Towards an in vivo understanding of RNase H1 regulation in Saccharomyces cerevisiae
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Abstract
RNA:DNA hybrids are structures, in which RNA base pairs with DNA. A special kind of RNA:DNA hybrid is called R-loop. R-loops are three-stranded structures, in which one DNA strand is displaced by the RNA portion of the hybrid. These structures have multiple functions in cells that can be beneficial, like regulating transcription or allowing error-free DNA repair, or detrimental, like stalling of the replication fork or mutagenesis of the displaced ssDNA strand. Due to the positive and negative functions of RNA:DNA hybrids, these structures have to be tightly regulated. Different kinds of enzymes exist for preventing or resolving RNA:DNA hybrids. Ribonucleases H (RNases H) for example can degrade the RNA portion of the hybrids, while Sen1 helicase and the human homologue Senataxin can unwind RNA:DNA hybrids and prevent their formation.
While RNase H2’s regulation has been studied, little is known about RNase H1 regulation. It has been shown that RNase H1 mainly acts in genomic regions with a high RNA:DNA hybrid density. The enzyme also binds to RNA:DNA hybrids outside these regions of high RNA:DNA hybrid density, but the probability for degradation is low in these regions.
The aim of this study is to gain a broader understanding of RNase H1 regulation. I could show, that RNase H1 does not influence regulatory RNA:DNA hybrids in the budding yeast S. cerevisiae as overexpression of the enzyme or the catalytic inactive allele does not change the transcriptome. Furthermore, in the absence of Sen1, RNase H1 function is important for viability. This importance of RNase H1 most likely is the reason for the upregulation of the enzyme. Sen1 is traveling with the replisome and there preventing transcription-replication conflicts. In this work I showed that RNase H1 is acting together with Sen1 in this function. If Sen1 is gone or a loss of function mutant was used, RNase H1 served as a backup. As transcription-replication conflicts arise during replication, it is not surprising, that for upregulation of RNase H1 cells have to go through S-phase. Furthermore, transcription-replication conflicts favor the formation of R-loops. I showed that these R-loops are triggering a higher expression of RNase H1. In summary, I found a new function of RNase H1 in the prevention and resolution of transcription-replication conflicts in this study that impacts the regulation of RNase H1.