The role of Sen1 in DNA replication

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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.