RNA IN DNA: FROM STRUCTURE TO GENOME INSTABILITY

The presence of RNA in the genome of living cells is one of the emerging topics of the last two decades and has been implicated in many biological processes. I focused my attention on ribonucleotides (rNMPs) embedded into DNA during genome duplication, as a threat to its integrity. In fact, rNMPs have been classified as the most frequent non-canonical nucleotides introduced during genome duplication by DNA polymerases. Such high incorporation frequency has been related to a physiological role in mismatch repair, but it can be easily turned into a source of genomic instability if rNMPs are not removed from DNA. This task is performed by RNase H activities that enable error-free repair of embedded single and multiple ribonucleotides. I first approached the issue of ribonucleotides incorporation into DNA from a physical point of view. Utilizing Atomic Force Microscopy I studied how ribonucleotides intrusions impact on DNA structure. The results obtained provided new insights on the structural changes imposed by ribonucleotides persistence into DNA. The other part of my Ph.D. project concerned the study of rNMPs incorporation in vivo, using the budding yeast S. cerevisiae as a model organism. The second aim was to unravel the function of the Translesion Synthesis polymerase η (Pol η) when the genome contains residual ribonucleotides and when deoxyribonucleotides (dNTPs) pools are depleted. We found that DNA polymerase η is responsible for the cell lethality observed when dNTPs are scarce and RNase H activities are defective. Therefore, I explored and characterized this unexpected toxic activity. We propose a model where Pol η supports cell survival in low dNTPs conditions by promoting DNA replication using ribonucleotides. While this activity is normally beneficial to wild type cells, it is highly toxic to cells defective for RNase H activities.