RNA:DNA hybrids are transient physiological intermediates that arise during several cellular processes such as DNA replication. Although these structures have physiological relevance, their stable accumulation perturbs DNA replication, inducing replication stress and genome instability. Cells possess two enzymes that process these structures restoring the correct DNA:DNA sequence: RNase H1, which just handles stretches of multiple rNMPs, and RNase H2 that processes either single or multiple ribonucleotides hybridized with DNA. Mutations in the human RNase H2 lead to carcinogenesis and a severe auto-inflammatory disease known as Aicardi-Goutières syndrome. S.cerevisiae yeast cells lacking RNases H enzymes (rnh1Δ rnh201Δ) accumulate ribonucleotides in their genome, becoming sensitive to different replication-stress inducing agents such as hydroxyurea (HU), a compound that decreases the dNTP pools, stalling replication forks. Unexpectedly, we found that this HU-sensitivity is completely suppressed by removing the RAD30 gene, coding for the translesion DNA polymerase η. In this study, I characterized this novel activity for the yeast Pol η under HU-induced replication stress, and the toxicity observed in RNase H depleted-cells, also focusing on possible regulatory mechanisms. Our data indicate that Pol η acts at HU-stressed replication forks, with recruitment that seems to depend on PCNA mono-Ub, and might also be regulated by the Pol η-SUMOylation levels. We proved that the catalytic reaction performed by Pol η is detrimental for RNase H deficient cells, causing DNA damage checkpoint activation and G2/M arrest. These harmful effects arise during the first replication cycle in HU and seem to be due to the incorporation of stretches of ribonucleotides promoted by Pol η. In agreement, a Pol η mutant allele with enhanced ribonucleotide incorporation further exacerbates the sensitivity to HU of cells lacking RNases H enzymes. All these data are compatible with a model in which Pol η promotes DNA replication from stalled replication forks, inducing the formation or stabilization of RNA:DNA hybrids. These hybrids could result from either direct incorporation of rNMPs into DNA or incorrect Okazaki fragments maturation or R-loops stabilization. However, in a scenario where RNase H activity fails to restore DNA, these hybrids become toxic for cells. Finally, performing an SGA screening, I identified some negative interactors of RAD30 that might compensate for the role played by the polymerase at HU-stalled replication forks.

CHARACTERIZATION OF TLS POLYMERASE ETA FUNCTION UNDER REPLICATION STRESS INDUCED BY LOW DNTP POOLS / G.m. Nava ; supervisiore: F. Lazzaro. - : . Dipartimento di Bioscienze, 2021 May 21. ((33. ciclo, Anno Accademico 2020.

CHARACTERIZATION OF TLS POLYMERASE ETA FUNCTION UNDER REPLICATION STRESS INDUCED BY LOW DNTP POOLS

G.M. Nava
2021

Abstract

RNA:DNA hybrids are transient physiological intermediates that arise during several cellular processes such as DNA replication. Although these structures have physiological relevance, their stable accumulation perturbs DNA replication, inducing replication stress and genome instability. Cells possess two enzymes that process these structures restoring the correct DNA:DNA sequence: RNase H1, which just handles stretches of multiple rNMPs, and RNase H2 that processes either single or multiple ribonucleotides hybridized with DNA. Mutations in the human RNase H2 lead to carcinogenesis and a severe auto-inflammatory disease known as Aicardi-Goutières syndrome. S.cerevisiae yeast cells lacking RNases H enzymes (rnh1Δ rnh201Δ) accumulate ribonucleotides in their genome, becoming sensitive to different replication-stress inducing agents such as hydroxyurea (HU), a compound that decreases the dNTP pools, stalling replication forks. Unexpectedly, we found that this HU-sensitivity is completely suppressed by removing the RAD30 gene, coding for the translesion DNA polymerase η. In this study, I characterized this novel activity for the yeast Pol η under HU-induced replication stress, and the toxicity observed in RNase H depleted-cells, also focusing on possible regulatory mechanisms. Our data indicate that Pol η acts at HU-stressed replication forks, with recruitment that seems to depend on PCNA mono-Ub, and might also be regulated by the Pol η-SUMOylation levels. We proved that the catalytic reaction performed by Pol η is detrimental for RNase H deficient cells, causing DNA damage checkpoint activation and G2/M arrest. These harmful effects arise during the first replication cycle in HU and seem to be due to the incorporation of stretches of ribonucleotides promoted by Pol η. In agreement, a Pol η mutant allele with enhanced ribonucleotide incorporation further exacerbates the sensitivity to HU of cells lacking RNases H enzymes. All these data are compatible with a model in which Pol η promotes DNA replication from stalled replication forks, inducing the formation or stabilization of RNA:DNA hybrids. These hybrids could result from either direct incorporation of rNMPs into DNA or incorrect Okazaki fragments maturation or R-loops stabilization. However, in a scenario where RNase H activity fails to restore DNA, these hybrids become toxic for cells. Finally, performing an SGA screening, I identified some negative interactors of RAD30 that might compensate for the role played by the polymerase at HU-stalled replication forks.
LAZZARO, FEDERICO
DNA damage; translesion synthesis; RNA:DNA hybrids; yeast
Settore BIO/11 - Biologia Molecolare
CHARACTERIZATION OF TLS POLYMERASE ETA FUNCTION UNDER REPLICATION STRESS INDUCED BY LOW DNTP POOLS / G.m. Nava ; supervisiore: F. Lazzaro. - : . Dipartimento di Bioscienze, 2021 May 21. ((33. ciclo, Anno Accademico 2020.
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/841241
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