Eukaryotic cells prevent genomic instability by activating a complex network of safeguard pathways called DNA Damage Response (DDR). S. cerevisiae Mec1 and Tel1 protein kinases, orthologs of human ATR and ATM, play a central role in the DDR. These proteins activate a checkpoint cascade which coordinates DNA damage repair with cell cycle progression. The role of Tel1 is particularly evident in the presence of DNA Double-Strand Breaks (DSBs), one of the most cytotoxic forms of DNA lesions. DSBs can be repaired by Homologous Recombination (HR), which requires the degradation of 5’-ended strands of the break (resection). Tel1 contributes to DSB repair by promoting resection initiation. Despite Tel1 functions in DDR, the absence of Tel1 confers a moderate sensitivity to camptothecin (CPT), an inhibitor of type I DNA topoisomerases. Since CPT derivatives are currently used in chemotherapy, understanding the molecular basis of tel1Δ mutant sensitivity to CPT is relevant for the development of anti-cancer therapies based on combined treatments with CPT derivatives and ATM inhibitors. In addition, Tel1 is important for the maintenance of telomeres, which are replicated by a reverse transcriptase called telomerase. In particular, Tel1 promotes the recruitment of telomerase and therefore telomere homeostasis. Telomerase is inactivated in most human tissues, which undergo progressive telomere shortening. When telomeres become critically short, a block of cell division, known as replicative senescence, limits cell proliferation, thus acting as a cancer-suppressor mechanism. Senescence is triggered by the activation of a checkpoint response governed by Mec1/ATR and Tel1/ATM. While Mec1/ATR is known to block cell division in the presence of extended ssDNA, the molecular mechanism by which Tel1/ATM triggers senescence is still unclear. During my PhD I have managed two different projects with the aim to shed light into the molecular mechanisms that involve Tel1 in response to CPT and in the induction of replicative senescence. Regarding the first project, in both yeast and mammals, CPT induces replication fork reversal, which has been proposed to stabilize stalled replication forks, thus providing time for the repair of CPT-induced lesions and supporting replication restart. tel1∆ cells have a reduced amount of CPT-induced reversed forks compared to wild type cells. The lack of Mre11 nuclease activity restores wild-type levels of reversed forks in CPT-treated tel1Δ cells, without affecting fork reversal in wild-type cells. Moreover, Mrc1 inactivation prevents fork reversal in wild-type, tel1Δ, and mre11 nuclease-deficient cells and relieves the hypersensitivity of tel1Δ cells to CPT. Altogether, these data indicate that Tel1 stabilizes Mrc1-dependent reversed forks generated in the presence of CPT by counteracting Mre11 nucleolytic activity at these structures. Regarding the second project, to studying the role of Tel1/ATM in the induction of senescence, I took advantage of telomerase-deficient yeast cells, which are considered a reliable model of replicative senescence, and the TEL1-hy184 allele, previously identified because it was able to suppress the checkpoint defects of Mec1-deficient cells. Upon telomerase inactivation, Tel1-hy184 accelerates senescence compared to wild type Tel1, while the lack of Tel1 was found to delay senescence. The enhanced senescence in telomerase-negative TEL1-hy184 cells depends on the activation of a checkpoint that is completely Rad9-dependent and only partially dependent on Mec1. Furthermore, Tel1-hy184 does not appear to increase ssDNA at DNA ends, suggesting that Tel1 induces replicative senescence by directly contributing to checkpoint signaling at dysfunctional telomeres. Taken together, the results that I have obtained during my PhD allow to better understand the functions of Tel1/ATM in the maintenance of genome stability.

Le cellule eucariotiche prevengono l’instabilità genomica attivando un complesso sistema biochimico chiamato Risposta ai Danni al DNA (DDR). Le proteine chinasi Mec1 e Tel1 di S. cerevisiae, ortologhe di ATR ed ATM umane, giocano un ruolo centrale nella DDR. Queste proteine attivano il checkpoint da danni al DNA il quale coordina la riparazione dei danni al DNA con la progressione del ciclo cellulare. Il ruolo della proteina Tel1 è particolarmente evidente in presenza di rotture a doppia elica del DNA (DSB). I DSB possono essere riparati tramite ricombinazione omologa la quale richiede la degradazione dell’estremità 5’ della rottura (resection). Tel1 contribuisce alla riparazione dei DSB promuovendo l’inizio della resection. Nonostante le funzioni di Tel1 nella DDR, l’assenza di Tel1 conferisce alle cellule di lievito solo una moderata sensibilità alla camptotecina (CPT), un inibitore delle topoisomerasi di tipo I. Dato che derivati della CPT sono attualmente usati in chemioterapia, comprendere il ruolo di Tel1 in risposta alla CPT è rilevante per lo sviluppo di nuove terapie anticancro. Oltre a ciò, Tel1 è importante per il mantenimento dei telomeri, i quali vengono replicati grazie ad una trascrittasi inversa chiamata telomerasi. In particolare, Tel1 promuove il reclutamento della telomerasi e quindi l’omeostasi dei telomeri. La telomerasi è inattivata nella maggior parte dei tessuti umani che di conseguenza sono soggetti ad un progressivo accorciamento dei telomeri. Quando i telomeri divengono criticamente corti, si ha il blocco della divisione cellulare in un processo noto come senescenza replicativa che limita la proliferazione cellulare agendo da meccanismo oncosoppressore. La senescenza è innescata dall’attivazione del checkpoint da danni al DNA governato da Mec1/ATR e Tel1/ATM. In particolare, il meccanismo attraverso il quale Tel1/ATM induce la senescenza è ancora ignoto. Durante il mio dottorato ho quindi seguito due distinti progetti allo scopo di far luce sui meccanismi molecolari che coinvolgono Tel1 in risposta alla CPT e nell’induzione della senescenza. Riguardo al primo progetto, sia in lievito che in mammifero la CPT induce la reversione delle forche replicative. Cellule tel1∆ sono caratterizzate da un ridotto livello di forche regresse indotte dalla CPT rispetto a cellule selvatiche. In risposta alla CPT, l’assenza dell’attività nucleasica di Mre11 ripristina livelli normali di forche regresse in cellule tel1∆. Inoltre, l’inattivazione della proteina Mrc1 mitiga la sensibilità a CPT di cellule tel1∆ e previene la reversione delle forche in cellule selvatiche, in cellule tel1∆ e in cellule senza l’attività nucleasica di Mre11. Nel loro insieme questi risultati indicano che Tel1 stabilizza le forche regresse generate da Mrc1 in presenza della CPT inibendo l’attività nucleasica di Mre11 a livello di queste strutture di DNA. Riguardo al secondo progetto, per studiare il ruolo di Tel1 nell’induzione della senescenza ho sfruttato cellule di lievito senza telomerasi e l’allele mutante TEL1-hy184 identificato precedentemente come soppressore dei difetti di checkpoint di cellule mec1∆. Subito dopo l’inattivazione della telomerasi la variante Tel1-hy184 accelera la senescenza rispetto a cellule che esprimono la forma selvatica di Tel1. L’aumentata senescenza indotta da Tel1-hy184 è causata dall’attivazione di un checkpoint completamente dipendente dalla proteina Rad9 e solo in parte dipendente da Mec1. Inoltre, Tel1-hy184 non sembra aumentare il livello di ssDNA alle estremità di DNA. Ciò suggerisce che Tel1 induce la senescenza replicativa contribuendo direttamente all’attivazione del checkpoint in presenza di telomeri disfunzionali. Nel complesso, i risultati che ho ottenuto durante il mio dottorato permettono di comprendere meglio le funzioni di Tel1/ATM nel mantenimento della stabilità genomica.

(2019). Role of Tel1/ATM in protecting and signaling abnormal replication forks and short telomeres. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2019).

Role of Tel1/ATM in protecting and signaling abnormal replication forks and short telomeres

MENIN, LUCA
2019

Abstract

Eukaryotic cells prevent genomic instability by activating a complex network of safeguard pathways called DNA Damage Response (DDR). S. cerevisiae Mec1 and Tel1 protein kinases, orthologs of human ATR and ATM, play a central role in the DDR. These proteins activate a checkpoint cascade which coordinates DNA damage repair with cell cycle progression. The role of Tel1 is particularly evident in the presence of DNA Double-Strand Breaks (DSBs), one of the most cytotoxic forms of DNA lesions. DSBs can be repaired by Homologous Recombination (HR), which requires the degradation of 5’-ended strands of the break (resection). Tel1 contributes to DSB repair by promoting resection initiation. Despite Tel1 functions in DDR, the absence of Tel1 confers a moderate sensitivity to camptothecin (CPT), an inhibitor of type I DNA topoisomerases. Since CPT derivatives are currently used in chemotherapy, understanding the molecular basis of tel1Δ mutant sensitivity to CPT is relevant for the development of anti-cancer therapies based on combined treatments with CPT derivatives and ATM inhibitors. In addition, Tel1 is important for the maintenance of telomeres, which are replicated by a reverse transcriptase called telomerase. In particular, Tel1 promotes the recruitment of telomerase and therefore telomere homeostasis. Telomerase is inactivated in most human tissues, which undergo progressive telomere shortening. When telomeres become critically short, a block of cell division, known as replicative senescence, limits cell proliferation, thus acting as a cancer-suppressor mechanism. Senescence is triggered by the activation of a checkpoint response governed by Mec1/ATR and Tel1/ATM. While Mec1/ATR is known to block cell division in the presence of extended ssDNA, the molecular mechanism by which Tel1/ATM triggers senescence is still unclear. During my PhD I have managed two different projects with the aim to shed light into the molecular mechanisms that involve Tel1 in response to CPT and in the induction of replicative senescence. Regarding the first project, in both yeast and mammals, CPT induces replication fork reversal, which has been proposed to stabilize stalled replication forks, thus providing time for the repair of CPT-induced lesions and supporting replication restart. tel1∆ cells have a reduced amount of CPT-induced reversed forks compared to wild type cells. The lack of Mre11 nuclease activity restores wild-type levels of reversed forks in CPT-treated tel1Δ cells, without affecting fork reversal in wild-type cells. Moreover, Mrc1 inactivation prevents fork reversal in wild-type, tel1Δ, and mre11 nuclease-deficient cells and relieves the hypersensitivity of tel1Δ cells to CPT. Altogether, these data indicate that Tel1 stabilizes Mrc1-dependent reversed forks generated in the presence of CPT by counteracting Mre11 nucleolytic activity at these structures. Regarding the second project, to studying the role of Tel1/ATM in the induction of senescence, I took advantage of telomerase-deficient yeast cells, which are considered a reliable model of replicative senescence, and the TEL1-hy184 allele, previously identified because it was able to suppress the checkpoint defects of Mec1-deficient cells. Upon telomerase inactivation, Tel1-hy184 accelerates senescence compared to wild type Tel1, while the lack of Tel1 was found to delay senescence. The enhanced senescence in telomerase-negative TEL1-hy184 cells depends on the activation of a checkpoint that is completely Rad9-dependent and only partially dependent on Mec1. Furthermore, Tel1-hy184 does not appear to increase ssDNA at DNA ends, suggesting that Tel1 induces replicative senescence by directly contributing to checkpoint signaling at dysfunctional telomeres. Taken together, the results that I have obtained during my PhD allow to better understand the functions of Tel1/ATM in the maintenance of genome stability.
CLERICI, MICHELA
reversione forche; camptotecina; Tel1; telomero; senescenza
fork reversal; camptothecin; Tel1; telomere; senescenza
BIO/18 - GENETICA
English
8-feb-2019
BIOLOGIA E BIOTECNOLOGIE - 93R
31
2017/2018
open
(2019). Role of Tel1/ATM in protecting and signaling abnormal replication forks and short telomeres. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2019).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/241165
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