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，包含我们的遗传指令。为了将这些信息传递给我们细胞的机器，短区域(基因)被复制(转录)成RNA。这就像是复印了一页操作手册。相反，当一个细胞复制成两个细胞时，必须完全复制(复制)这46个DNA分子，以便将完整的遗传说明手册传递给两个新细胞。这些过程，即转录和复制，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？细胞有方法将T-R冲突的数量降至最低，但它们仍然大量出现在健康细胞中，甚至更多发生在癌细胞中(一些减少T-R冲突的方法已不再起作用)。尽管如此，我们的细胞包含了解决这些T-R冲突并防止它们造成问题的机制。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。这一点很重要，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将导致子细胞具有不正确的指令-这一结果被称为“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：酿酒酵母和庞贝葡萄球菌)来探索Senataxin如何发挥作用来实现T-R冲突的解决。在酿酒酵母中，已知一定比例的Senataxin与复制机器有关。我们使用S.pombe的初步数据显示，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一种方法(Pu-seq)观察到了这一点，该方法允许我们跟踪数百万细胞种群中复制机器的运动。有趣的是，一个基因必须以高复制速度复制才能显示出对复制的影响，但并不是所有以高复制速度复制的基因都显示出这种效果。这意味着DNA的这些区域一定还有其他特征决定了Senataxin是解决T-R冲突所必需的。我们的一个目标是了解我们的“说明手册”中的什么特征使得Senataxins解决T-R冲突是必要的。为了实现这一点，我们将应用第二种跟踪DNA复制机器的方法，该方法提供关于复制机器从单个细胞在单个DNA分子上移动的非常高分辨率的信息。当我们收集了足够的数据后，这将使我们能够更详细地了解是什么导致了这些T-R冲突，以及为什么它们会被Senataxin解决。例如，转录水平真的是一个预测指标吗，或者只是有必要在人口研究中可视化这种影响？是否有与T-R冲突部位相关的特定DNA序列或序列模式？这些数据还将使我们能够确定这一现象是否在这两种模式生物中是保守的。如果是这样的话，它很可能在人类细胞中也有类似的操作。我们还将讨论Senataxins取代转录机器的机制。目前有两种可能的模式。其一是，当T-R冲突发生时，被复制的RNA与被复制的DNA“缠绕”在一起(称为R-环)，这触发了感冒素的作用。我们有初步数据再次证明这一点，因此我们支持第二种模型；复制叉子上的Senataxin识别被复制的RNA，并在T-R冲突出现任何问题之前使用它来定位和取代转录机器。我们提出了一系列有助于区分这两种模型的基因实验。

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.