Role of Senataxins in resolving transcription-replication conflicts

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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.