The maintenance of genome stability is essential for every living organism to preserve and transmit the correct genetic information to the progeny. Every day in fact, cells are exposed to various DNA damaging agents that can cause nucleotide modifications, mismatches, double strand breaks (DSBs) and transcription and replication problems. Unrepaired DNA lesions lead to genome instability which has been found in cancer and genetic diseases. Cells counteract damages thanks to a multi-proteins pathway, known as the DNA damage response (DDR) that couples DNA repair with the activation of the DNA damage checkpoint. In Saccharomyces cerevisiae, the master regulators of this pathway are the kinases Tel1 and Mec1, ATM and ATR in human cells. They phosphorylate the effector kinases Rad53/CHK2 and Chk1, thus allowing the cell to slow down the cell-cycle progression to repair DNA damage. ATM and ATR are mutated in different types of tumours and genetic diseases. Moreover, nowadays ATM and ATR inhibitors are in clinical trials to be used as monotherapy or in combination with chemotherapy. Therefore, studying these two proteins is important to better understand their roles in the DDR and this could have also important implications in biomedical research. While Mec1 promotes checkpoint activation in the presence of long single-strand DNA stretches, Tel1 is recruited to DSBs where it promotes the processing (resection) of DNA ends and DSBs repair. Tel1 is involved in telomere homeostasis and replication fork maintenance after replication stress, even if this latter function is not totally clear. The aim of this project consists in identifying novel players that participate together with Tel1 and Mec1 in the DDR to increase the knowledge upon their function and to suggest new potential targets for cancer therapy. We initially focused on Tel1. Cells lacking TEL1 are sensitive only to the Top1-inhibitor camptothecin (CPT). We were interested in finding mutations that enhance or suppress this sensitivity. We have found that the inactivation of the nuclease Exo1 exacerbates tel1Δ cells sensitivity to CPT. We demonstrated that tel1Δ exo1Δ hypersensitivity is not caused by a defect in DSBs repair, but by a defect in the recovery of functional replication forks in the presence of CPT. We proposed that Exo1 is required to generate 3’-ended single-strand DNA tails that are necessary to restart replication after CPT poisoning in Tel1-deficient cells. In order to identify also mutations that suppress the CPT sensitivity of tel1Δ exo1Δ cells, we performed a genomic screening, and we have found loss of function mutations in genes that codify for proteins involved in Fe-S clusters biogenesis. They are able to insert Fe-S clusters in different proteins which regulate aspects of DNA metabolism. Their human orthologues are also mutated in different tumours. This suggests a link between Fe-S clusters biogenesis and genome stability and for this reason we decided to better investigate the function of these proteins in the DDR and their interplays with Tel1 and Mec1. Even if some aspects remain to be elucidated, this work has laid the groundwork for a better understanding of Tel1 and Mec1 functions and their interplays with Exo1 and Fe-S clusters biogenesis proteins. Since these proteins are conserved in human cells, the knowledge of these processes is useful to suggest new therapeutic strategies. In conclusion, it's important to remember that genome stability is a key factor to keep in mind even when developing industrial processes with microorganisms. I also participated in a project in which Saccharomyces cerevisiae and other yeasts were used as cell factories. We designed a zero-waste biorefinery concept to valorise food wastes and consequently produce new materials and chemicals. In this way we have proposed a solution for a current global environmental issue.
La stabilità del genoma è essenziale per i viventi per preservare e trasmettere correttamente l'informazione genetica alla progenie. Ogni giorno le cellule sono esposte a vari agenti che danneggiano il DNA e che causano rotture del doppio filamento (DSB) e problemi di replicazione. Le lesioni non riparate portano all'instabilità genomica, che è stata riscontrata in cancro e malattie genetiche. In presenza di danni al DNA, le cellule attivano un pathway noto come risposta al danno al DNA (DDR), che unisce la riparazione del DNA all'attivazione del checkpoint. In Saccharomyces cerevisiae, i principali regolatori di questa risposta sono le chinasi Tel1 e Mec1, rispettivamente ATM e ATR nelle cellule umane. Queste proteine fosforilano le chinasi effettrici Rad53/CHK2 e Chk1, permettendo alla cellula di rallentare il ciclo cellulare per riparare i danni al DNA. Le mutazioni in ATM e ATR sono presenti in vari tipi di tumori e malattie genetiche. Inoltre, attualmente gli inibitori di ATM e ATR sono in fase di sperimentazione clinica, da utilizzare nella terapia antitumorale. Pertanto, lo studio di queste due proteine è cruciale per comprendere meglio i loro ruoli nella DDR e potrebbe avere importanti implicazioni nella ricerca biomedica. Mec1 promuove l'attivazione del checkpoint in presenza di tratti di DNA a singolo filamento, mentre Tel1 viene reclutato ai DSB, dove favorisce il processamento delle estremità e la riparazione delle rotture. Tel1 opera anche ai telomeri e nel mantenimento della forca di replicazione dopo stress replicativo, sebbene questa funzione sia ancora poco chiara. L'obiettivo di questo progetto è identificare nuovi attori che partecipano con Tel1 e Mec1 nella DDR, per aumentare la conoscenza delle loro funzioni e così suggerire nuovi potenziali bersagli per la terapia del cancro. Inizialmente ci siamo concentrati su Tel1. Le cellule prive di TEL1 sono sensibili solo alla camptotecina (CPT), un inibitore della topoisomerasi I. Ci siamo occupati di cercare mutazioni che potessero aumentare o sopprimere questa sensibilità. Abbiamo scoperto che l'inattivazione della nucleasi Exo1 peggiora la sensibilità delle cellule tel1Δ alla CPT. Abbiamo dimostrato che questa ipersensibilità non è causata da un difetto nella riparazione dei DSB, ma da un difetto nel recupero della replicazione in presenza di CPT. Abbiamo ipotizzato che Exo1 sia necessario per generare estremità di DNA a singolo filamento, utili per riavviare la replicazione dopo il trattamento con CPT nelle cellule prive di Tel1. Per identificare mutazioni che sopprimano la sensibilità alla CPT nelle cellule tel1Δ exo1Δ, abbiamo effettuato uno screening genomico, trovando mutazioni nei geni che codificano per proteine coinvolte nella biogenesi dei cluster Fe-S. Queste inseriscono cluster Fe-S in proteine che regolano aspetti del metabolismo del DNA e i loro ortologhi umani sono mutati in vari tumori. Ciò suggerisce un legame tra la biogenesi dei cluster Fe-S e la stabilità del genoma, motivo per cui abbiamo deciso di indagare meglio la funzione di queste proteine nella DDR e la loro interazione con Tel1 e Mec1. Sebbene alcuni aspetti debbano ancora essere chiariti, questo lavoro ha gettato le basi per una migliore comprensione delle funzioni di Tel1 e Mec1 e delle loro interazioni con Exo1 e le proteine della biogenesi dei cluster Fe-S. Poiché queste proteine sono conservate anche nelle cellule umane, la conoscenza di questi processi è utile per suggerire nuove strategie terapeutiche. È importante ricordare inoltre che la stabilità genomica è un fattore cruciale anche nello sviluppo di processi industriali con i microrganismi. Ho partecipato anche a un progetto in cui Saccharomyces cerevisiae e altri lieviti sono stati utilizzati come cell factories. Abbiamo progettato un concetto di bioraffineria per valorizzare gli scarti alimentari e produrre nuovi materiali e sostanze proponendo così una soluzione a un problema ambientale attuale.
Research and characterisation of new genetic interactors of the key DNA damage response kinases Tel1/ATM and Mec1/ATR
FRIGERIO, CHIARA
2025
Abstract
The maintenance of genome stability is essential for every living organism to preserve and transmit the correct genetic information to the progeny. Every day in fact, cells are exposed to various DNA damaging agents that can cause nucleotide modifications, mismatches, double strand breaks (DSBs) and transcription and replication problems. Unrepaired DNA lesions lead to genome instability which has been found in cancer and genetic diseases. Cells counteract damages thanks to a multi-proteins pathway, known as the DNA damage response (DDR) that couples DNA repair with the activation of the DNA damage checkpoint. In Saccharomyces cerevisiae, the master regulators of this pathway are the kinases Tel1 and Mec1, ATM and ATR in human cells. They phosphorylate the effector kinases Rad53/CHK2 and Chk1, thus allowing the cell to slow down the cell-cycle progression to repair DNA damage. ATM and ATR are mutated in different types of tumours and genetic diseases. Moreover, nowadays ATM and ATR inhibitors are in clinical trials to be used as monotherapy or in combination with chemotherapy. Therefore, studying these two proteins is important to better understand their roles in the DDR and this could have also important implications in biomedical research. While Mec1 promotes checkpoint activation in the presence of long single-strand DNA stretches, Tel1 is recruited to DSBs where it promotes the processing (resection) of DNA ends and DSBs repair. Tel1 is involved in telomere homeostasis and replication fork maintenance after replication stress, even if this latter function is not totally clear. The aim of this project consists in identifying novel players that participate together with Tel1 and Mec1 in the DDR to increase the knowledge upon their function and to suggest new potential targets for cancer therapy. We initially focused on Tel1. Cells lacking TEL1 are sensitive only to the Top1-inhibitor camptothecin (CPT). We were interested in finding mutations that enhance or suppress this sensitivity. We have found that the inactivation of the nuclease Exo1 exacerbates tel1Δ cells sensitivity to CPT. We demonstrated that tel1Δ exo1Δ hypersensitivity is not caused by a defect in DSBs repair, but by a defect in the recovery of functional replication forks in the presence of CPT. We proposed that Exo1 is required to generate 3’-ended single-strand DNA tails that are necessary to restart replication after CPT poisoning in Tel1-deficient cells. In order to identify also mutations that suppress the CPT sensitivity of tel1Δ exo1Δ cells, we performed a genomic screening, and we have found loss of function mutations in genes that codify for proteins involved in Fe-S clusters biogenesis. They are able to insert Fe-S clusters in different proteins which regulate aspects of DNA metabolism. Their human orthologues are also mutated in different tumours. This suggests a link between Fe-S clusters biogenesis and genome stability and for this reason we decided to better investigate the function of these proteins in the DDR and their interplays with Tel1 and Mec1. Even if some aspects remain to be elucidated, this work has laid the groundwork for a better understanding of Tel1 and Mec1 functions and their interplays with Exo1 and Fe-S clusters biogenesis proteins. Since these proteins are conserved in human cells, the knowledge of these processes is useful to suggest new therapeutic strategies. In conclusion, it's important to remember that genome stability is a key factor to keep in mind even when developing industrial processes with microorganisms. I also participated in a project in which Saccharomyces cerevisiae and other yeasts were used as cell factories. We designed a zero-waste biorefinery concept to valorise food wastes and consequently produce new materials and chemicals. In this way we have proposed a solution for a current global environmental issue.I documenti in UNITESI sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
https://hdl.handle.net/20.500.14242/193027
URN:NBN:IT:UNIMIB-193027