Дисертації з теми "RNases H"

Les RNAses H sont des enzymes qui hydrolysent spécifiquement le brin ARN d'un hybride ARN/ADN. Celles-ci sont retrouvées tout au long des règnes animaux et végétaux. Elles participent à l'enlèvement des fragments d'Okazaki lors de la synthèse discontinue de l'ADN. Un rôle dans la transcription est également suspecté. Il existe deux classes de RNases H, la classe I (type 2) et la classe II (type 1), la classification reposant sur des critères biochimiques généraux (sensibilité au NEM, concentration en cations divalents à l'optimum d'activité, poids moléculaire). Tandis que les enzymes de classe I eucaryotes sont localisées dans le noyau, les enzymes de classe II sont retrouvées dans le cytoplasme et la mitochondrie. Les RNases H cellulaires sont aussi connues pour leur action dans les effets d'oligodésoxyribonucléotides antisens. Dans un souci d'optimisation d'oligonucléotides antisens et pour détenir un nouveau critère de classification, nous avons décidé d'étudier le comportement de RNases H eucaryotes de différentes origines sur les hybrides de 20 nucléotides de long. Nous avons analysé les premières coupures de chaque hybride. Il s'est avéré que les RNases HI testées (origine bovine et humaine) préféraient l'extrémité 3' des ARN engagés dans la formation d'hybrides tandis que les RNases HII coupaient à 6 et 8 nucléotides de l'extrémité 5' de ces mêmes ARN. De plus, grâce à ce nouveau critère de nouvelles informations en faveur d'une localisation mitochondriale des RNases HII eucaryotes ont été obtenues. Par la suite nous avons tenté de cloner un gène de RNase HII chez le protozoaire parasite Leishmania mexicana amazonensis. Cette tentative a échoué. Actuellement, très peu d'inhibiteurs de RNases H existent et la stratégie SELEX constitue un bon moyen d'obtenir de tels ligands. Nous avons entrepris une sélection in vitro contre la RNase HII recombinante humaine. A l'issue de la sélection deux aptamères ont retenu notre attention. Le premier, b33, était un bon inhibiteur de la RNase HII avec une IC50 de 120 nM. Cette inhibition était spécifique des RNases HGII eucaryotes. Celui-ci peut se structurer en tige-boucle imparfaite. Le second, b12 n'inhibait que modestement la RNase HII humaine et celui-ci avait la possibilité de former plusieurs structures impliquant des tétrades de G
RNases H are ubiquitous enzymes that hydrolyse the RNA of a DNA/RNA hybrid. They are found in all kingdoms. They participate in the removal of RNA primers of Okazaki fragments. A role in transcription also suspected. RNases H are divided in two classes : class I and class II. RNases HI are nuclear whereasRNases HII are cytoplasmic and mitochondrial. RNases H are also known to be implicated in antisens effects of oligodeoxyribonucleotides. To help in designing new antisens molecules and to possess a new classification criterion, we have analysed the first cuts of these enzymes on various hybrids of 20 nucleotides in length. The tested RNases HI (from bovine and human origin) prefers the 3' end of the RNA engaged in a hybrid whereas RNases HII cut preferentially at 6 and 8 nucleotides from the 5' end of the same RNAs. Moreover informations on mitochondrial localisation of RNases HII has been obtained using this new classification criterion. After this, we have attempted to clone an RNase HII gene from the protozoan Leishmania mexicana amazonensis. This attempt did not succeed. Nowadays, only a few inhibitors of the RNase H activity are known. A good mean to obtain such inhibitors is to use SELEX strategies. We have made an in vitro selection of single stranded DNA aptamers against human recombinant RNase HII. One, b33 inhibited RNaseHII with an IC50 value of 120 nM. This inhibition was specific for eukaryotic RNases HII. B33 could fold into an imperfect stem-loop structure. The second aptamer, b12 poorly inhibited human RNase HII. Moreover several structures could be formed implicating G-quartet formation

Au cours de la phase S, la réplication de l’ADN est initiée au niveau de multiples origines réparties le long du génome. La machinerie de réplication, ou réplisome, peut rencontrer des obstacles ralentissant sa progression, comme des structures secondaires de l’ADN ou des protéines liées à l’ADN telles que les ARN polymérases, générant ainsi ce que l’on appelle un stress réplicatif. Les réplisomes bloqués par un obstacle sont des structures fragiles qui peuvent générer des cassures et conduire à l’instabilité du génome. Lorsque la progression de l’ARN polymérase est ralentie ou arrêtée, le brin d’ARN naissant peut potentiellement s’hybrider avec le brin d’ADN complémentaire en déplaçant le second brin d’ADN, formant ainsi une structure à trois brins appelée R-loop, pouvant entraver la progression du réplisome. La coordination des processus de réplication et de transcription limite les interférences entre la réplication et la transcription. Cependant, cette coordination n’est pas parfaite et même dans des conditions physiologiques, la transcription et l’accumulation de R-loops peuvent conduire à des évènements de recombinaison, notamment au cours de la phase S. Les Ribonucléases H (RNases H) de type 1 et 2 sont des protéines impliquées dans la résolution des R-loops par la dégradation spécifique du brin d’ARN au sein du duplexe ARN:ADN. Les cellules dépourvues de RNases H présentent une accumulation de R-loops et sont extrêmement sensibles à différents agents génotoxiques induisant du stress réplicatif (e.g. MMS : méthanesulfonate de méthyle ou HU : hydroxyurée). Le but de mes travaux de thèse est de déterminer le rôle des RNases H dans la réponse cellulaire au stress réplicatif. Dans deux modèles cellulaires, la levure S. cerevisiae et les cellules humaines, nous avons pu montrer que les cellules déplétées en RNases H présentent des défauts de prise en charge et de redémarrage des fourches de réplication arrêtées en condition de stress réplicatif induit. L'utilisation de mutants de séparation de fonction de RNase H2 suggère que l’élimination défectueuse des hybrides ARN:ADN est responsable de ces défauts. La mesure du taux d’hybrides ARN:ADN au cours du cycle cellulaire montre qu’il augmente en phase S en présence de stress réplicatif exogène dans des cellules sauvages et mutantes pour RNases H. De plus, nos résultats indiquent que l’inhibition de la transcription ou la surexpression de l’hélicase ARN:ADN Sénataxine restaure la prise en charge et le redémarrage des fourches de réplication arrêtées lors d’un stress induit par le MMS et en absence des RNases H. Ainsi, l’ensemble de nos résultats suggère une étroite coopération entre les Ribonucléases H et l’hélicase Sénataxine pour résoudre les interférences entre les ARN polymérases et/ou les hybrides ARN:ADN avec les machineries de réplication
During S phase, DNA replication starts at multiple origins distributed throughout the genome. As the replication machinery (or replisome) progresses throughout the DNA, it often encounters obstacles such as DNA secondary structures or transcription complexes, thereby generating what is called replication stress. Stalled replisomes are fragile structures that can give rise to chromosome breaks and trigger genome instability. When RNA polymerases stall, the nascent RNA can potentially anneal with the template DNA strand, creating a three-strand structure called R-loop. Coordination between replication and transcription in S phase limits the risks of collisions between the replisome and RNA polymerases. Even though, physiological transcription level and R-loops accumulation lead to recombination events in S phase. Type 1 and 2 ribonucleases H (RNase H) are specific proteins involved R-loops’ resolution through the degradation of the RNA strand within the RNA:DNA duplex. In the absence of RNases H, cells accumulate R-loops and are extremely sensitive to different replication stress-inducing genotoxic agents (e.g. MMS: methyl methanesulfonate or HU: hydroxyurea).The goal of my PhD project was to assess the roles of RNases H in the cellular response to replication stress. Using two cellular models, the budding yeast S. cerevisiae and mammalian cells, we demonstrated that RNases H mutations induce HU- and MMS-stalled replication forks processing and restart defects. Analysis of separation-of-function RNase H2 mutants suggests that it is the RNA:DNA hybrids removal activity of RNase H2 that is important for the correct processing of stalled forks experiencing replication stress. Indeed, quantification of RNA:DNA hybrids during the cell cycle reveals a higher level of hybrids in S phase in the presence of exogenous replication stress in both wild-type and RNases H-depleted cells. Moreover, our results demonstrate that the inhibition of transcrip tion or the overexpression of the RNA:DNA helicase Senataxin restore stalled replication fork processing and restart upon MMS treatment when cells lack RNase H2 activities. Altogether, our data indicate that Ribonucleases H1 and 2 and Senataxin helicase cooperate to resolve RNA polymerases and/or RNA:DNA hybrids interferences with replication

Antisense oligonucleotides (AON) offer a rational approach for drug design. The specificity of AONs towards a complementary messenger RNA (mRNA) target via Watson and Crick base pairing as well as their ease of synthesis render this technology very attractive. RNase H-degradation of mRNA via formation of a AON/mRNA hybrid is crucial to mainstream antisense technologies. Numerous studies have demonstrated the importance of the AON's structure and conformational flexibility for efficient induction of RNase H activity. However the precise mode of action and substrate specificity of the RNase H are not fully understood at present. Our Laboratory recently discovered that incorporation of flexible acyclic linkers (e.g. butanediol, 2'-seco-RNA) significantly amplifies enzyme activity. Unfortunatly incorporation of such linkers was accompanied by a drop in the thermal stability of the AON/RNA hybrids. This prompted us to incorporate a less flexible linker such as a peptide nucleic acid, with the hope to maintain similar enzymatic activity while increasing the duplex thermal stability.
This thesis highlights the synthesis of the 5'-amino nucleoside analogue required for the incorporation of the peptide nucleic acid in both 2'-fluoroarabinonucleic acid (2'F-ANA) and DNA. Circular dichroism experiments afforded information on the hybrid conformation in solution, whereas UV thermal melting studies provided a measure of the thermal stability of such hybrid duplexes. Finally, ability of various linker modified AON/RNA hybrids to activate the RNase H enzyme was evaluated in parallel with the corresponding native unmodified DNA/RNA hybrids.
Incorporation of a PNA residue within DNA or 2'-FANA did not afford improvement in neither thermal stability nor enzymatic cleavage (except for homopolymeric sequences vs DNA) as compared to control or butyl-sequences.

Ribonucleases (RNases) H1 and H2 are endonucleases that hydrolyze the RNA strand of RNA-DNA hybrids forming at the chromosomal level as well as extra-chromosomal hybrids. Extra-chromosomal RNA-DNA hybrids can frequently occur in cells as intermediate structures in the process of reverse transcription and generation of cDNA by retrotransposition. It is known that mutations in RNase H2 are found in Aicardi-Goutières syndrome (AGS) patients. AGS is a rare but severe immune-mediated neurodevelopmental disorder. Currently, the mechanism by which defects in RNase H2 cause AGS is still unclear. We hypothesized that defects in RNases H, including those associated with AGS can trigger the accumulation of extra-chromosomal RNA-DNA hybrids. Thus, we speculate that increased stability of such free RNA-DNA hybrid structures could be a likely trigger for stimulating the autoimmune system, mimicking a viral infection in AGS patients. RNase H2 protein subunits of human and yeast Saccharomyces cerevisiae RNase H2 proteins have conserved amino acid sequences. Based on the similarity between human and yeast RNase H2, we thought to utilize S. cerevisiae as a research model to generate and study several AGS-related mutants. Initially, we set up an assay to detect retrotransposition activity in the budding yeast by introducing a recombinant DNA which includes a Ty1 retrotransposable element fused to an inactive his3 marker gene. To test whether the retrotransposition assay works in our yeast strains, we treated yeast cells with phosphonoformic acid (PFA) or knocked out DBR1 gene coding for the RNA lariat debranching enzyme. Both approaches strongly reduced the frequency of retrotransposition in our strains, demonstrating that the system was working as expected. Next, we examined whether yeast cells with defective forms of RNases H or AGS-orthologous mutants of RNase H2 had altered retrotransposition activity compared with cells with wild-type RNases H. Results showed that the retrotransposition activity was repressed in the absence of both types of RNase H. In addition, AGS-related mutants showed decreased retrotransposition frequencies when RNase H1 was also knocked-out. These findings are relevant to uncover the mechanism of the AGS.

HIV-1 the Reverse Transcriptase (RT), the most renowned retroviral specific enzyme, was the first anti-HIV target to be exploited, as. HIV-1 RT combines two functions essential for viral replication: DNA polymerase, synthesis DNA either in a RNA dependent (RDDP) or DNA dependent (DDDP) manner, and Ribonuclease H (RNase H) . The RNase H activity catalyzes highly specific hydrolytic events on the RNA strand of the RNA/DNA replication intermediate, critical to the synthesis of integration-competent double-stranded proviral DNA. Because of its essential role, RNase H is a promising target for drug development. However, despite years of efforts, no RNase H inhibitor (RHI) has yet reached clinical approval. In this work we pursued the identification and characterization of new promising RHIs targeting either the RNase H active site itself (RNase H active site chelating agents) or both RNase H and RDDP activities (allosteric dual inhibitors). The first approach faced the challenging nature of the RNase H active site region, the morphology of wich is, more open than that of the relatively similar,HIV-1 integrase (IN). This hampers the identification of a druggable pocket. We initially used Foamy Virus RT as a tool, to perform NMR and docking analyses on the interaction between FV RT RNase H domain and a previously identified diketo acid (DKA) derivative, inhibitor RDS1643. The amino acid residues of the FV RNase H active site region (T641, I647, Y672 and W703) were established to be important for the interaction with the inhibitor and analogous residues were successfully identified in the HIV-1 RNase H domain using structural overlays. Further docking and site directed mutagenesis studies were performed using six couples of ester/acid DKA, derived from RDS1643, showing for the first time, a broad interaction between RHIs and conserved residues in the HIV-1 RNase H active site region (R448, N474, Q475, Y501 and R557). Moreover, ester and acid derivatives exhibited a different binding orientation, that reflected a different specificity for RNase H versus IN. Among the synthesised derivatives one, RDS1759, showed to be an RNase H selective active site inhibitor characterized also, for the first time,in cell-based assays. The second approach focused on the determination of the mechanism of action of a new isatine-derived RNase H/RDDP dual inhibitor, RMNC6. Docking analysis and site directed mutagenesis results suported the hypothesis of a two-sites mode of action, with an independent role for two pockets,to be further characterized for a rational optimization of the scaffold.

Replication of HIV-1 is inhibited by azidothymidine (AZT), which leads to chain termination and inhibition of DNA synthesis. Resistance to AZT is frequently the result of mutations that increase the ability of RT to remove the chain-terminating nucleotides after they have been incorporated. It has been proposed that RNase H cleavage of the RNA template can occur when RT is stalled near the site of chain termination and contributes to the inhibition by causing the dissociation of the primer-template before the chain terminator can be excised. Mutations in the connection and RNase H domains of RT have been shown to increase excision. It has long been known that resistance to thymidine analogs is conferred by the mutations M41L, D67N, K70R, L210W, T215F/Y and T219Q/E in RT and that this resistance is suppressed by the additional presence of the M184V mutation. Changes in excision activity on DNA templates have been observed with these mutant RTs, but effects on RNase H cleavage resulting in indirect effects on excision activity is also possible with RNA templates. We used a 5'-labeled -3'-chain-terminated DNA primer annealed to either a DNA or RNA template to evaluate primer rescue activities, a 5'-labeled RNA template to evaluate RNA cleavage activity and a biotin-tagged chain-terminated oligodeoxynucleotide to monitor primer-template dissociation. We first investigated differences between RNA and DNA templates when the primers were chain terminated and observed a correlation between RNase H activities and template/primer (T/P) dissociation. An inverse correlation was observed between excision rescue rates and RNase H cleavages leading to T/P dissociation. We observed that the chain terminator (i.e. AZTMP or ddAMP) affected RNase H cleavages and excision rates with RNA template and dNTPs. When we investigated mutations in the N-terminal domain of RT associated with nucleoside reverse transcriptase inhibitor (NRTI) resistance we found that primer rescue was decreased when M184V was present in combination with thymidine analog mutations (TAMs) and the template was RNA with either ATP or PPi as excision substrate. RNase H cleavage at secondary cleavage sites (-7, -8) was substantially reduced with M41L/T215Y RT in comparison with wild type RT, and primer-template dissociation was decreased. In contrast, when M184V was present, RNase H cleavage at the secondary cleavage sites and dissociation of the primer-template occurred at higher levels and excision rescue was decreased. The ability of RT to rescue an AZT terminated primer in the presence of the 184V mutation was restored when the RNase H activity was inactivated by the RNase H negative mutation E478Q. Electromobility shift assay (EMSA) analysis of AZT-resistant mutant RT with M184V showed an increased Kd for formation of the ternary complex. These results suggest that RNase H-mediated RNA-DNA template-primer dissociation is influenced by mutations associated with thymidine analog resistance, and that suppression of resistance to nucleoside RT inhibitors by M184V may be partly explained by effects on RNase H cleavage that decrease the time available for excision to occur. This is the first time that mutations in the polymerase domain are shown to affect excision rescue through an RNase H-dependent mechanism.

The HBV RNAse H has been cloned into the PET43a vector, which contains the NusA protein which works as a solubilizing fusion protein. The fusion NUS-RNAse H protein was cleaved by enterokinase; the cleaved RNAse H is about 17 Kda which remains soluble and active. A fluorescence assay utilizing a quenching mechanism was used to characterize the activity of NUS-RNAse H and cleaved RNAse H proteins. The beacon is a RNA:DNA hybrid oligonucleotide labeled with a 5'DABCYL and a 3'fluorescein, when RNAse H digests the RNA, DABCYL is released resulting in high fluorescence. The digestion of the RNA was also confirmed by gel analysis. The protein was identified by N-terminal amino acid sequence analysis of the fusion protein, SDS-PAGE, western blot utilizing HBV positive sera for primary antibodies, and enzyme immunoassay by peroxidase labeling of HBV RNAse H. Structural analysis of the protein was done by circular dichroism, tryptophan fluorescence, the generation of a model from HIV RNAse H and initial crystals which unfortunately did not diffract. The ability to produce good amounts soluble RNAse H, the development of a sensitive assay to test for activity and the solution of the crystal structure will help develop new anti-viral inhibitors.

A short oligonucleotide sequence as in a single-stranded antisense oligo nucleotides (AON) or in double-stranded small interfering RNAs (siRNA) can modulate the gene expression by targeting against the cellular mRNA, which can be potentially exploited for therapeutic purposes in the treatment of different diseases. In order to improve the efficacy of oligonucleotide-based drugs, the problem of target affinity, nuclease stability and delivery needs to be addressed. Chemical modifications of oligonucleotides have been proved to be an effective strategy to counter some of these problems. In this thesis, chemical synthesis of conformationally constrained nucleosides such as 7′-Me-carba-LNA-A, -G, -MeC and -T as well as 6′, 7′-substituted α-L-carba-LNA-T (Papers I-III) was achieved through a key free-radical cyclization. 1D and 2D NMR techniques were employed to prove the formation of bicyclic ring system by free-radical ring closure as well as to identify the specific constrained conformations in sugar moieties. These sugar-locked nucleosides were transformed to the corresponding phosphoramidites and incorporated into antisense oligonucleotides in different sequences, to evaluate their physicochemical and biochemical properties for potential antisense-based therapeutic application. AONs modified with 7′-Me-carba-LNA analogues exhibited higher RNA affinities (plus 1-4°C/modification) (Papers I & III), but AONs containing α-L-carba-LNA analogues showed decreased affinities (minus 2-3°C/ modification) (Paper II) towards complementary RNA compared to the native counterpart.  It has been demonstrated in Papers I-III that 7′-methyl substitution in α-L-carba-LNA caused the Tm drop due to a steric clash of the R-configured methyl group in the major groove of the duplex, whereas 7′-methyl group of carba-LNA locating in the minor groove of the duplex exerted no obviously negative effect on Tms, regardless of its orientation. Moreover, AONs containing 7′-Me-carba-LNA and α-L-carba-LNA derivatives were found to be nucleolytically more stable than native AONs, LNA modified AONs as well as α-L-LNA modified ones (Papers I-III). We also found in Paper II & III that the orientations of OH group in C6′ of α-L-carba-LNAs and methyl group in C7′ of 7′-Me-carba-LNAs can significantly influence the nuclease stabilities of modified AONs. It was proved that the methyl substitution in cLNAs which points towards the vicinal 3′-phosphate were more resistant to nuclease degradation than that caused by the methyl group pointing away from 3′-phosphate. Additionally, AONs modified with 7′-Me-carba-LNAs and α-L-carba-LNAs were found to elicit the RNase H mediated RNA degradation with comparable or higher rates (from 2-fold to 8-fold higher dependent upon the modification sites) as compared to the native counterpart. We also found that the cleavage patterns and rates by E. coli RNase H1 were highly dependent upon the modification sites in the AON sequences, regardless of the structural features of modifications (Papers II & III). Furthermore, we have shown that the modulations of Tms of AON/RNA duplexes are directly correlated with the aqueous solvation (Paper III).

The integrity of the genome is continuously jeopardized by endogenous reactive byproducts of cellular metabolism and genotoxic insults by environmental agents, as well as by the DNA transactions (replication, transcription and recombination) required for cell survival and proliferation. Failure of the mechanisms deputed to the maintenance of genome integrity leads to genome instability, which is a hallmark of cancer and a driving force of tumorigenesis. To fully understand the mechanisms leading to genome instability and the cellular pathways counteracting them, three basic tasks must be achieved: i) identify all the genes implicated in the control of genome integrity; ii) unravel their biological role; iii) define the mechanistic molecular details of the processes in which they are implicated. This thesis describes work performed in the budding yeast Saccharomyces cerevisiae to explore the genome stability landscape at all these three levels. This model system is extremely useful for two main reasons: a) its high genetic tractability allows the application of genome-wide genetic screenings; b) the large conservation of the genome integrity pathways allows to extend the findings obtained in yeast to other eukaryotic organisms. We performed a genome-wide screen, based on the overexpression of the DDC2 DNA damage checkpoint gene in the yeast deletion collection, to identify genome stability genes on the basis of spontaneous accumulation of endogenous DNA damage in the corresponding mutant strains. Our screen identified several genes implicated in the control of genome integrity, highlighting, in particular, a key role for pathways protecting against oxidative stress. We present here the preliminary characterization of a new genome integrity gene, VID22. We also investigated the mechanisms counteracting a newly discovered source of genome instability, namely ribonucleotides (rNTPs) incorporated in genomic DNA during replication. We uncovered a role for RNase H enzymes, template switch pathways and Pol ζ translesion polymerase in protecting from misincorporated rNTPs. Given that mutations in any of the three human RNase H2 subunits were proven to cause Aicardi-Goutiéres Syndrome, these results might contribute to shed light on the complex and largely unknown pathogenetic mechanism of this rare genetic disease. Finally, we studied the molecular details underlying the role of Rad9 mediator protein in DNA damage checkpoint activation, exploring the dynamics of Rad9 dimerization, chromatin binding, CDK-dependent phosphorylation and checkpoint activation in G1 and M phases of the cell cycle; in particular, we characterized an M-phase specific pathway for checkpoint activation which is relies on Rad9-Dpb11 interaction.

Synthetic and physicochemical studies on appropriately functionalized ODN-conjugates have been performed to evaluate their abilities to act as antisense agents against RNA or as intramolecular DNA cross-linking agents. Intercalating aromatic systems [phenazine (Pnz), dipyridophenazine (DPPZ)] and metallointercalators such as Ru²⁺(phen)₂(DPPZ) and Ru²⁺(tpy)(DPPZ)L [where L = chemically or photochemically labile ligand, phen = phenanthroline, tpy = terpyridine], which are covalently tethered to the oligo-deoxynucleotides (ODNs), have been chosen for this purpose. The ODN-conjugates were typically prepared by automated solid phase synthesis using phosphoramidite building blocks, or on solid supports, both functionalized with the chromophore groups. The photosensitive metal complex, Ru²⁺(tpy)(DPPZ)(CH₃CN), has been incorporated by post-synthetic coupling to the amino-linker modified ODNs via an amide bond. The intercalating ability of the tethered chromophores gave enhanced stability of the duplexes and triplexes formed with ODN-conjugates and their complementary targets: DNA, RNA, or double-stranded DNA. The conjugation of DPPZ chromophore to ODN (at 3', 5' or at the middle) led us to incorporate Ru²⁺(phen)₂(DPPZ) through the DPPZ ligand, for the first time. The corresponding (Ru²⁺-ODN)•DNA duplexes showed dramatic stabilization (ΔT_m = 19.4 – 22.0ºC). The CD and DNase I footprinting experiments suggest that the stabilization is owing to metallointercalation by threading of the Ru²⁺(phen)₂ moiety through the ODN•DNA duplex core, thus "stapling" the two helical strands from the minor to major groove. On the other hand, Ru²⁺(tpy)(DPPZ)(CH₃CN)-ODN conjugates represent a new class of oligonucleotides containing the photoactivatible Ru²⁺ complexes, which can successfully crosslink to the complementary strand. The mechanism of cross-linking upon photoirradiation of [Ru²⁺(tpy)(DPPZ)(CH₃CN)-ODN]•DNA involves in situ conversion to the reactive [Ru²⁺(tpy)(DPPZ)(H₂O)-ODN]•DNA which are subsequently cross-linked through the G residue of the complementary DNA strand. All starting materials and products have been purified by HPLC and/or by PAGE and subsequently characterized by MALDI-TOF as well as ESI mass spectroscopy. Terminal conjugation of the planar Pnz and DPPZ groups through the flexible linkers were also shown to improve thermal stability of the ODN•RNA hybrid duplexes without alteration of the initial AB-type global helical structure as revealed from CD experiments. As a result, RNase H mediated cleavage of the RNA strand in the intercalator-tethered ODN•RNA duplexes was more efficient compared to the natural counterpart. The RNase H cleavage pattern was also found to be dependent on the chemical nature of the chromophore. It appeared that introduction of a tether at the 3'-end of the ODN can be most easily tolerated by the enzyme regardless of the nature of the appending chromophore. The tethered DPPZ group has also been shown to chelate Cu²⁺ and Fe³⁺, like phenanthroline group, followed by the formation of redox-active metal complex which cleaves the complementary DNA strand in a sequence-specific manner. This shows that the choice of appropriate ligand is useful to (i) attain improved intercalation giving Tm enhancement, and (ii) sequence-specifically inactivate target RNA or DNA molecules using multiple modes of chemistry (RNase H mediated cleavage, free-radical, oxidative pathways or photocross-linkage).

Over one hundred types of chemical modifications have been characterized in cellular RNAs. Pseudouridines (Ψ) and 2’-O-methylation of ribose sugars are the two most widespread modifications present in rRNAs, tRNAs and snRNAs. These modifications can be either guide-RNA mediated or RNA-independent (enzyme only). The RNAs that guide pseudouridylations are called box H/ACA RNAs and the ones that carry out 2’-O-methylations modifications are called box C/D RNAs. Previously, we identified that sR-h45 is the box H/ACA guide RNA responsible for Ψ1940, 1942 and 2605 formation in 23S rRNA of Haloferax volcanii. This RNA has two stem loops – SL1 and SL2. SL1 acts as the guide for Ψ2605 formation and SL2 is responsible for guiding Ψ1940 and Ψ1942. We found that SL2 sequentially guides Ψ1940 and Ψ1942 formation in the unpaired "UNUN" target. Ψ1942 is produced after and only if Ψ1940 is produced. The requirement for conserved ACA box was determined by using variants of these two stem loops. We found that the ACA motif is not required either in vivo or in vitro for the activity of the typical variants of both SL1 and SL2 but required for the activity of the atypical variants of these guides. Cbf5 is the pseudouridine synthase involved in this box H/ACA RNA guided process. Mutants of Methanocaldococcus jannaschii Cbf5 were used with both typical and atypical guide variants in vitro and certain residues were found to be important only for the atypical reactions.We have also studied sR-h41, which is a unique single guide box C/D guide responsible for methylation of G1934 position of 23S rRNA of Haloferax volcanii. We have done in vitro assembly reactions using mutants of sR-h41 assembled with its cognate proteins from Methanocaldococcus jannaschii to study the structural determinants needed to convert it to a dual guide RNA. The assembly pattern of the core proteins on the conserved box C/D and box C’/D motifs steer the dual guide nature of these archaeal box C/D guide RNAs.Another aim of this study was to determine the role of pseudouridine synthases (Pus enzymes) for Ψ55 formation in mammalian tRNAs. We find that three Pus enzymes – TruB1 (in the nucleus), TruB2 (in the mitochondria) and Pus10 (in the cytoplasm) are responsible for this modification depending on the specific sub-cellular location in the cell. These enzymes exhibit different structural requirement for Ψ55 formation that are located on the TΨC loop of tRNAs. A subset of tRNAs like tRNAs for Trp and Gln are protected from the action of TRUB1 in the nucleus by binding to the nuclear version of Pus10 that lacks Ψ55 activity. Ψ55 in this subset of tRNAs is produced by the cytoplasmic version of Pus10.While studying pseudouridylation functions of Pus10, we also found that Pus10 regulates G1/S cell cycle progression in PC3 cells. It does so by directly repressing another protein c-Rel, that is a positive regulator of Cyclin D1 protein. Cyclin D1 is known to play a central role in transition of cell from G1 to S phase during cell cycle progression. c-Rel also regulates the levels of PUS10 by an unknown mechanism.

H-Ras es un protooncogen que codifica para dos proteínas por splicing alternativo la p19 y la p21 (1,2). La p21 presenta mayor tamaño y se localiza en la membrana en donde realiza la función de GTPasa que activa múltiples vías de señalización (3-8) en cambio la p19 de menor tamaño, atraviesa la membrana nuclear, en donde forma complejos proteicos con otras proteínas y desde ahí regula múltiples vías de señalización (9-10). Las mutaciones en H-Ras inducen a la carcinogénesis y han sido frecuentemente detectadas en los tumores de los pacientes (30%) con melanoma, cáncer oral, de riñón y vejiga Para nuestros experimentos evaluamos las siguientes líneas celulares: 1) Células HeLa transfectadas en forma transiente, y que sobreexpresaban las proteínas H-Ras de nuestro interés en forma separada (vectores PRK5); 2) Fibroblastos embrionarios de ratón knock-out H-Ras (-/-) o bien doble knock-out H-Ras (-/-), N-Ras (-/-), transfectados de manera estable con los vectores pEGFP-p19 ó pEGFP-p21 y 3) líneas mutantes de fibroblastos obtenidas de los tumores extraídos por cirugía de los pacientes con Síndrome de Costello, el cuál es un raro desorden congénito causado por la activación en la línea germinal del oncogen H-Ras que afecta tanto a la proteína p19 como a la p21 (11-13). Nuestros resultados mostraron que las proteínas H-Ras promueven el incremento en la expresión de los miRNAs evaluados, y en forma diferencial, la p19 incrementa la expresión de miR-206 y promueve un estado de quiescencia celular en la fase G0/G1 provocando una disminución en la proliferación celular, la capacidad invasiva y la capacidad formadora de tumores; además la sobreexpresión de p19 incrementa la expresión de la proteína NM23H1 la cuál confiere protección contra el daño producido al DNA por las especies reactivas de oxígeno. En cuanto a las líneas mutantes de los fibroblastos de los pacientes con Síndrome de Costello, detectamos que 1) la mutación G12S posee mayor capacidad invasiva que la G12A, y 2) una disminución en la expresión de miR-206, la cuál ha sido relacionada recientemente con la aparición de rabdomyosarcoma en los pacientes por lo que se ha propuesto como un marcador del pronóstico de la enfermedad. REFERENCIAS 1. Cohen J.B., Broz S.D., and Levinson A.D. (1989). Expression of the H-Ras proto-oncogene is controlled by alternative splicing. Cell. 58: 461-472 2. Guil S, de La Iglesia N, Fernández-Larrea J, Cifuentes D, Ferrer JC, Guinovart JJ, Bach-Elias M. (2003a). Alternative splicing of the human proto-oncogene c-H-ras renders a new Ras family protein that trafficks to cytoplasm and nucleus.Cancer Res. (2003 a) 1;63(17):5178-87 3. Malumbres M., and Barbacid M. (2003). Ras oncogenes: the first 30 years. Nature Reviews 3,7-13. 4. Rodriguez-Viciana P., Sabatier C., and McCormick F. (2004) Signalling specificity by Ras family GTPases is determined by the full spectrum of effectors they regulated. Mol. Cell. Biol. 24(11):4943-4954. 5. Mitin N., Rossman L. K., and Der C. J. (2005). Signaling interplay in Ras superfamily function. Current Biology15 (14): R563 - R574. 6. Malaney S., and Daly R.J. (2001). The Ras signalling pathway in mammary tumorigenesis and metastasis. J. Mammary Gland Biol Neoplasia. 6(1):101-113 7. Downward, J. (2002). Targeting Ras signalling pathways in cancer therapy. Nat. Rev. Cancer 3: 11-22 8. Colicelli J. (2004). Human Ras superfamily proteins and related GTPases. Sci.Signal 250: re13 9. Camats-Malet., Calin G.A., Heesom, K.J., Liu cG., Volinia S., Croce M., Ladomery M., and Bach-Elias M. (2008b). P19 activates telomerase, regulates expression of proteins of the tuberous sclerosis (TSC) pathway and upregulate miRNA’s expression. Submitted to Plos One. 10. Camats-Malet M. (2008a). Mecanismes de Senyalitzacio intracellular regulats per la proteina p19 H Ras. Tesis de Doctorat. Departament de Bioquimica I Biologia Molecular. Unitat de Ciencies.Universitat Autonoma de Barcelona. 11. Costello, J.M. (1977). A new syndrome: mental subnormality and nasal papillomata. Aust Paediat J. 13: 114-118. 12. Gripp K.W., Innes A.M., Axelrad M.E., Gillan T.L., Parboosingh J.S., Davies C., Leonard N.J., Doyle D., Catalano S., Nicholson l., Stabley D., and Sol-Church K. (2008). Costello syndrome associated with novel germline H-Ras mutations: An attenuated phenotype? American Journal of Medical Genetics Part A. 146 A:683-690 13. Gripp KW, Lin A.E, Stabley D., Nicholson L., Scott Jr. C.I., Doyle D., Aoki Y., Matsubara Y., Zachai E.H., Lapunzina P., Gonzalez-Meneses, A., Holbrook J., Agresta C.A., Gonzalesz I.L and Sol-Church K. (2006). HRAS mutation analysis in Costello Syndrome: Genotype and Phenotype correlation. Am J. Med. Genet.A 140 (1):1-7
Ras is an evolutionary and conserved family of genes present in many organisms, in humans, Ras is conformed by three members called N-Ras, K-Ras and H-Ras located on chromosomes 1, 11 and 12 respectively (1, 2). Ras proteins act as a molecular switch, activating many signalling pathways through phosphorylation of GTPases; so their punctual mutations promote a constitutive activation in their GTPase function that fosters carcinogenesis, loss of adhesion and invasion of malignant cells (3-9). In the case of H-Ras, 30% of the malignant tumours analyzed have showed mutations and they were frequently detected in melanoma, thyroid, oral, bladder, and kidney cancers (10-14). H-Ras gene renders two different proteins by their alternative splicing called p19 and p21 (15, 16); even though both proteins have the same origin, they differ in their size function and localization. P19 and p21 are similar in their first 150 aminoacids, but they differ in the C-terminal amino acid sequences, meanwhile p21 contains 152-165 residues that confer the GTPase function, p19 lacks of it. Nevertheless p19 protein is smaller than p21, is able to cross the nuclear membrane and then p19 can bind with other proteins as such as RACK1, PKCβII, p73 and NSE (neuron specific enolase), (17,18) forming protein complexes; which suggest that p19 protein indirectly orchestrates multiple cell functions from the nucleus (19). For the experimental development of this PhD thesis we decided to overexpress p19, p21, or their mutant protein variants transient and ectopically in HeLa cells. Three mutations were analyzed in these assays: Q61L, G12S, and G12A, the first of them induces a constitutive activation of GTPase activity and G12S, G12A are both a frequent mutations observed in Costello Syndrome, a rare congenital disorder caused by germ-line activation of H-Ras oncogenes that affects both p19 and p21(20-22). We also analyzed the contribution of p19, or p21 proteins in knock-out H-Ras (-/-) and double knock-out for H-Ras (-/-), N-Ras (-/-) murine embryonic fibroblasts (MEFs); these cell lines have the advantage that they do not show gene redundancy in their expression, so in other words this means that the absence of one member of Ras proteins does not cause that other Ras protein assume its functions. We also analyzed mutated fibroblasts obtained from tumours of Costello Syndrome patients in order to determinate the contribution of G12S and G12A mutations in this syndrome. A general increment in the miRNA expression profile was detected when p19, p21 and their mutant variants were overexpressed in HeLa cells, even though further experiments in our knock-out H-Ras (-/-) and double knock-out H-Ras (-/-), N-Ras (-/-) MEFs transfected with pEGFP-p19 or pEGFP-p21, we detected a differential expression of miR-206 and miR-342 when p19 and p21 were expressed respectively. In addition, miR-206 was consistently downregulated in our mutated fibroblasts of Costello Syndrome patients which is agree with recent reports that have correlated the misregulation of miR-206 with the development of rhabdomyosarcoma in these patients. In other hand, overexpression of p21 (G12S) protein in HeLa cells showed the highest rate of invasion, followed by p21, p19 (G12S) and p19 proteins; in further cotransfection assays (p19/p21 (G12S) proteins); p19 was able to diminished the invasion and in mutated fibroblasts of Costello Syndrome patients, the highest invasion capacity rate was conferred by G12S mutation. P19 protein showed a low rate proliferation in double knock-out H-Ras (-/-), N-Ras (-/-) MEFs, further analyses of cytometry revealed that p19 induces a quiescent arrest in G0/G1 phase cell cycle. The capacity of forming colonies was also evaluated in clonogenic anchorage agar assays in which the presence of (G12S) mutation in p19 and p21 proteins contributed to the formation of bigger and more number of colonies. Additionally, overexpression of p19 protein (pRK5-p19 vector) in HeLa cells also conferred protection against reactive oxygen species emission overexpressing NM23H1 protein, this effect was also detected in high ROS emission environment. REFERENCES: 1. Lowy D.R., and Willumsen B.M. (1993). Function and regulation of Ras. Ann. Rev. Biochem. 62:851-891. 2. Wennerber K., Rossman K.L., and Der C.L. (2005).The Ras superfamily at glance. J. Cell Sci.118:843-846. 3. Mori K., Hata M., Neya S., Hoshino T. (2002) A study on the role of Mg+” in a Ras protein by MD simulation. CBIJ. 2 (4): 147-155 4. Cullen P. J., and Lockyer P.J. (2006) Integration of calcium and Ras signalling. Nat. Rev. 3: 339-344. 5. Rehman H., and Bos J. (2004) Thumbs up for inactivation. Nature. 249: 138-139 Ricarte-Filho JC, Fuziwara CS, Yamashita AS, Rezende E, da-Silva MJ, Kimura ET.(2009). Effects of let-7 microRNA on Cell Growth and Differentiation of Papillary Thyroid Cancer. Transl Oncol. 200. (4):236-41. 6. Campbel S.L., Khosravi-Far R., Rossman K.L., Clark G.J. and Der C.J. (1998). Increasing complexity of Ras signaling. Oncogene. 17:1395-1413. Cancer Lett. 2008 Oct 18;270(1):10-8. doi: 10.1016/j.canlet.2008.03.035. Epub 2008 May 23. Review. 7. Malumbres M., and Barbacid M. (2003). Ras oncogenes: the first 30 years. Nature Reviews 3,7-13. 8. Rodriguez-Viciana P., Sabatier C., and McCormick F. (2004) Signalling specificity by Ras family GTPases is determined by the full spectrum of effectors they regulated. Mol. Cell. Biol. 24(11):4943-4954. 9. Mitin N., Rossman L. K., and Der C. J. (2005). Signaling interplay in Ras superfamily function. Current Biology15 (14): R563 - R574. 10. Malaney S., and Daly R.J. (2001). The Ras signalling pathway in mammary tumorigenesis and metastasis. J. Mammary Gland Biol Neoplasia. 6(1):101-113 11. Downward, J. (2002). Targeting Ras signalling pathways in cancer therapy. Nat. Rev. Cancer 3: 11-22 12. Colicelli J. (2004). Human Ras superfamily proteins and related GTPases. Sci.Signal 250: re13 13. Castro P., Soares P., Gusmao L., Seruca R., Sobrinho-Simoes. (2006). H-RAS 81 polymorphism is significantly associated with aneuploidy in follicular tumors of the thyroid. Oncogene. 25: 4620-4627 14. Castellano E, Santos E.(2011). Functional specificity of ras isoforms: so similar but so different.Genes Cancer. 2011 Mar;2(3):216-31. doi: 10.1177/1947601911408081. 15. Cohen J.B., Broz S.D., and Levinson A.D. (1989). Expression of the H-Ras proto-oncogene is controlled by alternative splicing. Cell. 58: 461-472 16. Guil S, de La Iglesia N, Fernández-Larrea J, Cifuentes D, Ferrer JC, Guinovart JJ, Bach-Elias M. (2003a). Alternative splicing of the human proto-oncogene c-H-ras renders a new Ras family protein that trafficks to cytoplasm and nucleus.Cancer Res. (2003 a) 1;63(17):5178-87 17. Jeong MH., Bae J., Kim WH., Yoo SM., Kim JW., Son PI, Choi KH. (2006). P19 ras interacts with and activates p73 by involving the MDM2 protein. The Journal of Biological Chemistry. 281(13):8707-8715 18. Camats-Malet., Calin G.A., Heesom, K.J., Liu cG., Volinia S., Croce M., Ladomery M., and Bach-Elias M. (2008b). P19 activates telomerase, regulates expression of proteins of the tuberous sclerosis (TSC) pathway and upregulate miRNA’s expression. Submitted to Plos One. 19. Camats-Malet M. (2008a). Mecanismes de Senyalitzacio intracellular regulats per la proteina p19 H Ras. Tesis de Doctorat. Departament de Bioquimica I Biologia Molecular. Unitat de Ciencies.Universitat Autonoma de Barcelona. 20. Costello, J.M. (1977). A new syndrome: mental subnormality and nasal papillomata. Aust Paediat J. 13: 114-118. 21. Gripp K.W., Innes A.M., Axelrad M.E., Gillan T.L., Parboosingh J.S., Davies C., Leonard N.J., Doyle D., Catalano S., Nicholson l., Stabley D., and Sol-Church K. (2008). Costello syndrome associated with novel germline H-Ras mutations: An attenuated phenotype? American Journal of Medical Genetics Part A. 146 A:683-690 22. Gripp KW, Lin A.E, Stabley D., Nicholson L., Scott Jr. C.I., Doyle D., Aoki Y., Matsubara Y., Zachai E.H., Lapunzina P., Gonzalez-Meneses, A., Holbrook J., Agresta C.A., Gonzalesz I.L and Sol-Church K. (2006). HRAS mutation analysis in Costello Syndrome: Genotype and Phenotype correlation. Am J. Med. Genet.A 140 (1):1-7

Thesis (Ph. D.)--University of Toledo, 2008.
Typescript. "Submitted as partial fulfillment of the requirements for the Doctor of Philosophy in Chemistry." Includes bibliographical references (leaves 305-309).

This thesis is based on six original research publications describing synthesis, structure and physicochemical and biochemical analysis of chemically modified oligonucleotides (ONs) in terms of their potential diagnostic and therapeutic applications. Synthesis of two types of bicyclic conformationally constrained nucleosides, North-East locked 1',2'-azetidine and North locked 2',4'-aza-ENA, is described. Study of the molecular structures and dynamics of bicyclic nucleosides showed that depending upon the type of fused system they fall into two distinct categories with their respective internal dynamics and type of sugar conformation. The physicochemical properties of the nucleobases in the conformationally constrained nucleosides found to be depended on the site and ring-size of the fused system. The incorporation of azetidine modified nucleotide units into 15mer ONs lowered the affinity toward the complementary RNA. However, they performed better than previously reported isosequential 1',2'-oxetane modified analogues. Whereas aza-ENA-T modification incorporated into ONs significantly enhanced affinity to the complementary RNA. To evaluate the antisense potential of azetidine-T and aza-ENA-T modified ONs, they were subjected to RNase H promoted cleavage as well as tested towards nucleolytic degradation. Kinetic experiments showed that modified ONs recruit RNase H, however with lower enzyme efficiency due to decreased enzyme-substrate binding affinity, but with enhanced turnover number. Both, azetidine-T and aza-ENA-T modified ONs demonstrated improved 3'-exonuclease stability in the presence of snake venom phosphodiesterase and human serum compared to the unmodified sequence. Oligodeoxynucleotides (ODNs) containing pyrene-functionalized azetidine-T (Aze-pyr X) and aza-ENA-T (Aza-ENA-pyr Y) modifications showed different fluorescence properties. The X modified ODNs hybridized to the complementary DNA and RNA showed variable increase in the fluorescence intensity depending upon the nearest-neighbor at the 3'-end to X modification (dA > dG > dT > dC) with high fluorescence quantum yield. However, the Y modified ODNs showed a sensible enhancement of the fluorescence intensity only with complementary DNA. Also, the X modified ODN showed decrease (~37-fold) in the fluorescence intensity upon duplex formation with RNA containing a G nucleobase mismatch opposite to the modification site, whereas a ~3-fold increase was observed for the Y modified probe.

The aim of this diploma thesis was to study interactions between human Rase H enzyme and a natural and modified substrate using molecular dynamics simulations (altogether 9 MD runs ere produced). Conformational preferences of internucleotide linkages (undergoing contacts with the RNase H enzyme) were studied using several versions of the AMBER force field. Either one or two copies of RNase H were included into the simulated system. As the most important DNA-binding residues were recognized Trp93 and Ser101 in the first DNA binding site and Thr49 and Arg47 in the second DNA binding site. Further, the AMBER force field was re-parameterized slightly using ab initio calculations to produce force constants for the modified phosphonate internucleotide linkage. Biologically active version of the modified internucleotide linkage C3-O3-P-C-O5-C5 was able to bind Arg47 using two hydrogen bonds within the 10 ns MD run (even more effciently than in the case of MD runs with natural internucleotide linkages). On the other hand, the biologically inactive C3-O3-C-P-O5-C5 internucleotide linkage lose contacts with Arg47 quickly.

RNA editing in trypanosomes is the post-transcriptional insertion or deletion of uridylates at specific sites in mitochondrial mRNAs. This process is catalyzed by a multienzyme, multisubunit complex through a series of enzymatic cycles directed by small, trans-acting RNA molecules. Despite impressive progress in our understanding of the mechanism of RNA editing and the composition of the editing complex, fundamental questions regarding RNP assembly and the regulation of catalysis remain. This dissertation presents studies of RNA-protein interactions between RNA editing complexes and substrate RNAs and the determination of substrate secondary structural determinants that govern them. Our results suggest that substrate association, cleavage and full-round editing by RNA editing complexes in vitro obey hierarchical determinants that increase in complexity as editing progresses and we propose a model for substrate recognition by RNA editing complexes. In addition, this dissertation also presents the characterization of a novel mitochondrial RNA helicase, named REH2 and its macromolecular interactions. Our data suggest that REH2 is intimately involved in interactions with macromolecular complexes that integrate diverse processes mediating mitochondrial gene expression. These results have implications for the mechanism of substrate RNA recognition by RNA editing complexes as well as for the integration of RNA editing to other facets of mitochondrial RNA metabolism.

碩士
國立中正大學
生物醫學研究所
101
SOX2 (Sex-determining region Y (SRY)-box protein 2) is a member of the HMG transcription factor family, functioning as a transcription factor and involving in cell self-renewal, differentiation, proliferation and apoptosis. Over-expression of SOX2 has been demonstrated in a least 60 cancer cell types. The study conducted in our laboratory indicated that SOX2 also regulates alternative splicing in bladder cancer, implicating that SOX2 is not only a DNA transcription factor but also as a RNA binding protein. In my study, I intend to determine the preferred binding sequence of SOX2 by employing oligomer-directed RNase H digestion-coupled CLIP assay. To demonstrate whether SOX2 direct regulated mRNA, We first expressed SOX2 and E1A in BFTC905, follow by splicing reporter CLIP assay. The data suggest SOX2 directly binding on E1A. Furthermore, we determined that SOX2 regulates splice site selection of E1A, indicating that SOX2 is a bona fide splicing factor. Furthermore, the binding sequence of SOX2 on the S100A14 mRNA also determined through similar approach. Those data suggest that SOX2 regulates translation of the S100A14 mRNA by direct binding on the S100A14 mRNA Keywords: CLIP、 SOX2、 RNA binding protein

Mitotic genome instability can occur during the repair of double-strand breaks (DSBs) in DNA, which arise from endogenous and exogenous sources. Studying the mechanisms of DNA repair in the budding yeast, Saccharomyces cerevisiae has shown that Homologous Recombination (HR) is a vital repair mechanism for DSBs. HR can result in a crossover event, in which the broken molecule reciprocally exchanges information with a homologous repair template. The current model of double-strand break repair (DSBR) also allows for a tract of information to non-reciprocally transfer from the template molecule to the broken molecule. These “gene conversion” events can vary in size and can occur in conjunction with a crossover event or in isolation. The frequency and size of gene conversions in isolation and gene conversions associated with crossing over has been a source of debate due to the variation in systems used to detect gene conversions and the context in which the gene conversions are measured.

In Chapter 2, I use an unbiased system that measures the frequency and size of gene conversion events, as well as the association of gene conversion events with crossing over between homologs in diploid yeast. We show mitotic gene conversions occur at a rate of 1.3x10-6 per cell division, are either large (median 54.0kb) or small (median 6.4kb), and are associated with crossing over 43% of the time.

DSBs can arise from endogenous cellular processes such as replication and transcription. Two important RNA/DNA hybrids are involved in replication and transcription: R-loops, which form when an RNA transcript base pairs with the DNA template and displaces the non-template DNA strand, and ribonucleotides embedded into DNA (rNMPs), which arise when replicative polymerase errors insert ribonucleotide instead of deoxyribonucleotide triphosphates. RNaseH1 (encoded by RNH1) and RNaseH2 (whose catalytic subunit is encoded by RNH201) both recognize and degrade the RNA in within R-loops while RNaseH2 alone recognizes, nicks, and initiates removal of rNMPs embedded into DNA. Due to their redundant abilities to act on RNA:DNA hybrids, aberrant removal of rNMPs from DNA has been thought to lead to genome instability in an rnh201Δ background.

In Chapter 3, I characterize (1) non-selective genome-wide homologous recombination events and (2) crossing over on chromosome IV in mutants defective in RNaseH1, RNaseH2, or RNaseH1 and RNaseH2. Using a mutant DNA polymerase that incorporates 4-fold fewer rNMPs than wild type, I demonstrate that the primary recombinogenic lesion in the RNaseH2-defective genome is not rNMPs, but rather R-loops. This work suggests different in-vivo roles for RNaseH1 and RNaseH2 in resolving R-loops in yeast and is consistent with R-loops, not rNMPs, being the the likely source of pathology in Aicardi-Goutières Syndrome patients defective in RNaseH2.

Dissertation

Submiltted in fulfillment of the requirements for the degree of Master of Science in the faculty of Science, University of the Witwatersrand, Johannesburg • Johannesburg 1993.
The roles of the highly conserved aspartic acid residues found at positions 443 and 498 within the RNase H domain of Human Immunodeficiency Virus type-1 reverse transcription were investigated by the defined substitution of these residues using site-directed mutagenesis. [Abbreviated Abstract. Open document to view full version]
MT2016