The role of Sen1 in DNA replication

染色体上稳定结合的DNA-RNA杂交体是基因组不稳定的一大来源。首先，暴露的单链DNA更容易发生化学反应和内源性损伤剂，导致突变。此外，DNA-RNA杂交体采用A-DNA构型，在热力学上比双链DNA更稳定，从而为DNA上发生的其他可能的过程创造了障碍，如DNA复制。在原核和真核细胞中的许多研究表明，由RNA转录产生的DNA-RNA杂交体(R-环)是复制叉前进的障碍，因此构成细胞基因组不稳定的来源。在模式生物酿酒酵母中，有几种途径可以防止R-环在DNA上的积累，要么是通过促进RNA的及时成熟和从细胞核中移除(通过THO复合体)，要么是通过从DNA中移除RNA(通过RNA核酸酶RNH1和RNH2)。DNA/RNA解旋酶Sen1在去除R-环的过程中也发挥了关键作用。Sen1在整个进化过程中是保守的，人类同源基因senataxin在神经退行性遗传性疾病共济失调伴动眼运动性失用2型(AOA2)和肌萎缩侧索硬化症4(ALS4)中发生突变。Sen1在转录终止中起关键作用，通过解开DNA/RNA杂交体和从DNA中移除RNA聚合酶。染色质免疫沉淀分析间接地表明Sen1与复制叉一起旅行。我们以前的工作发现Sen1与复制体相互作用，我们绘制了在Forks招募Sen1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发挥着重要作用。然而，许多问题仍然没有得到回答。我的博士学位的目的是解决一些悬而未决的问题，关于在福克斯招聘参议员1的问题。特别是，我将分析细胞周期是如何调节这种相互作用的，并测试在高等真核生物的S阶段是否保持了SEN1和复制体之间的相互作用。这将是特别感兴趣的，因为赛那他辛与人类健康相关。最后，如果时间允许，我将探索Sen1和参与DNA修复的蛋白质之间的新的相互作用。

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.