Chez la levure à bourgeon, l’établissement de domaines silencieux pour la transcription nécessite la formation d’une structure, de type hétérochromatine, formée par le complexe SIR (Silencing Information Regulator). Les gènes soumis à la répression transcriptionnelle par ce complexe se trouvent aux sites cryptiques de détermination du type sexuel (HM) et dans les régions subtélomériques localisées à la périphérie nucléaire. Le recrutement des protéines Sir à ces sites nécessite la présence de séquences en cis comme les silencers ou les répétitions télomériques. Mon travail de thèse s’est attaché à l’étude d’une nouvelle voie d’établissement de la répression transcriptionnelle des gènes. En effet, nous avons démontré que la répétition en tandem de protéines fortement liées à l’ADN constitue un stress pour la fibre de chromatine. Ce stress induit le recrutement du complexe SIR favorisant ainsi la formation d’hétérochromatine et la mise en silence des gènes dans des régions normalement actives du génome. De plus, nous avons observé qu’en absence de l’ADN hélicase Rrm3, dont la fonction est de faciliter la progression de la fourche de réplication le long de la fibre de chromatine, la répression induite par ces complexes est exacerbée. Ce lien entre stress réplicatif et établissement de la répression transcriptionnelle a été observé, dans un premier temps, grâce à l’utilisation de systèmes artificiels (systèmes d’étiquetage des gènes : lacO/LacI et tetO/TetR). En outre, nous avons montré qu’un site naturel de pause de la réplication, tel qu’un gène codant un ARN de transfert, peut également favoriser la répression par les protéines Sir. De manière intéressante, à l’échelle du génome, nous avons pu observer le recrutement des protéines Sir dans des régions où la progression de la fourche de réplication est ralentie. Ainsi, nos données révèlent une nouvelle voie de mise en silence des gènes liant stress réplicatif et répression transcriptionnelle. / In budding yeast, the heterochromatin-like structure formed by the SIR complex (Silencing Information Complex) represses transcription. SIR mediated repression occurs at the cryptic mating type loci (HM) and subtelomeric regions localized at the nuclear periphery. The recruitment of the Sir proteins is induced by the presence of cis-acting elements as silencers or telomeric repeats.My doctorate work was focused on the characterization of a novel pathway of silencing establishment. Indeed, we have shown that arrays of tight DNA-proteins complexes lead to a chromatin stress. This stress induces the recruitment of the SIR complex and the establishment of stable heterochromatin-like domain at ectopic sites in the budding yeast genome. Moreover, this heterochromatinization is enhanced in cells mutated for Rrm3, a specialized DNA helicase acting ahead the fork to remove replication-impeding structures. Thus, we first observed a link between replication stress and silencing establishment by using artificial systems (gene tagging systems: lacO/LacI and tetO/TetR). Further, we have shown that tRNA genes, which are known to act as replication pause sites, can favor SIR-mediated repression. Interestingly, we found that Sir proteins are recruited where the replication fork progression is impeded at the genome wide scale. All together, these data reveal a novel mechanism for heterochromatin formation linking replication stress with gene repression.

Double-strand breaks and stalled forks arise when the replication machinery encounters damage from exogenous sources like DNA damaging agents or ionizing radiation, and require specific DNA helicases to resolve these structures. Sgs1 of Saccharomyces cerevisiae is a member of the RecQ family of DNA helicases and has a role in DNA repair and recombination. The RecQ family includes human genes BLM, WRN, RECQL4, RECQL1, and RECQL5. Mutations in BLM, WRN, and RECQL4 result in genetic disorders characterized by developmental abnormalities and a predisposition to cancer. All RecQ helicases have common features including a helicase domain, an RQC domain, and a HRDC domain. In order to elucidate the role of these domains and to identify additional regions in Sgs1 that are required for the maintenance of genome integrity, a series of systematic truncations to the C terminus of Sgs1 were created. We found that ablating the HRDC domain does not cause an increase in accumulating gross chromosomal rearrangements (GCRs). But deleting the RQC domain and leaving the helicase domain intact resulted in a rate similar to that of a helicase-defective mutant. Additionally, we exposed these truncation mutants to HU and MMS and demonstrated that losing up to 200 amino acids from the C terminus did not increase sensitivity to HU or MMS, whereas losing 300 amino acids or more led to sensitivity similar to that of an sgs1∆ cell. These results suggest that the RQC domain, believed to mediate protein-protein interactions and required for DNA recognition, is important for Sgs1’s role in suppressing GCRs and sensitivity to HU and MMS, whereas the HRDC domain that is important for DNA binding is not necessary. RecQL5 is a RecQ-like helicase that is distinct from the other members through its three different isoforms, RecQL5α, RecQL5β, and RecQL5ɣ. It has a helicase domain and an RQC domain, but lacks the HRDC domain that other RecQ-like helicases possess. In contrast to Blm, Wrn, and RecQL4, no human disorder has been associated with defects in RecQL5. For this reason the role of RecQL5 in the cell has remained largely unknown. To try to elucidate the pathways RecQL5 may be involved in we performed a yeast two hybrid to identify RecQL5-interacting proteins. We found that RecQL5 interacts with Hlp2, an ATP-dependent RNA helicase, and Ube2I, a SUMO-conjugating enzyme. These novel interactions shed light on a potential role of RecQL5 in the cell as a transcriptional regulator. Saccharomyces cerevisiae, Rrm3, is a 5’-3’ DNA helicase that is part of the Pif1 family of DNA helicases and is conserved from yeast to humans. It was initially discovered as a suppressor of recombination between tandem arrays and ribosomal DNA (rDNA) repeats. In its absence there are increased rates of extra-chromosomal rDNA circles, and cells accumulate X-shaped intermediates at stalled forks. Rrm3 may be involved in displacing DNA-protein blocks and unwinding DNA to facilitate fork progression. We used stable isotope labeling by amino acids in cell culture (SILAC)- based quantitative mass spectrometry in order to determine proteins that deal with the stalled fork in the absence of Rrm3. We found that in the absence of Rrm3 and increased replication fork pausing, there is a requirement for the error-free DNA damage bypass factor Rad5 and the homologous recombination factor Rdh54 for fork recovery. We also report a novel role for Rrm3 in controlling DNA synthesis upon exposure to replication stress and that this requirement is due to interaction with Orc5, a subunit of the origin recognition complex. Interaction of Orc5 was found to be located within a 26-residue region in the unstructured N-terminal tail of Rrm3 and loss of this interaction resulted in lethality with cells devoid of the replication checkpoint mediator Mrc1, and DNA damage sensitivity with cells lacking Tof1. In this study we describe two independent roles of Rrm3, a helicase-dependent role that requires Rad5 and Rdh54 for fork recovery, and a helicase-independent role that requires Orc5 interaction to control DNA synthesis. Our data provides novel insight into the role of DNA helicases and their role in protecting the genome. Through yeast genetics it was possible to determine the importance of the C terminus of Sgs1 and elucidate new RecQL5 interacting partners that shed light onto roles for RecQL5 distinct from other RecQ like helicases. Quantitative mass spectrometry allowed us to take on a more global view of the cell and determine how it responds to replication fork pausing in the absence of Rrm3. Using both proteomics and yeast genetics we were able to better understand how these DNA helicases contribute to maintaining genome stability.