的特定 DNA 序列或序列模式?这些数据还将使我们能够确定这种现象在两种模式生物中是否保守。如果是这样,它很可能在人类细胞中发挥类似的作用。我们还将探讨 Senataxin 取代转录机器的机制。目前有两种可能的模型。一是当T-R冲突发生时,正在复制的RNA与正在复制的DNA“纠缠在一起”(称为R环),这会触发Senataxin发挥作用。我们有初步数据再次证明这一点,因此我们倾向于第二种模型;复制叉上的 Senataxin 可以识别正在复制的 RNA,并在 T-R 冲突出现任何问题之前利用它来定位和取代转录机器。我们提出了一系列基因实验来帮助区分这两种模型。

项目成果

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Antony Carr其他文献

Antony Carr的其他文献

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{{ truncateString('Antony Carr', 18)}}的其他基金

Replication fork stability and fork restart
复制分叉稳定性和分叉重启
  • 批准号:
    G1100074-E01/1
  • 财政年份:
    2011
  • 资助金额:
    $ 49.75万
  • 项目类别:
    Research Grant
Genome Damage and Stability Centre
基因组损伤与稳定中心
  • 批准号:
    G0801130-E01/1
  • 财政年份:
    2009
  • 资助金额:
    $ 49.75万
  • 项目类别:
    Research Grant
Which DNA polymerase functions during HR-dependent fork restart?
哪种 DNA 聚合酶在 HR 依赖性分叉重启期间发挥作用?
  • 批准号:
    G0801078/1
  • 财政年份:
    2009
  • 资助金额:
    $ 49.75万
  • 项目类别:
    Research Grant
DNA damage response mechanisms
DNA损伤反应机制
  • 批准号:
    G0600233/1
  • 财政年份:
    2006
  • 资助金额:
    $ 49.75万
  • 项目类别:
    Research Grant

相似海外基金

Role of Senataxins in resolving transcription-replication conflicts
Senataxins 在解决转录复制冲突中的作用
  • 批准号:
    BB/W01520X/1
  • 财政年份:
    2022
  • 资助金额:
    $ 49.75万
  • 项目类别:
    Research Grant
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