The role of Sen1 in DNA replication

染色体上稳定结合的DNA-RNA杂合体是基因组不稳定性的重要来源。首先，暴露的单链DNA更容易接近化学反应和内源性破坏剂，导致诱变。此外，DNA-RNA杂交体呈现A-DNA构型，其在化学上比双链DNA更稳定，从而对DNA上发生的其他可能过程（例如DNA复制）造成障碍。在原核和真核细胞中的许多研究表明，由于RNA转录而产生的DNA-RNA杂合体（R环）代表了复制叉进展的障碍，因此构成了细胞中基因组不稳定性的来源。在模式生物酿酒酵母（Saccharomyces cerevisiae）中，有几种途径可以防止R环在DNA上的积累，或者通过促进RNA的及时成熟和从细胞核中去除（通过THO复合物），或者通过从DNA中去除RNA（通过RNA核酸酶RNH 1和RNH 2）。DNA/RNA解旋酶Sen 1也在去除R环中发挥关键作用。Sen 1在整个进化过程中是保守的，并且人类直系同源物Senataxin在神经退行性遗传性疾病共济失调伴眼用不能2型（AOA 2）和肌萎缩性侧索硬化4型（ALS 4）中突变。Sen 1在转录终止中起着关键作用，通过解旋DNA/RNA杂交体并从DNA中去除RNA聚合酶。有趣的是，染色质免疫沉淀分析显示Sen 1与复制叉一起旅行。我们以前的工作确定了Sen 1与复制体相互作用，我们绘制了Sen 1在叉处招募所需的蛋白质结构域。我们已经证明，这种招募在维持基因组稳定性方面起着重要作用。然而，许多问题仍然没有答案。我的博士学位的目的是解决一些悬而未决的问题，关于森1在福克斯招聘。特别是，我将分析细胞周期如何调节这种相互作用，并测试Sen 1和复制体之间的相互作用是否维持在S期在高等真核生物。由于Senataxin与人类健康的相关性，这将是特别感兴趣的。最后，如果时间允许的话，我将探索Sen 1和参与DNA修复的蛋白质之间的新型相互作用。

Stably associated DNA-RNA hybrids on chromosomes are a great source of genomic instability. Firstly, exposed single strand DNA is more easily accessible to chemical reactions and endogenous damaging agents, leading to mutagenesis. In addition, the DNA-RNA hybrids assume an A-DNA configuration, which is thermodynamically more stable than double stranded DNA, thus creating an obstacle for other possible processes occurring on DNA, such as DNA replication. Many studies, in prokaryotic and eukaryotic cells, have shown that DNA-RNA hybrids (R-loops), generated as a consequence of RNA transcription, represent an obstacle to the progression of replication forks, hence constituting a source of genomic instability in the cell. In the model organism Saccharomyces cerevisiae, several pathways prevent the accumulation of R-loops on DNA, either by promoting the timely maturation and removal of the RNA from the nucleus (via the THO complex) or by removing the RNA from the DNA (via the RNA nucleases RNH1 and RNH2). A key role in the removal of R-loops is also played by the DNA/RNA helicase Sen1. Sen1 is conserved throughout evolution and the human orthologue Senataxin is mutated in the neurodegenerative genetic disorders ataxia with oculomotor apraxia type 2 (AOA2) and amyotrophic lateral sclerosis 4 (ALS4). Sen1 plays a key role in transcription termination, by unwinding the DNA/RNA hybrid and removing the RNA polymerases from the DNA.Intriguingly, chromatin immuno-precipitation analysis shows that Sen1 travels with replication forks. Our previous work identified that Sen1 interacts with the replisome, and we mapped the protein domains required for the recruitment of Sen1 at forks. We have shown that this recruitment plays an important role in the maintenance of genome stability. Many questions, however, are still unanswered. The aim of my PhD is to address some outstanding questions regarding the recruitment of Sen1 at forks. In particular I will analyse how the cell cycle regulates this interaction and test whether the interaction between Sen1 and the replisome is maintained during S phase in higher eukaryotes. This will be of particular interest due to the relevance of Senataxin in human health. Finally I will explore, time permitting, the novel interaction between Sen1 and proteins involved in DNA repair.