Preserving the entire set of hereditary instructions necessary for life of every organism, genome integrity and identity maintenance is a serious concern for cells. Genomic DNA is continuously endangered by exogenous and endogenous factors and one of the most common source of genome instability was recently detected to be the misincorporation of ribonucleotides (rNTPs) during DNA synthesis. RNA is more susceptible than DNA to spontaneous hydrolysis, so the presence of rNMPs in the genome may render chromosomes more unstable. Moreover, embedded rNMPs induce replication forks stalling and alter the B-conformation of a dsDNA, resulting in replication stress. Normally, rNMPs in DNA are processed by Ribonuclease H (RNase H) enzymes, which cleave the RNA of RNA:DNA hybrids allowing the reconstruction of a dsDNA molecule. Cells lacking RNase H activity can survive thanks to the Post-Replication Repair mechanism, which includes an error free pathway, the Template Switch, and a more mutagenic pathway that relies on the ability of special enzymes to replicate across damaged DNA, the Translesion Synthesis (TLS). Here, we focus on the contribution of the three TLS Polymerases (Rev1, Pol-zeta and Pol-eta ) to genomic-rNMPs tolerance and incorporation. We previously demonstrated that Pol-zeta efficiently replicates rNMPs-containing DNA and that Rev1 plays a non-catalytic role in supporting this function (Lazzaro et al. 2012). In this study we observed that Rev1 has also a non-catalytic role in preventing Pol-eta function. Surprisingly, the polymerase activity of Pol-eta appeared to be toxic for cells where dNTP pools, necessary for replication, are downregulated by hydroxyurea (HU), and lead to cell death when the RNase H is missing. Furthermore, we provide evidence that, in our sperimental conditions, Pol-eta toxicity is due to its tendency to introduce rNMPs during the TLS when the dNTP levels are low. Our findings describe an unexpected mechanism for TLS that could be relevant to replication stress in cells defective in RNase H, including humans stricken from diseases associated with RNase H defects like the Aicardi-Goutieres Syndrome (AGS). AGS is an autoimmune disease characterized by high levels of interferon-alpha in the serum and cerebrospinal fluid. Due to the fact that identified mutations fall in genes implicated in nucleic acid metabolism or signalling, and that an emerging source of immunostimulatory nucleic acids are fragments derived from endogenous retroelements, a recent hypothesis states that the pathological immune response could be driven by aberrant accumulation of retroelements intermediates. We then decide to use S.cerevisiae as model system to investigate the role of RNase H2 in retroelements mobility and try to link it to the possible molecular origin of AGS. We found that yeast RNase H can block the retrotransposition process and, once replaced yeast enzymes with wild type or AGS mutated forms of human RNase H2, we noticed that one of the three tested mutations causes the loss of the RNase H protective function against the mutagenic potential of retroelements. Even if preliminary, these findings could guide the way to better understand the molecular causes of AGS.
|Titolo:||EFFECT OF THE LOSS OF RIBONUCLEASE H ACTIVITY ON GENOME INSTABILITY: FROM DNA DAMAGE TOLERANCE TO RETROTRANSPOSITION|
|Relatore:||MUZI FALCONI, MARCO|
|Data di pubblicazione:||22-mag-2015|
|Parole Chiave:||Ribonuclease H; Translesion Synthesis; ribonucleotides; retrotransposition; Aicardi-Goutières Syndrome|
|Settore Scientifico Disciplinare:||Settore BIO/11 - Biologia Molecolare|
|Citazione:||EFFECT OF THE LOSS OF RIBONUCLEASE H ACTIVITY ON GENOME INSTABILITY: FROM DNA DAMAGE TOLERANCE TO RETROTRANSPOSITION ; tutor: M. Muzi Falconi. - Milano : Università degli studi di Milano. DIPARTIMENTO DI BIOSCIENZE, 2015 May 22. ((27. ciclo, Anno Accademico 2014.|
|Appare nelle tipologie:||Tesi di dottorato|