Dissertationen zum Thema „RNA: DNA hybrides“

Les gènes transcriptionellement actifs peuvent être la source de l'instabilité du génome via de nombreux mécanismes. Ces gènes sont caractérisés par la formation de structures secondaires telles que les hybrides ADN : ARN. Ils se forment lorsque l'ARN sortant l'ARN polymérase II s'hybride au simple brin d'ADN. De nombreuses études ont montrées que l'accumulation de ces hybrides peut mener à la création de dommages à l'ADN. Parmi ces dommages, les Cassures Double Brins (CDB) sont les plus dangereuses pour la cellule puisqu'elles peuvent produire des mutations et des réarrangements chromosomiques. Il existe deux mécanismes de réparation majeurs dans la cellule : la Jonction Non-Homologue des Extrémités (NHEJ) et la Recombinaison Homologue (HR). Mon équipe a récemment montré que les CDB localisées dans les gènes transcrits sont préférentiellement réparés par HR. De plus, de nombreuses études ont montrées une interaction entre transcription et réparation des CDB. Au vue de ces résultats, nous avons donc émis l'hypothèse que les gènes transcriptionellement actifs pourraient être réparés par un mécanisme spécifique nécessitant l'activité de protéines associées à la transcription : "Réparation couplée à la transcription". Durant ma thèse, je me suis intéressée au rôle de deux protéines dans la réparation des régions transcrites en utilisant la lignée cellulaire DIvA (DSB Induction via AsiSI) qui permet l'induction de cassures annotées sur tout le génome. Premièrement, nous avons montré que la réparation des CDB dans des loci transcrits nécessitent une hélicase ADN : ARN connue : sénataxine (SETX). Après induction d'une cassure dans un gène, SETX est recrutée ce qui permet la résolution d'hybride ADN : ARN (cartographié par DRIP-seq). Nous avons aussi montré que SETX permet le recrutement de RAD51 et limite les jonctions illégitimes des CDB et par conséquent promeut la survie des cellules après induction des cassures. Cette étude montre que les CDB dans les loci transcrits requièrent la résolution spécifique des hybrides ADN : ARN par SETX pour permettre une réparation précise et est absolument indispensable pour la survie cellulaire. Deuxièmement, nous avons montré une interaction entre SETX et Bloom (BLM) une G4 DNA hélicase dans la réparation des CDB dans les régions transcrites. Nous avons montré que BLM est aussi recrutée au CDB dans les loci transcrits où elle est nécessaire à la résection et à la fidélité de réparation. De façon importante, nous avons montré que la déplétion de BLM restaure le défaut de survie cellulaire observé dans les cellules déplétées pour SETX après induction des CDB. La déplétion d'autres hélicases G4 (RTEL1, FANCJ) promeut aussi la survie des cellules déplétées pour SETX après dommages. Ces résultats suggèrent une interaction entre les hélicases G4 et la résolution des hybrides ADN : ARN dans la réparation des gènes actifs. En conclusion, ces études permettent une meilleure compréhension de la spécificité de la réparation des régions transcrites du génome, et notamment l'identification de protéines impliquées dans la "Réparation couplée à la Transcription"
Actively transcribed genes can be the source of genome instability through numerous mechanisms. Those genes are characterized by the formation of secondary structures such as RNA-DNA hybrids. They are formed when nascent RNA exiting RNA polymerase II hybridizes single stranded DNA. Numerous studies have shown that RNA-DNA hybrids accumulation can lead to DNA damages. Among those damages, DNA double strand breaks (DSB) are the most deleterious for cells since they can generate mutations and chromosomal rearrangements. Two major repair mechanisms exist in the cell: Non-Homologous End-Joining (NHEJ) and Homologous recombination (HR). My lab showed recently that DSB occurring in transcribed genes are preferentially repaired by HR. Moreover, multiple studies have shown a cross talk between transcription and DSB repair. Those results led us to propose that actively transcribed genes could be repaired by a specific mechanism implicating proteins associated with transcription: "Transcription-coupled DSB repair". During my PhD, using the DIvA (DSB Induction via AsiSI) cell line allowing the induction of annotated DSB through the genome, I worked on 2 projects focusing on DSB repair in transcribed genes. First, we showed that DSB repair in transcribed loci requires a known RNA: DNA helicase: senataxin (SETX). After DSB induction in an active gene, SETX is recruited which allows RNA-DNA hybrid resolution (mapped by DRIP-seq). We also showed that SETX activity allows RAD51 loading and limits DSB illegitimate rejoining and consequently promotes cell survival after DSB induction. This study shows that DSB in transcribed loci require specific RNA-DNA hybrids removal by SETX for accurate repair. Second, we showed an interplay between SETX and Bloom (BLM) a G4 DNA helicase in DSB repair induced in transcribed loci. We showed that BLM is also recruited at DSB in transcribed loci where it promotes resection and repair fidelity. Strikingly, we showed that BLM depletion rescued the survival defects observed in SETX depleted cells following DSB induction. Knock down of other G4-helicases (RTEL1, FANCJ) also promoted cell survival in SETX depleted cells upon damage. Those data suggest an interplay between G4 helicases and RNA: DNA resolution for DSB repair in active genes. Altogether, these studies promote a better understanding of the specificity of DSB repair in transcriptionally active genes, and notably identification of proteins involved in "Transcription-coupled DSB repair"

Les machineries de réplication et de transcription peuvent provoquer des conflits entre transcription et réplication (TRC), qui se produisent de manière frontale ou co-directionnelle. La collision frontale est considérée comme étant la plus délétère et peut conduire à de l’instabilité génomique au travers des R-loops qui se composent un hybride ADN-ARN et un brin d'ADN déplacé. En analysant les données multi-omiques, nous avons révélé avec succès que la pause transitoire de la fourche de réplication aux 3' des gènes enrichis en R-loops avec collision frontale affecte la stabilité génomique d'une manière dépendante de Topoisomérase1 (Nat.Communs. 2020) puis j'ai développé le premier outil bio-informatique pour analyser de données de réplication (OKseqHMM, disponible sur GitHub, Liu et al. BioRxiv. 2022). Finalement, il a été montré récemment que dans les cellules cancéreuses du sein, les R-loops colocalisent fortement avec une augmentation des cassures de l'ADN, de manière dépendante de la réplication. Nous visons à étudier le TRC dans des cellules cancéreuses et des échantillons de patients cancéreux pour déterminer comment le stress réplicatif induit de l'instabilité génomique dans le développent de cancer, ce qui pourront contribuer à l’établissement de nouvelles stratégies thérapeutiques contre le cancer
Replication and transcription machinery can cause transcription-replication conflicts (TRCs), which occur either frontally or co-directionally. The head-on collision is considered to be the most deleterious and can lead to genomic instability through R-loops that consist of a DNA-RNA hybrid and a displaced DNA strand. By analyzing multi-omics data, we successfully revealed that transient replication forks pause at the 3' of genes enriched in R-loops with more head-on collisions affects genomic stability in a Topoisomerase1-dependent manner (Nat. Commons . 2020) then I developed the first bioinformatics tool to analyze replication data (OKseqHMM, available on GitHub, Liu et al. BioRxiv. 2022). Finally, it has recently been shown that in breast cancer cells, R-loops strongly colocalize with an increase in DNA breaks, in a replication-dependent manner. We aim to study TRC in cancer cells and samples from cancer patients to determine how replicative stress induces genomic instability in cancer development, which may contribute to the establishment of new therapeutic strategies against cancer

The stability of our genome is constantly challenged by several genotoxic threats. DNA double-strand breaks (DSBs) are the most dangerous DNA lesions that, if not repaired, can lead to cancer initiation and progression and/or ageing. These detrimental consequences can only be avoided if cells promptly recognize the lesions and signal their presence, thus promoting either efficient repair and transient cell cycle arrest or cell death and cellular senescence. This is the role of the DNA damage response (DDR) proteins and the newly identified damage-induced non coding RNAs. We recently discovered that RNA polymerase II is recruited to DSBs and synthetizes damage-induced non-coding RNAs (dilncRNAs). DROSHA- and DICER-mediated processing of dilncRNAs generates small RNA species, named DNA damage response RNA (DDRNAs) (Francia, 2012), that localize to DSBs via pairing with dilncRNAs and promote DDR signaling (Michelini et al., in press). Similar small non-coding RNA species discovered in plants are involved in DNA repair by homologous recombination (HR) (Wei, 2012, Gao, 2014, Wang, 2016). In line with these results, I report that transcriptional inhibition impairs recruitment of the HR proteins BRCA1, BRCA2, and RAD51 to DSBs, while partially promoting DNA end resection. Moreover, I show DNA:RNA hybrids accumulation at DSBs in mammalian cells by both DRIP analyses and imaging techniques. Damage-induced DNA:RNA hybrids form upon the hybridization of RNA species, likely dilncRNAs, to the resected DSBs DNA ends generated during the S/G2 cell cycle phase. I also report that purified recombinant BRCA1 binds DNA:RNA hybrids in vitro; moreover, DNA:RNA hybrids in vivo contribute to BRCA1 recruitment to DSBs. Consistent with the need to tightly regulate DNA:RNA hybrid levels, I demonstrate that RNase H2, the major RNase H activity in mammalian nuclei, is recruited to DSBs through direct interaction with RAD51. In summary, I report for the first time that DNA:RNA hybrids accumulate at DSBs in mammalian cells in a cell-cycle- and DNA end resection-depended way. At DSBs, BRCA1 directly recognizes DNA:RNA hybrids and likely controls their turn-over by mediating the recruitment of RNase H2 via RAD51.

Dividing cells are constantly under threat from both endogenous and exogenous DNA damaging stresses that can lead to mutations and structural variations in DNA. One contributor to genome instability is three-stranded DNA:RNA hybrid structures called R-loops. Though R-loops are known to induce DNA damage and DNA replication stress, it is unclear whether they are recognized and processed by an established DNA repair pathway prior to inducing DNA breaks. Canonically, DNA repair proteins work downstream of R-loop-induced DNA damage to stimulate repair and suppress genome instability. Recently, the possibility that some DNA repair pathways actively destabilize R-loops, thus preventing unscheduled DNA damage has emerged. Here we identify the helicase SGS1 as a suppressor of R-loop stability. Our data reveals that SGS1 depleted cells accumulate R-loops. In addition, we define a role for transcription in genome instability of cells lacking SGS1, which is consistent with an R-loop based mechanism. Hyper-recombination in SGS1 mutants is dependent on transcript length, transcription rate, and active DNA replication. Also, rDNA instability in sgs1Δ can be suppressed by ectopic expression of RNaseH1, a protein that degrades DNA:RNA hybrids. Interestingly, R-loops are known to form at rDNA loci. We favour a model in which SGS1 contributes to the stabilization of stalled replication forks associated with transcription complexes, and unresolved DNA:RNA hybrids. Finally, we showed that knockdown of the human Sgs1 orthologue BLM in HCT116 cells also led to the accumulation of more R-loops than control HCT116 cells. In summary, our data supports the idea that some DNA repair proteins involved in replication fork stabilization might also prevent and process R-loops.
Science, Faculty of
Graduate

The activation of the innate immune system is the first line of host defence against infection. Nucleic acids can potently stimulate this response and trigger a series of signalling cascades leading to cytokine production and the establishment of an inflammatory state. Mutations in genes encoding nucleases have been identified in patients with autoimmune diseases, including Aicardi-Goutières syndrome (AGS). This rare childhood inflammatory disorder is characterised by the presence of high levels of the antiviral cytokine interferon-α in the cerebrospinal fluid and blood, which is thought to be produced as a consequence of the activation of the innate immunity by unprocessed self-nucleic acids. This thesis therefore aimed to define the role of one of the AGS nucleases, the Ribonuclease H2 (RNase H2) complex, in innate immunity, and to establish if nucleic acid substrates of this enzyme were able to induce type I interferon production in vitro. The AGS nucleases may function as components of the innate immune response to nucleic acids. Consistent with this hypothesis, RNase H2 was constitutively expressed in immune cells, however, its expression was not upregulated in response to type I interferons. RNase H2-deficient cells responded normally to a range of nucleic acid PAMPs, which implied that a role for RNase H2 as a negative regulator of the immune response was unlikely, in contrast to the reported cellular functions of two other AGS proteins, TREX1 and SAMHD1. Therefore, no clear evidence was found for the direct involvement of RNase H2 in the innate immune response to nucleic acids. An alternative model for the pathogenesis of disease hypothesises that decreased RNase H2 activity within the cell results in an accumulation of RNA:DNA hybrids. To investigate the immunostimulatory potential of such substrates, RNA:DNA hybrids with different physiochemical properties were designed and synthesised. Methods to purify the hybrids from other contaminating nucleic acid species were established and their capacity as activators of the innate immune response tested using a range of in vitro cellular systems. A GU-rich 60 bp RNA:DNA hybrid was shown to be an effective activator of a pro-inflammatory cytokine response exclusively in Flt3-L bone marrow cultures. This response was completely dependent on signalling involving MyD88 and/or Trif, however the specific receptor involved remains to be determined. Reduced cellular RNase H2 activity did not affect the ability of Flt3-L cultures to mount a cytokine response against the RNA:DNA hybrid. These in vitro studies suggested that RNA:DNA hybrids may be a novel nucleic acid PAMP. Taken together, the data in this thesis suggest that the cellular function of RNase H2 is in the suppression of substrate formation rather than as a component of the immune response pathways. Future studies to identify endogenous immunostimulatory RNA:DNA hybrids and the signalling pathways activated by them should provide a detailed understanding of the molecular mechanisms involved in the pathogenesis of AGS and related autoimmune diseases.

Telomeres are repetitive sequences of DNA at the ends of chromosomes that become progressively shorter during cell division, acting as a form of “biological clock” causing cell death once they have reached a certain length. Almost 90% of cancer cells overexpress the enzyme telomerase which can lengthen telomeres and confer immortality to the tumour cells. Thus, telomerase has become an important target for drug discovery in the oncology area, and there is also interest from researchers investigating the aging process. During the catalytic cycle of telomerase, a unique DNA/RNA hybrid duplex (DRH) forms that is typically between 6-11 base pairs long and is key to extending the telomere. There is interest in discovering small drug-like molecules that can recognize and bind to this hybrid duplex to inhibit selectively telomerase, either by stabilizing the structure and thereby preventing telomerase dissociation (a key step in the catalytic cycle) or by sufficiently distorting the hybrid duplex to cause the misalignment of key catalytic groups. This project began by using oligonucleotides representing DNA/RNA hybrid duplex (DRH), telomeric G-quadruplex and control duplex DNA sequences to screen against the National Cancer Institute compound libraries (i.e., Diversity Set II, Mechanistic Set and Natural Product Set) using a high throughput Fluorescent Resonance Energy Transfer (FRET)-based DNA thermal denaturation assay to determine binding affinity and specificity. Thirteen novel chemical scaffold families were identified in the assay, compounds which showed a >5 °C selective stabilization of the DNA/RNA hybrid duplex at a 1 μM ligand concentration. Chemical modifications were then made to these scaffolds to generate focused libraries of analogues to improve selectivity, potency and drug-likeness, and to provide Structure-Activity Relationship (SAR) information. A total of 49 novel molecules were synthesized and then screened against an expanded range of four different nucleic acid constructs including telomeric and DNA/RNA hybrid duplex sequences. A number of compounds showed selective DNA/RNA hybrid stabilization potential with some compounds also showing notable telomeric G-quadruplex stabilization without significant affinity for promoter G-quadruplexes (i.e., c-Kit1, c-Kit-2 and c-Myc) and control duplex DNA sequences. The compounds from library-1 provided DNA/RNA hybrid duplex stabilization in the 0.5-7.2 C range and telomeric G-quadruplex stabilization in the 0.2-6.5 C range at a 1 μM ligand concentration. Molecular modelling and molecular dynamics studies confirmed that the methylene spacer between the benzimidazole and phenylene moieties of molecules within library-1 is perfectly shaped to fit within the DRH sequence. In addition, it was confirmed that minor-groove binding and simultaneous intercalation between the nucleobases of a DNA/RNA hybrid duplex requires a linker of specific length (i.e., an eight methylene spacer as in compound 3.3). Selected compounds were then studied further using a variety of biological techniques to confirm selective telomerase inhibition and cell-based assays to utilize their potential as antitumour agents.

O gene supressor tumoral RASSF1A tem sido associado com redução da proliferação celular em diversos tumores. Sua expressão é regulada por eventos epigenéticos que envolvem o complexo repressivo polycomb (PRC2), no entanto os mecanismos moleculares da modulação do recrutamento deste modificador epigenético para este locus ainda são desconhecidos. Neste trabalho identificamos e caracterizamos ANRASSF1, um RNA não codificador longo (lncRNA) intrônico unspliced, que é transcrito na fita oposta do gene RASSF1A, em várias linhagem celulares e tecidos, e se liga a PRC2. ANRASSF1 é transcrito pela RNAPII, possui cap-5´ e cauda poli-A, além de localizar-se no núcleo e possuir uma meia-vida em média quatro vezes menor comparada com outros lncRNAs ligados à PRC2. A super-expressão ectópica de ANRASSF1 reduziu os níveis de RASSF1A e aumentou a taxa de proliferação em células HeLa, enquanto seu silenciamento provocou efeito oposto. Essas mudanças nos níveis de ANRASSF1 não afetaram a abundância da isoforma RASSF1C em nenhuma das condições. A super-expressão de ANRASSF1 provocou um grande aumento tanto da ocupação de PRC2 como da marca de histona repressiva H3K27me3 especificamente na região promotora RASSF1A. Nenhum efeito da super-expressão de ANRASSF1 foi detectado na ocupação de PRC2 e na histona H3K27me3 nas regiões promotoras de RASSF1C e de outros quatro genes vizinhos, incluindo dois genes supressores tumorais bem caracterizados. Além disso, foi demonstrado que ANRASSF1 forma um híbrido de RNA/DNA e recruta SUZ12, um componente do PRC2, para o promotor de RASSF1A. Notavelmente, foi detectado pelo ensaio de RNase-ChIP que a degradação de ANRASSF1 diminui a ocupação de PRC2 neste promotor. Esses resultados demonstram um novo mecanismo de repressão epigenética do supressor tumoral RASSF1A, envolvendo um lncRNA unspliced antissenso, onde ANRASSF1 reprime seletivamente a expressão da isoforma de RASSF1 que sobrepõe o transcrito antissenso de modo local e específico. Considerando uma perspectiva mais ampla, nossos resultados sugerem que outros lncRNAs intrônicos unspliced não caracterizados no genoma humano podem contribuir para uma modulação epigenética local e específica de cada região em que os lncRNAs são transcritos.
Tumor-suppressor RASSF1A gene down-regulation has been implicated in increasing cell proliferation in several tumors. Its expression is regulated by epigenetic events involving polycomb repressive complex 2 (PRC2), however the molecular mechanisms modulating recruitment of this epigenetic modifier to the locus remain largely unknown. Here, we identify and characterize ANRASSF1, an endogenous unspliced long noncoding RNA (lncRNA) that is transcribed from the opposite strand of RASSF1 gene in several cell lines and tissues, and binds to PRC2. ANRASSF1 is transcribed by RNA Polymerase II, 5\'-capped, polyadenylated, displays nuclear localization, and has on average a four-fold shorter half-life compared to other lncRNAs that bind PRC2. ANRASSF1 ectopic overexpression decreases RASSF1A abundance and increases the proliferation rate of HeLa cells, whereas its silencing causes opposite effects. These changes in NRASSF1 levels do not affect RASSF1C isoform abundance. ANRASSF1 overexpression causes a marked increase both in PRC2 occupancy and in histone H3K27me3 repressive mark specifically at the RASSF1A promoter region. No effect of ANRASSF1 overexpression is detected on PRC2 occupancy and on histone H3K27me3 at the promoter regions of RASSF1C and of four other neighbor genes, including two well-characterized tumor suppressor genes. Additionally, we demonstrate that ANRASSF1 forms an RNA/DNA hybrid, and recruits SUZ12, a PRC2 component, to the RASSF1A promoter. Notably, depletion of ANRASSF1 disrupts SUZ12 occupancy on RASSF1A promoter as measured by RNAse-ChIP assay. Together, these results show a new mechanism of epigenetic repression of RASSF1A tumor suppressor gene involving an antisense unspliced lncRNA, in which ANRASSF1 selectively represses expression of the RASSF1 isoform overlapping the antisense transcript in a location-specific manner. In a broader perspective, our findings suggest that other non-characterized unspliced intronic lncRNAs transcribed in the human genome may contribute to a location-specific epigenetic modulation of genes.

During DNA replication, forks often stall upon encountering obstacles blocking their progression. Cells will act to speedily remove or overcome such barriers, thus allowing complete synthesis of chromosomes. This is the case for R-loops, DNA/RNA hybrids that arise during transcription. One mechanism to remove such R-loops involve DNA/RNA helicases. Here, I have shown that one such helicase, Sen1, associates with replisome components during S phase in the model organism S. cerevisiae. I demonstrate that the N-terminal domain of Sen1 is both sufficient and necessary for the interaction of the protein with the replisome. I also identified Ctf4 as one of at least two replisome interactors of Sen1. By mutational analysis, a mutant of Sen1 (Sen1-3) that cannot interact with the replisome was created. This mutant is healthy on its own but is lethal in the absence of both RNase H1 and H2. Overexpression of the sen1-3 allele from the constitutive ACT1 promoter is able to suppress this synthetic lethality, suggesting that Sen1 travels with replisomes in order to be quickly recruited at sites of R-loops that impair fork progression so as to remove those R-loops. In some cases, cells exploit fork stalling for biologically important processes. This is the case in Sz. pombe, where an imprint prevents complete DNA replication, triggering cell-type switching. This imprint is dependent on Pol1, a component of the replisome. Importantly, a single imprinting-defective allele of pol1 has been identified to date. Using in vitro assays, I have shown that this Pol1 mutant has reduced affinity for its substrates and is a correspondingly poor polymerase. By generating novel alleles of pol1, I have also demonstrated that switching-deficiency correlates with the affinity of Pol1 for its substrates in vivo. Finally, two interactors of Pol1 (Mcl1Ctf4 and Spp1Pri1 ) have been shown to have switching defects. S. cerevisiae and Sz. pombe have similar yet distinct genetic nomenclature conventions. Given that both model organisms were used in this study, it is important to highlight the conventions for both organisms to prevent confusion. In S. cerevisiae, wildtype gene names are expressed as a three letter, uppercase and italic name followed by a number (e.g. SEN1). The three letter name often corresponds to the screen through which the gene in question was originally identified. Mutants are generally designated with the same three letter but in lower case (unless the mutant is dominant) and with an allele designation (e.g. sen1∆, sen1-1 and sen1-2). Because of historical context, the allele designations vary in format (e.g. leu2-3,112 is a mutant of LEU2). Protein names are given as a three letter name with the first letter in uppercase (e.g. Sen1). This is also true for mutant proteins, with the added allele designation (e.g Sen1-1 and Sen1-2). In this study, I have generated constructs of the SEN1 gene and these constructs are referred to as SEN1 (X-Y), where X and Y refer to the first and last residues being encoded for. The corresponding proteins are referred to as Sen1 (X-Y). Different promoters have been used and, where appropriate, the promoters are expressed similarly to their wildtype gene names (e.g. GAL1, SEN1 and ACT1). In Sz. pombe, wildtype gene names are expressed as a three letter, lowercase and italic name followed by a number (e.g. pol1). Mutants are generally designated in the same format but with an allele designation. Like in S. cerevisiae, the allele designation varies widely (e.g. pol1-1, pol1-H4 and pol1-ts13). Additionally, because of the historical context, some (but not all) alleles of pol1 are referred to as swi7 to reflect the fact that they are defective for cell-type switching. Similar to the situation in S. cerevisiae, proteins names are given as a three letter name with the first letter in uppercase for both wildtype and mutants (e.g. Pol1 and Swi7-1). Sometimes, for the sake of comparison, genes or proteins are referred to their S. cerevisiae orthologues (e.g. swi1TOF1 and Swi1Tof1 , respectively). Several protein tags have been used in this study. When written in gene form, they were written in capital letters and italicized, irrespective of the host (e.g. 5FLAG) and when in protein form, they were written in capital, irrespective of the host (e.g. 5FLAG).

Faithful transmission of genetic material is challenged by the presence of natural impediments affecting replication fork progression that jeopardize genome integrity. Transcription, which competes with DNA replication for the same template, is a common barrier to replication in both prokaryotes and higher eukaryotes. Multiple mechanisms minimize the consequences of DNA replication and transcription collisions in order to prevent torsional stress accumulation that occurs when replication fork encounters the transcription machinery. Defects in resolving topological problems during chromosome replication lead to fork reversal, R loop formation and recombination-induced genome rearrangements. Our interest is focused on the processes that coordinate replication with transcription at TERs (termination sites) and on the molecular pathways involved in termination of DNA replication. We investigated the roles of Rrm3, a DNA helicase that assists replication fork progression, and of Sen1, a DNA/RNA helicase that resolves the conflicts between replication and transcription. We found that Rrm3 and Sen1 mediate replication termination at specific TERs, preventing aberrant events ultimately leading to chromosome fragility. Our results contribute to the elucidation of mechanisms coordinating replication and transcription at TER zones in eukaryotes.

Un nombre croissant d’études soutiennent le fait que les protéines majeures du métabolisme des ARN, telles que les hélicases ARN, sont impliquées dans la réponse aux dommages à l’ADN. Cette activité est généralement accomplie par leur interaction avec des facteurs de réparation de l’ADN. BRCA2, une protéine suppressive de tumeurs, joue un rôle crucial dans la réparation des cassures double-brin (CDB) de l'ADN par recombinaison homologue (RH) et donc, est un facteur essentiel pour l’intégrité du génome. Les cellules déficientes pour BRCA2 accumulent des hybrides ADN-ARN ou R-loops, une source de dommage à l'ADN, suggérant ainsi l’importance de cette protéine dans la prévention ou la suppression de ces structures. Toutefois, le rôle spécifique de BRCA2 dans la résolution des hybrides ADN-ARN reste inconnu.Afin de connaître des potentiels partenaires de BRCA2, une analyse par spectrométrie de masse réalisée dans notre laboratoire a révélé un enrichissement en protéines impliquées dans le métabolisme de l'ARN, comme les hélicases ARN. Ces résultats nous ont menés à examiner la coopération entre BRCA2 et les hélicases ARN dans la séparation des structures ADN-ARN. Nous avons d’abord confirmé l'interaction entre l'hélicase ARN DDX5 et BRCA2, qui est améliorée dans les cellules exposées à γ-irradiation. Ensuite, nous avons réduit l’interaction aux premiers 250 aa de BRCA2 (BRCA2T1) et avons constaté que celle-ci est directe en utilisant des protéines purifiées. En collaboration avec le laboratoire du docteur A. Aguilera (Cabimer, SP), nous avons montré que la déplétion de DDX5 conduit à une accumulation des hybrides ADN-ARN dans l’entièreté du génome, particulièrement aux sites de dommages à l’ADN. De plus, nos résultats indiquent que DDX5 localise aussi aux hybrides ARN-ADN qui se forment à proximité de CDB.De manière intéressante, nous avons constaté que BRCA2 est important pour la rétention de DDX5 aux sites de dommage à l’ADN induit par l’irradiation laser. Notamment, des tests de déroulement de brins in vitro en utilisant les protéines purifiées DDX5 et BRCA2 ont révélé que BRCA2 stimule l’activité de déroulement des R-loops de DDX5.Un variant de signification inconnue (VSI) trouvé dans de patients atteints de cancer du sein situé dans la région BRCA2T1 (T207A) réduit l’interaction de BRCA2 avec DDX5 et conduit à l’accumulation des hybrides ADN-ARN. Les cellules exprimant stablement BRCA2-T207A montrent également une diminution de l’association de DDX5 avec les hybrides ARN-ADN, en particulier lors d’une exposition de cellules à l’irradiation. L’analyse de l’efficacité de la réparation des CDB par RH dans les cellules déficientes en DDX5 ou exprimant BRCA2-T207A, montre une cinétique retardée de l’apparition des foyers de réparation RAD51 lors de l’irradiation, ce qui suggère un rôle actif de l’interaction BRCA2-DDX5 pour assurer la réparation par RH efficacement. En accord avec cette hypothèse, la ribonucléase RNAseH1, qui dégrade spécifiquement la fraction d’ARN dans les structures d’ADN-ARN, restaure partiellement le phénotype de cinétique des foyers RAD51 dans les cellules BRCA2 T207A. De plus, les cellules portant le variant BRCA2-T207A ont également montré un nombre réduit de foyers RPA par rapport aux cellules qui expriment BRCA2 sauvage, témoins d’un défaut dans l’étape qui précède le chargement de RAD51 aux CDB.Ensemble, nos résultats suggèrent que les hybrides ADN-ARN représentent un obstacle à la réparation des CDB par RH et révèlent BRCA2 et DDX5 en tant que facteurs actifs dans leur suppression
Increasing evidence support the idea that proteins involved in RNA metabolism such as RNA binding proteins (RBPs) and RNA helicases are directly implicated in the DNA damage response (DDR). This activity is generally achieved through their interaction with DNA repair factors.BRCA2 is a tumor suppressor protein that plays an important role in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) as well as protecting stalled replication forks from unscheduled degradation; therefore, it is essential to maintain genome integrity. Interestingly, BRCA2 deficient cells accumulate DNA-RNA hybrids or R-loops, a known source of DNA damage and genome instability, providing evidence for its role in either R-loop prevention or processing. However, the specific role of BRCA2 on these structures remains poorly understood.A mass spectrometry screen to identify partners of BRCA2 performed in our laboratory revealed an enrichment of proteins involved in RNA metabolism such as RNA helicases. These findings led us to investigate whether BRCA2 could cooperate with these candidate interacting RNA helicases in processing DNA-RNA structures. First, we confirmed the interaction of BRCA2 and the DEAD-box RNA helicase DDX5, which we found is enhanced in cells exposed to -irradiation. Then, we narrowed down the interaction to the first 250 aa of BRCA2 (BRCA2T1) and found that it is direct using purified proteins. In collaboration with A. Aguilera lab (Cabimer, SP), we could show that depletion of DDX5 leads to a genome-wide accumulation of DNA-RNA hybrids that is particularly enriched at DNA damage sites. DDX5 associates with DNA-RNA hybrids that form in the vicinity of DSBs. Interestingly, we found that BRCA2 is important for the retention of DDX5 at laser irradiation-induced DNA damage. Notably, in vitro R-loop unwinding assays using purified DDX5 and BRCA2 proteins revealed that BRCA2 stimulates the R-loop helicase activity of DDX5.A breast cancer variant of unknown clinical significance (VUS) located in BRCA2T1 (T207A) reduced the interaction between BRCA2 and DDX5 and led to the accumulation of DNA-RNA hybrids. Cells stably expressing BRCA2-T207A also showed a decreased association of DDX5 with DNA-RNA hybrids, especially upon irradiation. Notably, monitoring RAD51 foci to evaluate HR-mediated DSBs repair efficiency in either DDX5-depleted cells or in BRCA2-T207A cells resulted in a delayed kinetics of appearance of RAD51 foci upon irradiation suggesting an active role of BRCA2-DDX5 interaction in ensuring timely HR repair. In agreement with this, overexpression of the RNAseH1 ribonuclease, that specifically degrades the RNA moiety in DNA-RNA structures, partially restored RAD51 kinetics phenotype of BRCA2-T207A cells. Moreover, cells bearing BRCA2-T207A variant also showed a reduced number of RPA foci compared to BRCA2 WT expressing cells, a step that precedes RAD51 loading at DSBs.Taken together, our results are consistent with DNA-RNA hybrids being an impediment for the repair of DSBs by HR and reveal BRCA2 and DDX5 as active players in their removal

Yes
Curiouser and curiouser! A biarylpyrimidine ligand (see picture: N blue, H cyan, S yellow) shows a marked structure and sequence selectivity for the poly(dA)⋅poly(rU) hybrid duplex. An intercalative binding site was discovered where the ligand occupies a surprising ten base pairs. A strong correlation between hybrid duplex and DNA triplex binding indicates new directions for ligand design.

R-loops are transient intermediates that are formed during the transcription and consist of an RNA-DNA hybrid that is formed between the nascent RNA strand and the template DNA strand and a displaced non-template DNA strand. Formation of R-loops have been detected in organisms from bacteria to humans. If R-loops form more frequently, they impact transcription affecting genome stability, genome integrity and cause a number of diseases. Recently, genome instability caused due to stable RNA-DNA hybrid in R-loop was shown to predominantly associate with expansion of trinucleotide repeats, leading to many incurable neurological and neuromuscular genetic disorders like Myotonic dystrophy1, Fragile X syndrome, Huntington's disease, Friedreich’s ataxia, Spinocerebellar ataxias etc. Till date, the structural information about RNA-DNA hybrids formed by trinucleotide repeat expansions (TREs) are unknown to elucidate the mechanism behind RNA-DNA hybrid in instigating TREs. In this context, we aim here to study the structures of RNA-DNA hybrids consisting of trinucleotide repeats such as dGAA-rUUC, rGAA-dTTC, rCAG-dCTG, rCUG-dCAG, rCGG-dCCG and rCCG-dCGG by employing molecular dynamics (MD) simulation technique. It’s noteworthy that trinucleotide repeat expansion disorders (TREDs) can also be treated at mRNA level, wherein, a short complimentary DNA oligonucleotide is targeted against the mRNA that is followed by the cleavage of the mRNA strand by RNase H1 enzyme. Thus, to understand the efficacy of RNase H1 against TRE containing RNA-DNA hybrid, the interaction of TRE containing RNADNA hybrid with RNase H1 is also probed. Such scenario comes into picture during replication, wherein a transient RNA-DNA hybrid is formed and subsequently, the RNA strand is cleaved by RNase H1. Thus, this investigation is expected to shed light on structural properties of TRE containing RNA-DNA hybrids as well as its complex with RNase H1 to understand the TRE mechanism at replication and transcription levels and their treatment. The study on structure and dynamics of RNA-DNA hybrids consisting of various TREs suggest that these hybrids have both A & B characteristics irrespectively of sequence. The study on HsRNase H1 complexed with TREs containing RNA-DNA hybrids suggest that the protein follow the same cleavage mechanism for all the hybrids irrespective of sequence and thus, antisense strategy can be utilized to treat TREDs at RNA level.

碩士
國立清華大學
生命科學系
88
We have determined the solution structure of DNA/RNA hybrid chimeric duplex [d(CGC)r(aaa)d(TTTGCG)]2 using high resolution NMR spectroscopy, simulated annealing and restraint molecular dynamics. The solution structure of this hybrid duplex differs from its DNA duplex analog d(CGCAAATTTGCG)2 solved by X-ray crystallography (Edwards, et al 1992) and its RNA duplex analog r(cgcaaauuugcg)2 solved by NMR (Conte et al., 1997) as well. However, the overall structure is closer to a typical B-form DNA with a narrow minor groove width of about 6 A in the central hybrid segment. Long-lived water molecules with water correlation time tc longer than 0.3 ns were observed around the H2 and H1' protons of three RNA adenine residues as well as the methyl group of the thymine at the 7 position by combination of the laboratory frame nuclear Overhauser effect spectroscopy (NOESY) and the rotating frame NOE spectroscopy (ROESY). The unusual hydration pattern of the 7T methyl group in the major groove has not been observed in the previous NMR studies. The sugar ribose of 7T in the RNA-DNA hybrid junction was found to adopt a O4'-endo conformation, while other DNA residues, including 3C in the DNA-RNA hybrid junction, adopted C1'-exo or C2'-endo conformations. X-ray crystallography study of RNA duplex hydration also suggested a hydration network bridged via the C2'-OH of the RNA ribose with a C3'-endo conformation (Egli et al., 1996). The special sugar conformation of the 7T together with the additional C2'-OH of the 5'-adjacent RNA adenine may provide a hydraulic environment and thus showed a different hydration pattern from the other two thymine residues, 8T and 9T. The exchange rate of the RNA C2'-OH was found to be ~ 5-20 s-1. This slow exchange rate may due to the narrow minor groove width which restricts the dynamic motion of the hydroxyl protons. We further examined the structural role of the C2'-OH of the RNA residues by a 2'-O-methylated analog [d(CGC)r(amamam)d(TTTGCG)]2. The structure was not altered significantly, but the hydration of the 7T methyl decreased and it became merely observable in the minor groove. These distinct structural features and hydration patterns reveal the importance of the C2'-OH in the DNA/RNA hybrid structure and therefore provide a potential target in molecular recognition.

A protein, B0238.11, was identified in a yeast one-hybrid screen to bind to the ribosomal intergenic spacer region (IGS) of Caenorhabditis elegans. Proteins interacting with this region of the DNA have been implicated in ribosome biogenesis in other model organisms, so it is also possible that B0238.11 plays a role in RNA transcription by interacting with RNA polymerase I or other transcription machinery. Thus, the goal of this study was to further characterize the structure and function of B0238.11. I used yeast two-hybrid experiments to identify proteins that interact with B0238.11 within the nucleus. RPS-0, K04G2.2, DPY-4, EFT-3, PAL-1, and B0238.11, itself, were found to bind to B0238.11. Additionally, I analysed the amino acid sequence of B0238.11 using in silico bioinformatics methods to determine its structure and putative function and also to identify and characterize the other interacting proteins. I found that B0238.11 contains a high-mobility group box domain, which is also found in HMO1P in yeast and UBF in vertebrates. These other proteins also bind to the IGS, are known to form homodimers and have been implicated in the initiation of ribosomal RNA transcription. Here I scrutinize the validity of the interaction between each protein and B0238.11. I conclude that B0238.11 is likely to be a C. elegans homolog of UBF and present an updated interactome map for B0238.11.
Synopsis: I carried out yeast two-hybrid assay to find proteins interacting with B0238.11 (O16487_CAEEL). I found that this protein's DNA-binding profile and protein interaction profile mimic other HMG-box containing proteins UBF and HMO1P which are involved in ribosomal RNA transcription initiation. Acknowledgements: I would like to thank my supervisor, Dr. Teresa J. Crease, for not only giving me the opportunity to investigate an interesting topic in Molecular Biology, but also for her patient guidance, encouragement and sound advice. I feel extremely lucky to have a supervisor who cared so much about my work, who responded to my questions and queries so promptly, and was always available to discuss project and career related matters. I would also like to thank Dr. Todd Gillis and Dr. Terry Van Raay for their careful consideration of this project and timely constructive criticisms that helped shape my project. I would like to thank all the members of my committee for helping me see things from different perspectives and helping me develop and critical and mature understanding of the scientific process. I must also express my gratitude to Dr. Robin Floyd for allowing me to build upon his work and Dr. Marian Walhout, at the University of Massachusetts, for providing the Caenorhabditis elegans complimentary DNA library. A large part of this project would not have been possible without the people at the genomics facility in the Department of Integrative Biology, I commend their professionalism and punctuality in delivering results. Completing this work would have been all the more difficult were it not for the support and friendship provided by my peers Shannon Eagle, Tyler Elliott, Nick Jeffery, Joao Lima, Sabina Stanescu, Fatima Mitterboeck and Paola Pierossi. And finally, I would like to thank my parents and siblings Sara Omar and Ali Omar for their continued support through good times and bad, and letting me use their laptops when mine broke down.