The maintenance of genome stability is essential for every living organism to preserve the genetic information and to transmit the correct genetic inheritance to the progeny. Every day, each cell in the human body is subjected to tens of thousands of DNA lesions, and the most cytotoxic ones are DNA double strand breaks (DSBs). DNA lesions that are not properly repaired lead to genome instability that can yield cancer, neurodegenerative diseases, and immunodeficiency. To counteract the DNA lesions, cells have evolved an evolutionary conserved multi-protein pathway, known as DNA damage response (DDR), that couples DNA repair with the activation of the DNA damage checkpoint, that slows down the cell cycle progression to repair DNA damage. In Saccharomyces cerevisiae the master regulators of the DNA damage checkpoint are the protein kinases Tel1 and Mec1 (ATM and ATR in human), that arrest the cell cycle through phosphorylation of the effector kinases Rad53/CHK2 and Chk1, whose activation also requires the adaptors Rad9/53BP1 and Mrc1/Claspin. Tel1/ATM recognizes DSBs, whereas Mec1/ATR is activated by single strand stretches (ssDNA) coated with Replication Protein A (RPA). Moreover, Tel1/ATM also participates in DSB repair. In Saccharomyces cerevisiae DSBs are repaired mainly by homologous recombination (HR), which uses the homolog sequence on the sister chromatid as a repair template and it is initiated by the nucleolytic degradation of the 5’ DSB ends, in a process called DNA end resection. Resection involves the coordinate action of several nucleases and helicases, such as the MRX/N complex (Mre11-Rad50-Xrs2/NBS1), the endonuclease Sae2 /CtIP in human, the nucleases Exo1 and Dna2, together with the RecQ helicase Sgs1/BLM. Beside its role at the DSB Tel1/ATM has a role in telomere maintenance and fork maintenance after replication stress. My PhD thesis aims at identifying novel players that participate together with Tel1/ATM in the DDR to increase the knowledge upon Tel1 functions and to reveal novel potential targets for cancer therapy. Indeed, we searched for mutations that enhanced the sensitivity of Tel1-deficient cells to the chemotherapy agent and Top1 inhibitor camptothecin (CPT), that induces replication stress by creating transient Top1-adducts on DNA. We have found that the exonuclease Exo1, supports Tel1/ATM functions, promoting cell survival to torsional stress induced by CPT in the absence of Tel1. In addition, our findings showed that in the presence of torsional stress induced by CPT, Tel1 and Exo1 synergistically counteract Mec1-dependent DNA damage checkpoint activation and RPA-coated ssDNA persistence. We directly evaluated HR repair at the HO-DSB and, we demonstrated that Tel1 and Exo1-deficient cells hypersensitivity does not depend on a DNA end resection or ectopic recombination defect. Since the CPT does not necessarily generate DSBs, whereas it can interfere with normal replication forks progression, we studied Tel1 and Exo1 role at early replication origins showing that Exo1 supports Tel1 kinase deficient cells viability by promoting HR-dependent fork recovery. In addition, to better define Tel1 and Exo1 functions in response to topoisomerase poisoning and to unveil novel antagonistic or synergistic players that act together with Tel1 and Exo1, we performed a genomic screening. We selected five suppressors with mutations in a single gene that belongs to different complementation groups. Among them we have identified one loss and two gain of function mutations in MET18 and DNA2 genes, respectively. Taken together these results show that the nucleases play an important action that supports Tel1/ATM functions in the presence of CPT-induced damage.
Il mantenimento della stabilità del genoma è essenziale per ogni organismo vivente al fine di preservare le informazioni genetiche e trasmettere una corretta eredità genetica alla progenie. Ogni giorno, ciascuna cellula è soggetta a decine di migliaia di lesioni al DNA, e le più citotossiche sono le rotture del DNA a doppio filamento (DSB). Le lesioni che non vengono riparate correttamente portano a instabilità genomica, che può causare cancro, malattie neurodegenerative e immunodeficienza. Per contrastare le lesioni al DNA, le cellule hanno sviluppato un pathway multi-proteico conservato noto come risposta al danno del DNA (DDR), che accoppia la riparazione del DNA con l'attivazione del checkpoint da DNA, rallentando la progressione del ciclo cellulare per riparare le lesioni. In Saccharomyces cerevisiae, i regolatori principali del checkpoint da danno sono le proteine chinasi Tel1 e Mec1 (ATM e ATR nell'uomo), che arrestano il ciclo cellulare attraverso la fosforilazione delle chinasi effettrici Rad53/CHK2 e Chk1, la cui attivazione richiede gli adattatori Rad9/53BP1 e Mrc1/Claspin. Tel1/ATM riconosce i DSB, mentre Mec1/ATR è attivato da tratti di DNA a singolo filamento (ssDNA) rivestiti dalla proteina RPA. Inoltre, Tel1/ATM partecipa anche alla riparazione delle estremità. In Saccharomyces cerevisiae, i DSB sono riparati principalmente attraverso la ricombinazione omologa (HR), che utilizza la sequenza omologa sul cromatidio fratello come come templato di riparazione e viene iniziata dalla degradazione nucleolitica delle estremità 5' dei DSB, in un processo chiamato resection. La resection coinvolge l'azione coordinata di diverse nucleasi ed elicasi, come il complesso MRX/N (Mre11-Rad50-Xrs2/NBS1), l'endonucleasi Sae2/CtIP, le nucleasi Exo1 e Dna2, insieme all'elicasi Sgs1/BLM. Inoltre, Tel1/ATM ha un ruolo nel mantenimento dei telomeri e della forca replicativa. La mia tesi di dottorato mira a identificare nuovi interattori che partecipano insieme a Tel1/ATM nella risposta al danno per aumentare la conoscenza delle funzioni di Tel1 e rivelare nuovi potenziali bersagli per la terapia contro il cancro. Abbiamo cercato mutazioni che aumentassero la sensibilità delle cellule prive di Tel1 all'agente chemioterapico e inibitore della topoisomerasi di tipo I, chiamato camptotecina (CPT), che induce stress replicativo creando addotti transienti di Top1 sul DNA. Abbiamo scoperto che l'esonucleasi Exo1 supporta le funzioni di Tel1/ATM, promuovendo la sopravvivenza cellulare allo stress torsionale indotto da CPT in assenza di Tel1. Inoltre, le nostre scoperte hanno mostrato che in presenza di CPT, Tel1 ed Exo1 contrastano sinergicamente l'attivazione del checkpoint da danno dipendente da Mec1 e la persistenza di foci di RPA. Abbiamo valutato direttamente la riparazione omologa al DSB e abbiamo dimostrato che l'ipersensibilità delle cellule prive di Tel1 ed Exo1 non dipende da un difetto di resection delle estremità del DNA o da una ricombinazione ectopica. Poiché la CPT può interferire con la normale progressione delle forche di replicazione, abbiamo studiato il ruolo di Tel1 ed Exo1 alle origini di replicazione mostrando che Exo1 promuove il recupero della forca stallata. Inoltre, per definire meglio le funzioni di Tel1 ed Exo1 per scoprire nuovi interratori antagonisti o sinergici che agiscono insieme a Tel1 ed Exo1, abbiamo effettuato uno screening genomico. Abbiamo selezionato cinque soppressori con mutazioni in un singolo gene appartenente a diversi gruppi di complementazione. Tra questi abbiamo identificato una mutazione per perdita e due mutazioni per guadagno di funzione nei geni MET18 e DNA2, rispettivamente. Insieme, questi risultati mostrano che le nucleasi svolgono un'importante azione che supporta le funzioni di Tel1/ATM in presenza di danni indotti da CPT.
(2024). Interplays between Tel1/ATM and nucleases in the DNA damage response. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2024).
Interplays between Tel1/ATM and nucleases in the DNA damage response
GALLI, MICHELA
2024
Abstract
The maintenance of genome stability is essential for every living organism to preserve the genetic information and to transmit the correct genetic inheritance to the progeny. Every day, each cell in the human body is subjected to tens of thousands of DNA lesions, and the most cytotoxic ones are DNA double strand breaks (DSBs). DNA lesions that are not properly repaired lead to genome instability that can yield cancer, neurodegenerative diseases, and immunodeficiency. To counteract the DNA lesions, cells have evolved an evolutionary conserved multi-protein pathway, known as DNA damage response (DDR), that couples DNA repair with the activation of the DNA damage checkpoint, that slows down the cell cycle progression to repair DNA damage. In Saccharomyces cerevisiae the master regulators of the DNA damage checkpoint are the protein kinases Tel1 and Mec1 (ATM and ATR in human), that arrest the cell cycle through phosphorylation of the effector kinases Rad53/CHK2 and Chk1, whose activation also requires the adaptors Rad9/53BP1 and Mrc1/Claspin. Tel1/ATM recognizes DSBs, whereas Mec1/ATR is activated by single strand stretches (ssDNA) coated with Replication Protein A (RPA). Moreover, Tel1/ATM also participates in DSB repair. In Saccharomyces cerevisiae DSBs are repaired mainly by homologous recombination (HR), which uses the homolog sequence on the sister chromatid as a repair template and it is initiated by the nucleolytic degradation of the 5’ DSB ends, in a process called DNA end resection. Resection involves the coordinate action of several nucleases and helicases, such as the MRX/N complex (Mre11-Rad50-Xrs2/NBS1), the endonuclease Sae2 /CtIP in human, the nucleases Exo1 and Dna2, together with the RecQ helicase Sgs1/BLM. Beside its role at the DSB Tel1/ATM has a role in telomere maintenance and fork maintenance after replication stress. My PhD thesis aims at identifying novel players that participate together with Tel1/ATM in the DDR to increase the knowledge upon Tel1 functions and to reveal novel potential targets for cancer therapy. Indeed, we searched for mutations that enhanced the sensitivity of Tel1-deficient cells to the chemotherapy agent and Top1 inhibitor camptothecin (CPT), that induces replication stress by creating transient Top1-adducts on DNA. We have found that the exonuclease Exo1, supports Tel1/ATM functions, promoting cell survival to torsional stress induced by CPT in the absence of Tel1. In addition, our findings showed that in the presence of torsional stress induced by CPT, Tel1 and Exo1 synergistically counteract Mec1-dependent DNA damage checkpoint activation and RPA-coated ssDNA persistence. We directly evaluated HR repair at the HO-DSB and, we demonstrated that Tel1 and Exo1-deficient cells hypersensitivity does not depend on a DNA end resection or ectopic recombination defect. Since the CPT does not necessarily generate DSBs, whereas it can interfere with normal replication forks progression, we studied Tel1 and Exo1 role at early replication origins showing that Exo1 supports Tel1 kinase deficient cells viability by promoting HR-dependent fork recovery. In addition, to better define Tel1 and Exo1 functions in response to topoisomerase poisoning and to unveil novel antagonistic or synergistic players that act together with Tel1 and Exo1, we performed a genomic screening. We selected five suppressors with mutations in a single gene that belongs to different complementation groups. Among them we have identified one loss and two gain of function mutations in MET18 and DNA2 genes, respectively. Taken together these results show that the nucleases play an important action that supports Tel1/ATM functions in the presence of CPT-induced damage.File | Dimensione | Formato | |
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phd_unimib_791676.pdf
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Descrizione: Tesi di Galli Michela - 791676
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