Genomic integrity is threatened by DNA damage that, if not properly repaired, can be converted into mutations, whose accumulation leads to genomic instability, one of the hallmarks of cancer. Eukaryotic cells deal with DNA damage by activating DNA damage response. DNA double strand breaks (DSBs) are among the most dangerous DNA lesions. In Saccharomyces cerevisiae, DSBs are mainly repaired by Homologous Recombination (HR), which exploits a homologous sequence as a template to repair the damage. HR requires the DSB ends to be nucleolytically degraded in order to generate single-strand DNA (ssDNA) tails, in a process known as DSB end resection. Resection initiates with an endonucleolytic cleavage by the MRX complex together with Sae2, while resection extension is carried out by the nucleases Exo1 and Dna2. DNA damage checkpoint is a signal transduction cascade that halts the cell cycle in order to give cells sufficient time to repair the damage. In S. cerevisiae, DNA damage checkpoint is activated by the kinases Tel1 and Mec1, orthologues of human ATM and ATR. Once activated, Mec1 and Tel1 phosphorylate different substrates including the adaptor Rad9 and the effector kinase Rad53, which allow signal amplification. Both DNA end resection and DNA damage checkpoint must be finely regulated to ensure efficient DSB repair, avoiding excessive ssDNA generation, and to properly coordinate repair with cell cycle progression. In this PhD thesis, we provide evidences of a new level of resection regulation, based on the modulation of Exo1 amount by the RNA-binding protein (RBP) Npl3. We have also studied the role of Sae2 in DNA damage repair and checkpoint activation. Npl3 is a S. cerevisiae RBP, which plays a central role in RNA metabolism and is highly conserved from yeast to humans. Since emerging evidences support strong connections between RNA metabolism and genome integrity, we investigated if Npl3 was involved in DSB response. We demonstrated that the absence of Npl3 impairs the generation of long ssDNA tails at DSB ends. In particular, Npl3 promotes resection extension by acting in the same pathway of Exo1. Moreover, both the lack of Npl3 and the inactivation of its RNA-binding domains cause the reduction of Exo1 protein level. So, Npl3 promotes resection extension by regulating EXO1 at the RNA level. Indeed, we proved that the decrease of Exo1 level is due to the presence of not properly terminated EXO1 RNA species. These findings, together with the observation that EXO1 overexpression partially suppresses the resection defect of npl3Δ cells, suggest that Npl3 participates in DSB end resection regulation by promoting the proper biogenesis of EXO1 mRNA. Concerning the second PhD project, Sae2 promotes MRX endonucleolytic activity during resection and negatively regulates Tel1-dependent checkpoint response. Indeed, Sae2 limits MRX accumulation at the damage site, thus reducing Tel1 recruitment and its signalling activity. How Sae2 functions in supporting DNA damage resistance and in inhibiting the DNA damage checkpoint are connected is still unclear. From a genetic screen, we identified the sae2-ms mutant that, similarly to Sae2 absence, upregulates Tel1 signalling activity by increasing both MRX and Tel1 recruitment to the DSBs. However, unlike SAE2 deletion, Sae2-ms does not cause any resection or tethering defect, nor any sensitivity to genotoxic agents. Moreover, Sae2-ms induces Tel1 but not Rad53 hyperactivation. Indeed Sae2 absence, but not Sae2-ms presence, increases Rad53-Rad9 interaction. These data indicate that Sae2 regulates checkpoint activation both by controlling MRX removal from the DSBs and by limiting Rad53-Rad9 interaction and that Rad53 downregulation is the main responsible for Sae2-promoted DNA damage resistance. Altogether, our results allow to better understand the molecular mechanisms involved in the control of DNA damage response processes.
L’integrità genomica è minacciata da danni al DNA che, se non adeguatamente riparati, si convertono in mutazioni, il cui accumulo causa instabilità genomica, una tipica caratteristica tumorale. Le cellule eucariotiche reagiscono ai danni attivando la risposta ai danni al DNA. Le rotture a doppia elica del DNA (DSB) sono tra i danni più pericolosi. In Saccharomyces cerevisiae i DSB sono principalmente riparati tramite ricombinazione omologa (HR), che sfrutta sequenze omologhe come stampo per riparare il danno. La HR necessita il processamento nucleolitico (resection) delle estremità del DSB così da generare code di DNA a singolo filamento (ssDNA). La resection inizia con un taglio endonucleolitico da parte del complesso MRX insieme a Sae2, mentre l’estensione della resection è eseguita dalle nucleasi Exo1 e Dna2. Il checkpoint da danno al DNA è una cascata di trasduzione del segnale che blocca il ciclo cellulare così che le cellule abbiano tempo sufficiente per riparare il danno. In S. cerevisiae il checkpoint è attivato dalle chinasi Tel1 e Mec1, ortologhe di ATM e ATR umane. Una volta attivate, Mec1 e Tel1 fosforilano diversi substrati, tra cui l’adattatore Rad9 e la chinasi effettrice Rad53, che amplificano il segnale. Sia la resection che il checkpoint devono essere finemente regolati per garantire una riparazione efficiente dei DSB, evitando di generare troppo ssDNA, e per coordinare la riparazione con la progressione del ciclo. In questa tesi di dottorato, abbiamo dimostrato un nuovo livello di regolazione della resection, basato sul controllo della quantità di Exo1 da parte della proteina che lega l’RNA (RBP) Npl3. Inoltre, abbiamo studiato il ruolo di Sae2 nella riparazione dei danni e nell’attivazione del checkpoint. Npl3 svolge un ruolo chiave nel metabolismo degli RNA ed è molto conservata nell’uomo. Poiché studi recenti mostrano forti connessioni tra metabolismo degli RNA e mantenimento dell’integrità genomica, abbiamo verificato se Npl3 fosse coinvolta nella risposta ai DSB. Abbiamo dimostrato che l’assenza di Npl3 provoca difetti nel processamento delle estremità del DSB. In particolare, Npl3 promuove la resection estesa, agendo nello stesso pathway di Exo1. Inoltre, sia l’assenza di Npl3 che l’inattivazione dei suoi domini di legame all’RNA causano una riduzione del livello di Exo1. Quindi, Npl3 promuove la resection estesa regolando EXO1 a livello dell’RNA. Infatti, in assenza di Npl3, abbiamo dimostrato la presenza di molecole di RNA di EXO1 non correttamente terminate. Questi dati, oltre al fatto che l’overespressione di EXO1 sopprime parzialmente il difetto di resection di cellule npl3Δ, suggeriscono che Npl3 partecipi alla regolazione della resection promuovendo la corretta biogenesi dell’mRNA di EXO1. Riguardo al secondo progetto, Sae2 promuove l’attività endonucleasica di MRX durante la resection e regola negativamente il checkpoint Tel1-dipendente. Infatti, Sae2 limita l’accumulo di MRX alla lesione, riducendo sia il reclutamento che l’attività di segnalazione di Tel1. Non è ancora chiaro come le funzioni di Sae2 nel promuovere la resistenza ai danni e nell’inibire il checkpoint siano collegate. Tramite screening genetico, abbiamo identificato il mutante sae2-ms che, come accade in assenza di Sae2, iperattiva il checkpoint Tel1-dipendente, aumentando il reclutamento ai DSB sia di MRX che di Tel1. A differenza della delezione di Sae2, Sae2-ms non causa difetti di resection né di tethering, e non provoca sensibilità agli agenti genotossici. Inoltre, Sae2-ms provoca iperattivazione di Tel1, ma non di Rad53. Infatti, l’assenza di Sae2, ma non la presenza di Sae2-ms, aumenta l’interazione tra Rad53 e Rad9. Questi dati dimostrano che Sae2 regola il checkpoint sia controllando la rimozione di MRX dai DSB che limitando l’interazione Rad53-Rad9, e che l’inibizione di Rad53 è la principale responsabile della resistenza ai danni promossa da Sae2.
(2019). New insights into the regulation of DNA end processing and DNA damage checkpoint. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2019).
New insights into the regulation of DNA end processing and DNA damage checkpoint
COLOMBO, CHIARA VITTORIA
2019
Abstract
Genomic integrity is threatened by DNA damage that, if not properly repaired, can be converted into mutations, whose accumulation leads to genomic instability, one of the hallmarks of cancer. Eukaryotic cells deal with DNA damage by activating DNA damage response. DNA double strand breaks (DSBs) are among the most dangerous DNA lesions. In Saccharomyces cerevisiae, DSBs are mainly repaired by Homologous Recombination (HR), which exploits a homologous sequence as a template to repair the damage. HR requires the DSB ends to be nucleolytically degraded in order to generate single-strand DNA (ssDNA) tails, in a process known as DSB end resection. Resection initiates with an endonucleolytic cleavage by the MRX complex together with Sae2, while resection extension is carried out by the nucleases Exo1 and Dna2. DNA damage checkpoint is a signal transduction cascade that halts the cell cycle in order to give cells sufficient time to repair the damage. In S. cerevisiae, DNA damage checkpoint is activated by the kinases Tel1 and Mec1, orthologues of human ATM and ATR. Once activated, Mec1 and Tel1 phosphorylate different substrates including the adaptor Rad9 and the effector kinase Rad53, which allow signal amplification. Both DNA end resection and DNA damage checkpoint must be finely regulated to ensure efficient DSB repair, avoiding excessive ssDNA generation, and to properly coordinate repair with cell cycle progression. In this PhD thesis, we provide evidences of a new level of resection regulation, based on the modulation of Exo1 amount by the RNA-binding protein (RBP) Npl3. We have also studied the role of Sae2 in DNA damage repair and checkpoint activation. Npl3 is a S. cerevisiae RBP, which plays a central role in RNA metabolism and is highly conserved from yeast to humans. Since emerging evidences support strong connections between RNA metabolism and genome integrity, we investigated if Npl3 was involved in DSB response. We demonstrated that the absence of Npl3 impairs the generation of long ssDNA tails at DSB ends. In particular, Npl3 promotes resection extension by acting in the same pathway of Exo1. Moreover, both the lack of Npl3 and the inactivation of its RNA-binding domains cause the reduction of Exo1 protein level. So, Npl3 promotes resection extension by regulating EXO1 at the RNA level. Indeed, we proved that the decrease of Exo1 level is due to the presence of not properly terminated EXO1 RNA species. These findings, together with the observation that EXO1 overexpression partially suppresses the resection defect of npl3Δ cells, suggest that Npl3 participates in DSB end resection regulation by promoting the proper biogenesis of EXO1 mRNA. Concerning the second PhD project, Sae2 promotes MRX endonucleolytic activity during resection and negatively regulates Tel1-dependent checkpoint response. Indeed, Sae2 limits MRX accumulation at the damage site, thus reducing Tel1 recruitment and its signalling activity. How Sae2 functions in supporting DNA damage resistance and in inhibiting the DNA damage checkpoint are connected is still unclear. From a genetic screen, we identified the sae2-ms mutant that, similarly to Sae2 absence, upregulates Tel1 signalling activity by increasing both MRX and Tel1 recruitment to the DSBs. However, unlike SAE2 deletion, Sae2-ms does not cause any resection or tethering defect, nor any sensitivity to genotoxic agents. Moreover, Sae2-ms induces Tel1 but not Rad53 hyperactivation. Indeed Sae2 absence, but not Sae2-ms presence, increases Rad53-Rad9 interaction. These data indicate that Sae2 regulates checkpoint activation both by controlling MRX removal from the DSBs and by limiting Rad53-Rad9 interaction and that Rad53 downregulation is the main responsible for Sae2-promoted DNA damage resistance. Altogether, our results allow to better understand the molecular mechanisms involved in the control of DNA damage response processes.File | Dimensione | Formato | |
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