Role of Senataxins in resolving transcription-replication conflicts

我们的DNA序列编码在46个DNA分子（染色体）上，包含了我们的遗传指令。为了将信息传递给我们的细胞机制，短区域（基因）被复制（转录）成RNA。这就像复印一页说明书。相反，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令手册传递给两个新细胞。这些过程，转录和复制，涉及到读取DNA序列的不同分子机器。当这两台机器试图读取相同的DNA片段（转录-复制冲突，或T-R冲突）时会发生什么？细胞有办法减少T-R冲突的数量，但它们在健康细胞中仍然大量发生，在癌细胞中甚至更多（其中一些减少T-R冲突的方法不再起作用）。尽管如此，我们的细胞包含解决这些T-R冲突并防止它们引起问题的机制。例如，一种叫做Senataxin的蛋白质可以取代将DNA复制（转录）成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，但是复制原始手册的错误会导致具有错误指令的子细胞——这种结果被称为“遗传不稳定”。在这里，我们建议使用两种模式生物（都是简单的单细胞真核生物：S. cerevisiae和S. pombe）来探索Senataxin如何发挥作用以实现T-R冲突的解决。众所周知，在酿酒酵母中，Senataxin的一部分与复制机器有关。我们使用S. pombe的初步数据表明，在一些高频率转录（复制）的基因中，Senataxin的丢失导致复制机器停止。我们使用我们最近开发的一种方法（Pu-seq）观察到这一点，该方法允许我们跟踪数百万细胞群体中复制机器的运动。有趣的是，一个基因必须以高速率复制才能显示出这种对复制的影响，但并不是所有高速率复制的基因都显示出这种影响。这意味着DNA的这些区域一定有其他特征决定了需要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有必要解决T-R冲突。为了实现这一目标，我们将应用第二种方法来跟踪DNA复制机器，这种方法提供了复制机器在单个细胞中单个DNA分子上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Senataxin可以解决这些冲突。例如，转录水平真的是一个预测因子吗，还是仅仅需要在人口研究中可视化效果？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是否在两种模式生物中守恒。如果是的话，它很可能在人类细胞中起类似的作用。我们还将讨论Senataxins取代转录机的机制。目前有两种可能的模式。一是当T-R冲突发生时，被复制的RNA与被复制的DNA“缠在一起”（称为r环），这就触发了Senataxin的作用。我们有初步的数据再次证明了这一点，因此我们倾向于第二种模型；复制叉上的Senataxin识别被复制的RNA，并在T-R冲突产生任何问题之前用它来瞄准和取代转录机器。我们提出了一系列的基因实验，将有助于区分这两种模式。

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.