Every living organism is programmed to reproduce and to pass genetic information to descendants. The information has to be carefully copied and accurately transferred to the next generation.  Therefore organisms have developed the network of conserved mechanisms to survey the protection and precise transfer of the genetic information. Such mechanisms are called checkpoints and they monitor the correct execution of different cell programs. The DNA damage and the replication blocks are surveyed by the conserved Mec1-Rad53 (human ATM/ATR and Chk2, respectively) protein kinase cascade. Mec1 and Rad53 are essential for survival and when activated orchestrate the multiple cellular responses, including the activation of the ribonucleotide reductase (RNR), to the genotoxic stress. RNR is an enzyme producing all four dNTPs - the building blocks of the DNA - and is instrumental for the maintenance both proper concentration and balance of each of dNTPs. The appropriate concentration of the dNTPs should be strictly regulated since inadequate dNTP production can impede many cellular processes and lead to higher mutation rates and genome instability. Hence RNR activity is regulated at many levels, including allosteric and transcriptional regulation and the inhibition at protein level. In our research, we addressed the question of the transcriptional regulation of RNR and the consequences of dNTP malproduction in the terms of the genomic stability. In yeast S. cerevisiae, four genes encode RNR: 2 genes encode a large subunit (RNR1 and RNR3) and 2 genes encode a small subunit (RNR2 and RNR4). All 4 genes are DNA-damage inducible: transcription of RNR2, RNR3 and RNR4 is regulated via Mec1-Rad53-Dun1 pathway by targeting the transcriptional repressor Crt1 (Rfx1) for degradation; on the contrary, RNR1 gene promoter does not contain Crt1-binding sites and is not regulated through the Mec1-Rad53-Dun1 pathway. Instead, we show that intrastrand cross (X)-link recognition protein (Ixr1) is required for the proper transcription of the RNR1 gene and maintenance of the dNTP pools both during unperturbed cell cycle and after the DNA damage. Thus, we identify the novel regulator of the RNR1 transcription. Next, we show that the depletion of dNTP pools negatively affects genome stability in the hypomorphic mec1 mutants: the hyper-recombination phenotype in those mutants correlates with low dNTP levels. By introducing even lower dNTP levels the hyper-recombination increased even further and conversely all the hyper-recombination phenotypes were suppressed by artificial elevation of dNTP levels. In conclusion, we present Ixr1 as a novel regulator of the RNR activity and provide the evidence of role of dNTP concentration in the genome stability.

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No description available.

From microscopic bacteria to the giant whale, every single living organism on Earth uses the same language of life: DNA. Deoxyribonucleoside triphosphates––dNTPs (dATP, dTTP, dGTP, and dCTP)––are the building blocks of DNA and are therefore the “alphabet of life”. A balanced supply of dNTPs is essential for integral DNA transactions such as faithful genome duplication and repair. The enzyme ribonucleotide reductase (RNR) not only synthesizes all four dNTPs but also primarily maintains the crucial individual concentration of each dNTP in a cell. In this thesis we investigated what happens if the crucial balanced supply of dNTPs is disrupted, addressing whether a cell has a mechanism to detect imbalanced dNTP pools and whether all pool imbalances are equally mutagenic. To address these questions, we introduced single amino acid substitutions into loop 2 of the allosteric specificity site of Saccharomyces cerevisiae RNR and obtained a collection of yeast strains with different but defined dNTP pool imbalances. These results directly confirmed that the loop 2 is the structural link between the substrate specificity and effector binding sites of RNR. We were surprised to observe that mutagenesis was enhanced even in a strain with mildly imbalanced dNTP pools, despite the availability of the two major replication error correction mechanisms: proofreading and mismatch repair. However, the mutagenic potential of different dNTP pool imbalances did not directly correlate with their severity, and the locations of the mutations in a strain with elevated dTTP and dCTP were completely different from those in a strain with elevated dATP and dGTP. We then investigated, whether dNTP pool imbalances interfere with cell cycle progression and if they are detected by the S-phase checkpoint, a genome surveillance mechanism activated in response to DNA damage or replication blocks. The S-phase checkpoint was activated by the depletion of one or more dNTPs. In contrast, when none of the dNTP pools was limiting for DNA replication, even extreme and mutagenic dNTP pool imbalances did not activate the S-phase checkpoint and did not interfere with the cell cycle progression. We also observed an interesting mutational strand bias in one of the mutant rnr1 strains suggesting that the S-phase checkpoint may selectively prevent formation of replication errors during leading strand replication. We further used these strains to study the mechanisms by which dNTP pool imbalances induce genome instability. In addition, we discovered that a high dNTP concentration allows replicative DNA polymerases to bypass certain DNA lesions, which are difficult to bypass at normal dNTP concentrations. Our results broaden the role of dNTPs beyond ‘dNTPs as the building blocks’ and suggest that dNTPs are not only the building blocks of DNA but also that their concentrations in a cell have regulatory implications for maintaining genomic integrity. This is important as all cancers arise as a result of some kind of genomic abnormality.

L'environnement des organismes vivants est par définition fluctuant, toutes variations aléatoires du milieu de vie constituent un stress pour les cellules. Au fil de l'évolution, une forte pression de sélection a façonné le fonctionnement cellulaire jusqu'aux réponses complexes élaborées par les organismes vivants. Mes travaux s'inscrivent autour des mécanismes moléculaires de la réponse au stress et plus particulièrement les stress génotoxiques. La première partie de l'étude décrit finement la réplication de l'ADN en condition de stress réplicatif. Ainsi, nous avons montré que les pools de dNTPs sont limitants pour la progression des fourches de réplication en phase S normale et en stress, et que leurs niveaux conditionnent le programme temporel de réplication. De plus, nous avons mis en évidence un mécanisme d'adaptation au stress réplicatif et aux dommages constitutifs dans des mutants caractérisés par de l'instabilité génétique (CIN) via l'activation du checkpoint de dommage conduisant à l'expansion des pools de dNTPs. Pour finir, nous montrons que l'augmentation des niveaux de dNTPs facilite la réplication en présence de lésions de l'ADN, d'une manière indépendante des ADN polymérases translésionnelles. Le second projet apporte de nouveaux éléments sur le rôle de Crt10 in vivo, préalablement identifié comme un régulateur transcriptionnel des gènes de la Ribonucléotide Réductase (RNR). Nos données indiquent que les mutants crt10Δ ont des niveaux de dNTP similaires à ceux des cellules sauvages, et que cette mutation a un très faible impact sur l'expression des gènes RNR, malgré un phénotype de vitesse de progression des fourches accrue. Nous montrons que le mutant crt10Δ est caractérisé par un défaut d'entrée en phase S et d'initiation des origines de réplication. L'origine de ce défaut pourrait résider dans les fonctions de Crt10 impliquant la régulation de la biosynthèse des ribosomes au sein du complexe Rtt101-Mms1. Le troisième projet identifie MRX (Mre11-Rad50-Xrs2) comme un acteur de la voie de terminaison des ARN non codants. MRX s'associe à des loci recrutant également le complexe de terminaison Nrd1-Nab3-Sen1 à l'échelle du génome entier. L'inactivation de RAD50 se traduit par une perte d'efficacité de terminaison et l'accumulation de transcrits bicistroniques, ainsi qu'une dérégulation du niveau d'ARNs non codants instables (CUT) et de leurs gènes associés. Tout comme Sen1, MRX pourrait intervenir dans la résolution des collisions entre les machineries de transcription et de réplication. / A fluctuating environment is a powerful mean of selection for living organisms, which evolved complex signaling networks to integrate these variations and direct swift and efficient cellular responses. The aim of my work is the identification and characterization of molecular mechanisms involved in the tolerance of replicative stress and DNA damage. First, we show that changes in dNTP pools affect several aspects of replication dynamics in budding yeast. dNTP levels are limiting for normal S-phase progression and determine the temporal program of replication during a replicative stress. Interestingly, we also observed that chromosomal instability (CIN) mutants display expanded dNTP pools due to the constitutive activation of the DNA damage checkpoint. Since increased dNTP levels promote forks progression in the presence of DNA lesions, we propose that CIN mutants adapt to chronic replicative stress by upregulating dNTP pools. Secondly, we bring new lights on the role of Crt10 in vivo, which has been initially identified as a negative regulator of Ribonucleotide Reductase (RNR) genes expression. Deletion of CRT10 neither leads to expanded dNTP pools, nor to a massive deregulation of RNR genes, although crt10Δ cells exhibit faster fork progression. The crt10Δ mutant accumulates at the G1/S transition and exhibits a strong defect of origin firing that could account for its replication phenotype. Moreover, we observed a global decrease in ribosome biogenesis in crt10Δ. The physical interaction of Crt10 with several members of the ribosome biogenesis pathway and its role in the Rtt101-Mms1 complex suggest that Crt10 may regulate ribosome levels in vivo. At last, we identified MRX (Mre11-Rad50-Xrs2) as a bona fide member of the transcription termination of non-coding RNA (ncRNA). ChIP-seq reveals that MRX localized at the same loci than the Nrd1-Nab3-Sen1 complex in vegetative growth. rad50Δ cells exhibit transcriptional read-through and upregulation of unstable cryptic transcripts (CUTs) leading to a misregulation of their associated gene. Finally, MRX seems to be involved in the resolution of branched structures emanating from collision between transcription and replication machineries, as it is the case for Sen1.

A prerequisite for a multicellular organism to survive is the ability to correctly replicate and repair DNA while minimizing the number of heritable mutations. To achieve this, cells need a balanced supply of deoxyribonucleoside triphosphates (dNTPs), the precursors for DNA synthesis. The rate-limiting step in de novo biosynthesis of dNTPs is catalyzed by the enzyme ribonucleotide reductase (RNR). The classic eukaryotic RNR enzyme consists of a large and a small subunit. Together, these subunits form a heterotetrameric RNR complex. The larger subunit harbours active sites whereas the smaller subunit contains a stable tyrosyl free radical. Both subunits are required for RNR activity. Since failure to correctly regulate de novo dNTP biosynthesis can lead to misincorporation of nucleotides into DNA, genetic abnormalities and cell death, RNR activity is tightly regulated. The regulation of RNR activity involves cell cycle-specific expression and degradation of the RNR proteins, as well as binding of allosteric effectors to the large RNR subunit. In this thesis, in vitro assays based on purified recombinant RNR proteins, in combination with in vivo assays, have been used successfully to study the regulation of RNR activity in response to DNA damage. I present new findings regarding the function of an alternative mammalian RNR small subunit, and on the role of a small RNR inhibitor protein of fission yeast, during normal growth and after DNA damage. I also show conclusively that there are fundamental differences in the regulation of dNTP biosynthesis between the cells of higher and lower eukaryotes after DNA damage.

To transfer the information in the genome from mother cell to daughter cell, the DNA replication must be carried out only once and with very high fidelity prior to every cell division. In yeast there are several different DNA polymerases involved in DNA replication and/or DNA repair. The two replicative DNA polymerases, DNA polymerase delta (Pol delta) and DNA polymerase epsilon (Pol epsilon), which both include a proofreading 3´→5´exonuclease activity, can replicate and proofread the genome with a very high degree of accuracy. The aim of this thesis was to gain a better understanding of how the enigmatic DNA polymerase epsilon participates in DNA transactions. To investigate whether Pol epsilon or Pol delta is responsible for the synthesis of DNA on the lagging strand, the processing and assembly of Okazaki fragments was studied. Pol delta was found to have a unique property called “idling” which, together with the flap-endonuclease (FEN1), maintained a ligatable nick for DNA ligase I. In contrast, Pol epsilon was found to lack the ability to “idle” and interact functionally with FEN-1, indicating that Pol epsilon is not involved in processing Okazaki fragments. Together with previous genetic studies, it was concluded that Pol delta is the preferred lagging strand polymerase, leaving Pol epsilon to carry out some other function. The structure of Pol epsilon was determined by cryo-electron microscopy, to a resolution of ~20 Å. Pol epsilon is composed of a globular “head” domain consisting of the large catalytic subunit Pol2p, and a “tail” domain, consisting of the small subunits Dpb2p, Dpb3p, and Dpb4p. The two separable domains were found to be connected by a flexible hinge. Interestingly, the high intrinsic processivity of Pol epsilon depends on the interaction between the tail domain and double-stranded DNA. As a replicative DNA polymerase, Pol epsilon encounters different lesions in DNA. It was shown that Pol epsilon can perform translesion synthesis (TLS) through a model abasic site in the absence of external processivity clamps under single-hit conditions. The lesion bypass was dependent of the sequence on the template and also on a proper interaction of the “tail”domain with the primer-template. Yeast cells treated with a DNA damaging agent and devoid of all TLS polymerases showed improved survival rates in the presence of elevated levels of dNTPs. These genetic results suggested that replicative polymerases may be engaged in the bypass of some DNA lesions. In vitro, Pol epsilon was found to bypass 8-OxoG at elevated dNTP levels. Together, the in vitro and in vivo results suggest that the replicative polymerases may be engaged in bypass of less bulky DNA lesions at elevated dNTP levels. In conclusion, the low-resolution structure presented represents the first structural characterization of a eukaryotic multi-subunit DNA polymerase. The replicative DNA polymerase Pol epsilon can perform translesion synthesis due to an interaction between the tail domain and double-stranded DNA. Pol epsilon may also bypass less bulky DNA lesions when there are elevated dNTP concentrations in vivo.

In order to build any structure, a good supply of materials, accurate workers and quality control are needed. This is even the case when constructing DNA, the so-called “Code of Life.” For a species to continue to exist, this DNA code must be copied with incredibly high accuracy when each and every cell replicates. In fact, just one mistake in the 12 million bases that comprise the genome of budding yeast, Saccharomyces cerevisiae, can be fatal. DNA is composed of a double strand helix made up of just four different bases repeated millions of times. The building blocks of DNA are the deoxyribonucleotides (dNTPs); dCTP, dTTP, dATP and dGTP. Their production and balance are carefully controlled within each cell, largely by the key enzyme Ribonucleotide Reductase (RNR). Here, we studied how the enzymes that copy DNA, the replicative polymerases α, δ and ε, cope with the effects of an altered dNTP pool balance. An introduced mutation in the allosteric specificity site of RNR in a strain of S. cerevisiae, rnr1-Y285A, leads to elevated dCTP and dTTP levels and has been shown to have a 14-fold increase in mutation rate compared to wild type. To ascertain the full effects of the dNTP pool imbalance upon the replicative polymerases, we disabled one of the major quality control systems in a cell that corrects replication errors, the post-replicative Mismatch Repair system. Using both the CAN1 reporter assay and whole genome sequencing, we found that, despite inherent differences between the polymerases, their replication fidelity was affected very similarly by this dNTP pool imbalance. Hence, the high dCTP and dTTP forced Pol ε and Pol α/δ to make the same mistakes. In addition, the mismatch repair machinery was found to correct replication errors driven by this dNTP pool imbalance with highly variable efficiencies. Another mechanism to protect cells from DNA damage during replication is a checkpoint that can be activated to delay the cell cycle and activate repair mechanisms. In yeast, Mec1 and Rad53 (human ATR and Chk1/Chk2) are two key S-phase checkpoint proteins. They are essential as they are also required for normal DNA replication and dNTP pool regulation. However the reason why they are essential is not well understood. We investigated this by mutating RAD53 and analyzing dNTP pools and gene interactions. We show that Rad53 is essential in S-phase due to its role in regulating basal dNTP levels by action in the Dun1 pathway that regulates RNR and Rad53’s compensatory kinase function if dNTP levels are perturbed. In conclusion we present further evidence of the importance of dNTP pools in the maintenance of genome integrity and shed more light on the complex regulation of dNTP levels.

Au cours de ma thèse au sein du laboratoire du Professeur Michelle Debatisse, je me suis intéressé aux mécanismes maintenant la stabilité du génome et contrôlant la dynamique de réplication dans les cellules de mammifères. J'ai étudié le rôle des kinases ATR (" Ataxia Telangectasia and Rad3 related ") et Chk1 (" Checkpoint Kinase 1 "), du point de contrôle intra-S (" checkpoint "), dans le contrôle de la dynamique de réplication. Cette première étude m'a amené à étudier la relation entre les dommages à l'ADN et la dynamique de réplication, dans des modèles cellulaires déficients pour des facteurs de la réponse aux dommages à l'ADN (DDR), appartenant soit au " checkpoint ", soit à la voie de réparation par recombinaison homologue (HR), tels que Rad51 et BRCA2. Je montre ici, que le ralentissement des fourches de réplication et l'augmentation de la densité d'événements d'initiation, observés dans des cellules déficientes pour Chk1 ou Rad51, sont la conséquence indirecte des lésions apparaissant spontanément dans de telles cellules. Le ralentissement des fourches dans ces cellules dépend d'une perturbation de la disponibilité en précurseurs de nucléotides qui dépend de la sur-expression et/ou de la re-localisation de la sous-unité p53R2 de la ribonucléotide réductase (RNR). De plus, contrairement à ce qui était proposé, je montre que Chk1 n'a pas de rôle actif dans la répression des origines latentes, mais que c'est la vitesse des fourches qui détermine l'espacement entre les origines actives, par un mécanisme de compensation découvert auparavant au laboratoire (Anglana, 2003 ; Courbet, 2008). L'ensemble de mes résultats permet de proposer un mécanisme général de communication entre la réplication et la réparation. Ce mécanisme confère un avantage aux cellules, puisque le ralentissement des fourches stabilise la machinerie de réplication qui voyage sur une matrice endommagée, et l'activation d'origines latentes procure une source de sauvetage pour les fourches bloquées.