<p>The action of Mung Bean Nuclease (MBN) on DNA makes it possible to clone intact gene fragments from genes of the malaria parasite, Plasmodium. This &ldquo / genease&rdquo / activity has provided a foundation for further investigation of the coding elements of the Plasmodium genome. MBN has been reported to cleave genomic DNA of Plasmodium preferentially at positions before and after genes, but not within gene coding regions. This mechanism has overcome the difficulty encountered in obtaining genes with low expression levels because the cleavage mechanism of the enzyme yields sequences of genes from genomic DNA rather than mRNA. However, as potentially useful as MBN may be, evidence to support its genease activity comes from analysis of a limited number of genes. It is not clear whether this mechanism is specific to certain genes or species of Plasmodia or whether it is a general cleavage mechanism for Plasmodium DNA .There have also been some projects (Nomura et al., 2001 / van Lin, Janse, and Waters, 2000) which have identified MBN generated fragments which contain fragments of genes with both introns and exons, rather than the intact genes expected from MBN-digestion of genomic DNA, which raises concerns about the efficiency of the MBN mechanism in generating complete genes.</p> <p><br /> Using a large-scale, whole genome mapping approach, 7242 MBN generated genome survey sequences (GSSs) have been mapped to determine their position relative to coding sequences within the complete genome sequences of the human malaria parasite Plasmodium falciparum and the incomplete genome of a rodent malaria parasite Plasmodium berghei. The location of MBN cleavage sites was determined with respect to coding regions in orthologous genes, non-coding /intergenic regions and exon-intron boundaries in these two species of Plasmodium. The survey illustrates that for P. falciparum 79% of GSSs had at least one terminal mapping within an ortholog coding sequence and 85% of GSSs which overlapped coding sequence boundaries mapped within 50 bp of the start or end of the gene. Similarly, despite the partial nature of P.berghei genome sequence information, 73% of P.berghei GSSs had at least one terminal mapping within an ortholog coding sequence and 37% of these mapped between 0-50 bp of the start or end of the gene. This indicates that a larger percentage of cleavage sites in both P.falciparum and P.berghei were found proximal to coding regions. Furthermore, 86% of P.falciparum GSSs had at least one terminal mapping within a coding exon and 85% of GSSs which overlapped exon-intron boundaries mapped within 50bp of the exon start and end site. The fact that 11% of GSSs mapped completely to intronic regions, suggests that some introns contain specific cleavage sites sensitive to cleavage and this also indicates that MBN cleavage of Plasmodium DNA does not always yield complete exons.</p> <p><br /> Finally, the results presented herein were obtained from analysis of several thousand Plasmodium genes which have different coding sequences, in different locations on individual chromosomes/contigs in two different species of Plasmodium. Therefore it appears that the MBN mechanism is neither species specific nor is it limited to specific genes.</p>

Magister Scientiae - MSc / The action of Mung Bean Nuclease (MBN) on DNA makes it possible to clone intact gene fragments from genes of the malaria parasite, Plasmodium. This &ldquo;genease&rdquo; activity has provided a foundation for further investigation of the coding elements of the Plasmodium genome. MBN has been reported to cleave genomic DNA of Plasmodium preferentially at positions before and after genes, but not within gene coding regions. This mechanism has overcome the difficulty encountered in obtaining genes with low expression levels because the cleavage mechanism of the enzyme yields sequences of genes from genomic DNA rather than mRNA. However, as potentially useful as MBN may be, evidence to support its genease activity comes from analysis of a limited number of genes. It is not clear whether this mechanism is specific to certain genes or species of Plasmodia or whether it is a general cleavage mechanism for Plasmodium DNA .There have also been some projects (Nomura et al., 2001;van Lin, Janse, and Waters, 2000) which have identified MBN generated fragments which contain fragments of genes with both introns and exons, rather than the intact genes expected from MBN-digestion of genomic DNA, which raises concerns about the efficiency of the MBN mechanism in generating complete genes.Using a large-scale, whole genome mapping approach, 7242 MBN generated genome survey sequences (GSSs) have been mapped to determine their position relative to coding sequences within the complete genome sequences of the human malaria parasite Plasmodium falciparum and the incomplete genome of a rodent malaria parasite Plasmodium berghei. The location of MBN cleavage sites was determined with respect to coding regions in orthologous genes, non-coding intergenic regions and exon-intron boundaries in these two species of Plasmodium. The survey illustrates that for P. falciparum 79% of GSSs had at least one terminal mapping within an ortholog coding sequence and 85% of GSSs which overlapped coding sequence boundaries mapped within 50 bp of the start or end of the gene. Similarly, despite the partial nature of P.berghei genome sequence information, 73% of P.berghei GSSs had at least one terminal mapping within an ortholog coding sequence and 37% of these mapped between 0-50 bp of the start or end of the gene. This indicates that a larger percentage of cleavage sites in both P.falciparum and P.berghei were found proximal to coding regions. Furthermore, 86% of P.falciparum GSSs had at least one terminal mapping within a coding exon and 85% of GSSs which overlapped exon-intron boundaries mapped within 50bp of the exon start and end site. The fact that 11% of GSSs mapped completely to intronic regions, suggests that some introns contain specific cleavage sites sensitive to cleavage and this also indicates that MBN cleavage of Plasmodium DNA does not always yield complete exons. Finally, the results presented herein were obtained from analysis of several thousand Plasmodium genes which have different coding sequences, in different locations on individual chromosomes/contigs in two different species of Plasmodium. Therefore it appears that the MBN mechanism is neither species specific nor is it limited to specific genes. / South Africa

C2H2-zinc fingers (ZFs) are commonly found in transcription factors that code for nearly 3% of gene products in the human genome. ZF proteins are commonly involved in gene regulation during development, cell differentiation, and tumor suppression. Each "finger" is a domain composed of approximately 30 amino acids. Since the discovery of these domains over 25 years ago, several groups have contributed to the structural and biochemical knowledge to understand their DNA-binding properties. Taking advantage of the simplicity of manipulating the DNA-binding potential of a ZF, the technology has now evolved to make sequence-specific Zinc Finger Nucleases (ZFNs), Artificial Transcription Factors (ATFs), Zinc Finger Recombinases, and DNA detection tools. ZFPs have been used for various applications, ranging from regulating genes by ZF-ATFs to manipulating genomes in diverse organisms. ZFNs have remarkably revolutionized the field of genome engineering. ZFN-modified T-cells have now advanced into human clinical trials for cell-based therapies as a treatment against HIV. Despite the advances in the ZFN technology, one of the challenges in the field is obtaining effective ZFNs using publicly available tools. The traditional method of synthesizing custom ZF arrays was using modular assembly (MA). In this method, preselected ZFs from publicly available one-finger archives can be assembled modularly to make long arrays. MA of ZFNs provides a rapid method to create proteins that can recognize a broad spectrum of DNA sequences. However, three- and four-finger arrays often fail to produce active nucleases. The low success rate of MA ZF arrays was attributed to the fact that they suffer from finger-finger incompatibility referred to as context-dependent effects. However, we hypothesized that the low affinity of MA arrays was the limiting factor. The work presented in this dissertation describes our efforts at addressing these fundamental methodological challenges. We developed the Extended Modular Assembly method that overcomes the limitations of both the previous Modular Assembly. We performed a systematic investigation of number and composition of modules on ZFN activity and analyzed ZFN specificity both in vitro and in vivo. Our current experiments apply the ZFNs produced by our method to study the role of genetic variation in human disease.

The phage-derived Lambda Red recombination system utilizes exogenous DNA in order to generate precise insertion, deletion, and point mutations in Escherichia coli and other bacteria. Due to its convenience, it is a frequently-used tool in genetics and molecular biology, as well as in larger-scale genome engineering projects. However, limited recombination frequency constrains the usefulness of Lambda Red for several important applications. In this work, I utilize a mechanism-guided approach in order to improve the power and utility of Lambda Red recombination.

CARVALHO, Edinilton Muniz. Reatividade do Nitrosilo Complexo de Ferro trans-[Fe(cyclam)(NO)Cl]Cl2. 2015. 169 f. Dissertação (Mestrado em Química)-Universidade Federal do Ceará, Fortaleza, 2015. / Submitted by Jairo Viana (jairo@ufc.br) on 2017-04-27T23:10:01Z No. of bitstreams: 1 2015_dis_emcarvalho.pdf: 6115679 bytes, checksum: c7683c81230542c04a08aa6928f54ec7 (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2017-04-27T23:10:16Z (GMT) No. of bitstreams: 1 2015_dis_emcarvalho.pdf: 6115679 bytes, checksum: c7683c81230542c04a08aa6928f54ec7 (MD5) / Made available in DSpace on 2017-04-27T23:10:16Z (GMT). No. of bitstreams: 1 2015_dis_emcarvalho.pdf: 6115679 bytes, checksum: c7683c81230542c04a08aa6928f54ec7 (MD5) Previous issue date: 2015 / Nitric oxide (NO), produced endogenously, is responsible for vasodilation of blood vessels and other physiological processes. The nitroxyl ion (NO-), a species generated by monoeletronic reduction of NO, has rised the interest of the scientific community due to its distinct biological activity of nitric oxide. Sodium nitroprusside (SNP) is part of a class of compounds that release NO, being the only metal complex used clinically, but there are problems associated with their use, including photosensitivity and release of cyanide. Therefore, alternative compounds including iron-based ones have been studied, such as trans-[Fe(cyclam)(NO)Cl]Cl2, whose studies of chemical reactivity as well as its precursor (trans-[Fe(cyclam)Cl2](PF6)), were investigated in this work. In addition, we evaluated the potential anticancer and microbicides these complexes. The nitrosyl complex showed the ability to release NO under physiological conditions and spontaneously when stimulated by light. The NO released by nitrosyl complex thermally and photochemically was probed by the reaction with selective traps at different. The half-life values (t1/2) for reaction of nitrosyl complex with cPTIO were 115 (k = 0.1 x 10-3 s-1) and 385 (k = 3.0 x 10-5 s-1) minutes to photochemical and thermal reactions, respectively, being well known that light accelerates the process of release. The photochemical test using myoglobin is indicated release of NO, however it was not possible to determine the rate of this reaction. The reaction of the complex with glutathione (GSH) was monitored by electron paramagnetic resonance (EPR) and suggested the production of HNO. Kinetic results for this reaction indicates that there is the formation of a reactive intermediate with band at 541 nm, likely the adduct trans-[Fe(cyclam)(Cl)N(O)SG]+. The rate constants indicate that the first intermediate rapidly forms (kobs = 2.79 s-1) and decays slowly (kobs = 7.17 x 10-5 s-1). The high stability of this intermediate can be related to its ability to engage in intramolecular interactions. Vasodilation assay showed that complex trans-[Fe(cyclam)(NO)Cl]2+ has vasodilating activity with EC50 = 910 nmol L-1. It was also found that the nitrosyl complex presented scavenging activity toward O2•⎯ ions, thus having antioxidant potential. Electrophoresis experiments employing plasmid DNA and the complex trans-[Fe(cyclam)Cl2]+ and trans-[Fe(cyclam)(NO)Cl]2+, showed nuclease activity stimulated by light (350 and 460 nm) or stimulated by biologically relevant agents (H2O2 and GSH). However, cell viability assays with cancer cells showed that both complexes have low cytotoxic activity, the complex trans-[Fe(cyclam)Cl2]+ and trans-[Fe(cyclam)(NO)Cl]2+, respectively, showed IC50 values equal to: 8.5 and 144.2 mol L-1 against strain B16-F10 cells, and 130.4 and 142.9 mol L-1 against HUH-7 cell line. Additionally, preliminary tests of bactericidal activity in Escherichia coli strains indicated that the compounds were effective at inhibiting bacterial growth. These studies support a synergistic action as releasing NO/HNO and vasodilating action, anticancer and microbicide, which deserve further pharmacological and biochemical investigations for a better description of the mechanisms of action. / O óxido nítrico (NO), espécie produzida endogenamente, é responsável pela vasodilatação dos vasos sanguíneos entre outros processos fisiológicos. O íon nitroxila (NO-), espécie gerada por redução monoeletrônica do NO, tem despertado o interesse da comunidade científica graças às suas atividades biológicas distintas do óxido nítrico. O nitroprussiato de sódio (SNP) faz parte de uma classe de compostos que liberam NO espontaneamente, sendo o único complexo metálico usado clinicamente, porém existem problemas associados ao seu uso, incluindo fotossensibilidade e liberação de cianeto. Desta forma, compostos alternativos a base de ferro tem sido estudados, tal como o trans-[Fe(cyclam)(NO)Cl]Cl2, cujos estudos de reatividade química, bem como de seu precursor (trans-[Fe(cyclam)Cl2](PF6)), foram investigados nesta dissertação. Adicionalmente, avaliou-se as potencialidades anticancerígenas e microbicidas destes complexos. O nitrosilo complexo exibiu a capacidade de liberar NO espontaneamente em condições fisiológicas e quando estimulado por luz. O NO liberado termicamente e fotoquimicamente reagiu com traps seletivos, em diferentes taxas de velocidade. Os valores de meia-vida (t1/2) para reação do nitrosilo complexo com cPTIO foram 115 (k = 0,1 x 10-3 s-1) e 385 (k = 3,0 x 10-5 s-1) minutos para reação fotoquímica e térmica, respectivamente, sendo notório que a luz acelera o processo de liberação. O ensaio fotoquímico empregando mioglobina indicou liberação de NO, contudo não foi possível determinar a taxa de reação entre as espécies. A reação do complexo de NO com glutationa (GSH), monitorada por espectroscopia de ressonância paramagnética electrónica (EPR), sugeriu a produção de HNO. Os resultados cinéticos para essa reação indicam que há a formação de um intermediário reativo com banda em 541 nm, referente ao aduto trans-[Fe(cyclam)(Cl)N(O)SG]+. As constantes de velocidade indicaram que o primeiro intermediário se forma rapidamente (kobs = 2,79 s-1) e decai lentamente (kobs = 7,17 x 10-5 s-1). A grande estabilidade desse intermediário pode estar relacionada a capacidade do mesmo apresentar interações intramoleculares que o estabilizam. Ensaios de vasodilatação, mostraram que o complexo trans-[Fe(cyclam)(NO)Cl]2+ possui atividade vasodilatadora com EC50 = 910 nmol L-1. Também foi verificado que o nitrosilo complexo apresentou atividade sequestradora de íons O2•⎯, tendo assim potencial anti-oxidante. Experimentos de eletroforese, empregando DNA plasmidial e os complexos trans-[Fe(cyclam)Cl2]+ e trans-[Fe(cyclam)(NO)Cl]2+, mostraram atividade nuclease estimulados por luz (350 e 460 nm), ou estimulados por agentes biologicamente relevantes (H2O2 e GSH). Todavia, ensaios de viabilidade celular com células cancerígenas indicaram que os dois complexos possuem baixa atividade citotóxica, os complexos trans-[Fe(cyclam)Cl2]+ e trans-[Fe(cyclam)(NO)Cl]2+, respectivamente, apresentaram valores de IC50 iguais a: 8,5 e 144,2 mol L-1 frente a linhagem de células B16-F10, e 130,4 e 142,9 mol L-1 frente a linhagem de células HUH-7. Adicionalmente, ensaios preliminares de atividade bactericida em cepas de Escherichia coli mostraram que os complexos foram eficientes na inibição do crescimento bacteriano. Estes estudos dão suporte a uma ação sinergística como liberador de NO/HNO e ação vasodilatadora, anticancerígena e microbicida, os quais merecem posteriores investigações farmacológicas e reatividade bioquímica para uma melhor descrição dos mecanismos de ação dos complexos.

Simple and novel nuclease models have been synthesized. These involve metal-binding ligand 1,4,7,10-tetraazlcyclododecane (cyclen) tethered to an acridine ring (a DNA-binding group) by amide linkers of various lengths. Binding of these probes to DNA was studied by monitoring changes in their UV-visible spectra affected by the presence of DNA. Titration of these compounds with increasing amounts of pBR322 DNA caused hypochromic effects and shifted the acridine absorption at 360nm to a longer wavelength. Under biologically relevant conditions (37°C and pH 7.4), specific transition metal complexes of these compounds are found to be highly effective catalysts toward the hydrolysis of plasmid DNA. This is demonstrated by their ability to convert the super-coiled DNA (form I) to open-circular DNA (form II). Structure-activity correlation studies show that hydrolytic activity depends on both the structure of ligand (L1>L2>L3) and the nature of metal ion cofactor (Co3+>Zn2+>Cr2+>Ni2+>Cu2+>Fe3+).

Fundação de Amparo a Pesquisa no Estado do Rio Grande do Sul / Despite the large number of antimicrobials and anti-tumor drugs, therapeutic arsenal is limited due to the emergence of bacterial resistance mechanisms and refractoriness, besides the toxicity of certain drugs. This has contributed to advances in scientific research, especially those aimed at obtaining new active agents against infectious diseases and cancer, with innovative and less toxicity mechanisms. From this perspective, due to the great versatility and diverse pharmacological properties demonstrated by triazenes (TZCs) This study aimed to evaluate in vitro biological activity of two compounds unpublished TZCs complexed with platinum (II): {trans [1- (2-bromophenyl ) -3- (2-nitrophenyl) triazenido) bis (pyridine) (chloro) platinum (II)} (C1) and cis- {[1- (2-nitrophenyl) -3- (2-bromofenil) triazenido)] (ditrifenilfosfina ) (chloro) platinum (II)} (C2). The antibacterial activity was performed by the conventional method of broth microdilution, by evaluating the minimum inhibitory concentration (MIC) against the bacterial strains benchmark American Type Culture Collection (ATCC) and clinical isolates with multiple resistance to drugs (RMD). Cytotoxicity was assessed by colorimetric assay based on reduction of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) compared to the bone marrow cells from patients diagnosed with leukemia treated at the University Hospital of Santa Maria. The chemical nuclease activity was verified by electrophoresis in agarose gel. According to the results showed that both compounds have antibacterial activity TZCs. The C1 complex showed activity against E. faecalis 51299, 15305 S. saprophyticus, E. faecalis 29212, S. aureus ATCC 25923 and S. aureus ATCC 29213. The C2 complex was able to inhibit growth of a bacterium RMD S. epidermidis 27. The studied complexes showed narrow spectrum of activity being active only against Gram positive bacteria. In addition, C1 promoted high percentage of cell death in the face of patient cells with Chronic Myeloid Leukemia (LMC) of 50.23% 75.59% will. With respect to nuclease activity, in investigated conditions, the compounds were not able to cleave the plasmid DNA. This study reflects the broad antimicrobial and cytotoxic activity of TZCs compounds. / Apesar do grande número de medicamentos antimicrobianos e antitumorais existentes, o arsenal terapêutico encontra-se limitado em virtude do surgimento de mecanismos de resistência bacteriana e refratariedade, além da toxicidade de alguns fármacos. Tal fato tem contribuído para os avanços nas investigações científicas, especialmente, aquelas que visam a obtenção de novos agentes ativos contra doenças infecciosas e o câncer, com mecanismos inovadores e menor toxicidade. Nessa perspectiva, devido a grande versatilidade e diversas propriedades farmacológicas demonstradas pelos triazenos (TZCs) este estudo teve como objetivo a avaliação in vitro da atividade biológica de dois compostos TZCs inéditos complexados com Platina (II): {trans[1-(2-bromofenil)-3-(2-nitrofenil)triazenido)-bis-(piridina)(cloro)platina(II)} (C1) e {cis[1-(2-nitrofenil)-3-(2-bromofenil)triazenido)](ditrifenilfosfina)(cloro)platina(II)} (C2). A atividade antibacteriana foi realizada pelo método convencional da microdiluição em caldo, através da avaliação da concentração inibitória mínima (CIM), frente às cepas bacterianas padrão de referência American Type Culture Collection (ATCC) e isolados clínicos com resistência múltipla às drogas (RMD). A citotoxicidade foi investigada através do ensaio colorimétrico baseado na redução do brometo de 3-(4,5-dimetiltiazol-2-il)-2,5 difeniltetrazólio (MTT) frente às células de medula óssea provenientes de pacientes diagnosticados com leucemia, atendidos no Hospital Universitário de Santa Maria. A atividade de nuclease química foi verificada por meio da técnica de eletroforese em gel de agarose. De acordo com os resultados, observou-se que ambos os compostos TZCs possuem atividade antibacteriana. O complexo C1 demonstrou atividade frente a E. faecalis 51299, S. saprophyticus 15305, E. faecalis 29212, S. aureus ATCC 25923 e S. aureus ATCC 29213. O complexo C2 foi capaz de inibir o crescimento de uma bactéria RMD, S. epidermidis 27. Os complexos estudados demonstraram atividade de estreito espectro sendo ativos somente frente a bactérias Gram positivas. Além disso, C1 promoveu elevada porcentagem de morte celular frente às células de paciente com Leucemia Mieloide Crônica (LMC) de 50,23% à 75,59%. No que se refere à atividade de nuclease, nas condições investigadas, os compostos não foram aptos a clivar o DNA plasmidial. Esse estudo reflete a ampla atividade antimicrobiana e citotóxica dos compostos TZCs.

Recombination Activating Genes (RAGs) orchestrate the process called V (D) J recombination, which enables the vertebrate adaptive immune system to specifically recognize millions of antigens. During this recombination process, V (variable), D (diversity) and J (joining) gene segments of antibody (B cell receptor) and TCR (T cell receptor) join by different possible combinations to generate antigen receptor diversity. This unique site specific recombination process is actuated by lymphoid specific proteins called RAG1 and RAG2 (RAGs or RAG complex). RAGs recognize a conserved sequence motif flanking the above subexons called Recombination Signal Sequence (RSS). There are two types of RSS known as 12-RSS and 23-RSS, where a conserved heptamer sequence and nonameric sequence is separated by 12 or 23 bp, respectively. RAGs specifically bind to RSS by RAG1 Nonamer Binding Domain (NBD) and generate nicks which are converted to DSBs via a hairpin intermediate and finally repaired by Non-Homologous DNA End Joining (NHEJ), a major DSB repair pathway in eukaryotes. Thus, RAGs act as a sequence specific endonuclease, and is unique to higher eukaryotes. Therefore, reduced or loss of RAG activity could result in immune deficiency syndromes like Omenn Syndrome (OS) and Severe Combined Immunodeficiency (SCID). Apart from acting as a sequence specific nuclease, RAGs have been shown to cleave on altered DNA structures like mismatches (bubbles), hairpins, flaps, gaps, triplexes and 3’ overhangs. This structure specific nuclease activity is implicated in causing genomic instability in B and T cells, particularly leading to generation of chromosomal translocations in certain lymphoid cancers. However, unlike the sequence- specific cleavage activity, this novel property of RAGs is poorly understood. Structure-specific nuclease activity of RAGs was characterized by using heteroduplex DNA substrate containing bubble region. RAG proteins were overexpressed and purified from human cell line and used for the assay. Results showed that RAGs cleave different bubble substrates with different efficiency. The role of DNA sequence at single-stranded region of heteroduplex DNA on RAG cleavage was investigated by synthesizing the substrate DNA having either adenineguanine/ thymine/ cytosine in the bubble sequence. Interestingly, efficient RAG cleavage was observed only when cytosines were present at single-stranded region, while thymine bubbles were cleaved with much lower efficiency. Adenine and guanine containing bubbles were not cleaved by RAGs. This was the first observation showing sequence specificity during structure-specific nuclease activity of RAGs. Besides, it was observed that RAG cleavage on bubbles with cytosines resulted in DSB formation, which is essential for generation of chromosomal translocations. Further, such specificity and cytosine preference was observed, even when RAGs acted on other altered DNA substrates like hairpin loops, 3’ overhangs and gaps. When the role of flanking duplex region on RAG cleavage was tested, only the 5’ duplex nucleotide was critical for RAG cleavage reaction and cytosine was the most preferred nucleotide. By systematic mutation of bubble region, it was observed that the two cytosines present at the double strand-single strand junction are critical for RAG cleavage. A single nucleotide bubble (mismatch) with cytosines was cleaved by RAGs with low but detectable efficiency. A bubble with at least 2 nt length possessing cytosine was cleaved with higher efficiency resulting in both single-stranded nicks and DSBs. Based on these studies, “C(d)C(s)C(s)” was proposed as a novel recognition motif for RAG cleavage, on altered DNA structures, where“d” and “s” represent double- and single-strand region, respectively. To be targeted by RAGs in vivo, the altered DNA substrates have to compete with RSS, the physiological substrate. It is not known whether such structures will be cleaved by RAGs, when present along with RSS. Besides, the regulation of the both structure and sequence specific nuclease activities are not studied. To address the above questions, RAG cleavage on bubble substrates was performed in presence of RSS either in cis or trans configuration. Results showed that both bubble substrates and RSS were cleaved with similar efficiency by RAGs. In fact, they can compete out each other in a concentration dependent manner. When kinetics of RSS and bubble cleavage were performed, RSS cleavage reaction was faster and saturated within 10-15 min, while bubbles cleavage started slow and went on increasing with time. This difference in kinetics persisted when both substrates were present together. This could be a regulatory mechanism for targeting RAGs to RSS sites and limiting bubble cleavage which can be deleterious to cells. HMGB1, a DNA binding protein which is shown to enhance RSS binding and synapsis, does not affect RAG action on bubble substrates. RAG postcleavage complex is formed during V(D)J recombination process where RAGs remain bound to cleaved RSS after cleavage, which limits further RAG action on other sites. Such cleavage complex was not detected on bubble substrates, which suggests that after cleavage RAGs were not associated to DSBs of bubble cleavage. Finally, the nonamer binding domain of RAG1 involved in RSS binding in V(D)J recombination, was found to be dispensable for the structure-specific nuclease activity and it appears that RAGs bind to bubble substrates using a different domain. In summary, in this study, the structure-specific nuclease activity of RAGs was characterized. A novel sequence motif that can regulate this activity of RAGs was identified. Though altered structures can be equally favored substrates as RSS, differences in reaction kinetics, cleavage complex formation and separate DNA binding domains regulate RAG cleavage, when it acts as a structure-specific nuclease. Thus, this study may help in the better understanding of RAG induced genomic instabilities in lymphoid tissues.

Interstrand cross-links (ICLs) are a type of DNA damage that prevents strand separation required for basic cellular processes. ICL-based anti-cancer therapies exploit the cytotoxic consequences of replication and transcription inhibition, however, they are limited by the ability of the cell to repair DNA crosslinks. The challenge of ICL repair involves coordinating multiple DNA repair pathways to remove damage occurring on both strands of DNA. Participation of factors that are both exclusive and essential to crosslink repair suggests a pathway requirement to process unique structures and/or intermediates arising only in ICL repair. SNM1A is a nuclease required for survival of human cells in response to ICL exposure, but the specific function and role of SNM1A remain unclear. Here we show that, in addition to known 5’-3’exonuclease activity, SNM1A possesses single-strand specific endonuclease activity. Furthermore, SNM1A exhibits translesion nuclease activity on crosslinks which deform the helical backbone, but not non-distorting stable ICLs. We report the identification and characterization of nine small molecules inhibitors of SNM1A, isolated from an in vitro high-throughput screen of nearly 4,000 bioactive compounds. Finally, we demonstrate that inhibitors of SNM1A potentiate the cytotoxicity of ICL-inducing agent cisplatin in HeLa cells. The work in this thesis expands the possible roles of SNM1A in ICL repair and lays the groundwork for SNM1A inhibition in ICL sensitization efforts. / Thesis / Doctor of Philosophy (PhD)

Les télomères sont des structures nucléo-protéiques présentes à l'extrémité des chromosomes. Ils sont un des facteurs garant de la stabilité génomique. Ils assurent la protection des extrémités des chromosomes et leur entière réplication. Les dysfonctionnements du télomère sont impliqués dans la tumorigénèse et le vieillissement.Un des rôles majeurs des télomères est d'éviter que les extrémités des chromosomes ne soient reconnues comme des cassures double brin de l'ADN et traitées comme telles par la machinerie de réparation. Cependant, de nombreuses protéines impliquées dans la reconnaissance et le métabolisme des cassures double brin, comme la protéine Tel1 et le complexe MRX par exemple, sont présentes au niveau des télomères et participent au maintien de leur taille par la télomérase. En s'appuyant sur cette analogie, j'ai étudié le rôle télomérique de l'endonucléase Sae2 et de l'hélicase Sgs1, impliquées dans l'étape de dégradation du brin 5' qui précède la réparation des cassures double brin de l'ADN par recombinaison.Les rôles des protéines Sae2 et Sgs1 ont été étudiés sur les télomères natifs et sur les télomères érodés lors de la sénescence réplicative. L'ensemble de mes résultats suggèrent que, bien que les télomères érodés en absence de télomérase soient reconnus comme une cassure double brin de l'ADN et traités comme tels par les nucléases et hélicases, le rôle majeur de Sae2 et Sgs1 au niveau des télomères natifs serait de les protéger contre des recombinaisons illégitimes au cours de leur réplication. / Telomeres are nucleoprotein complexes that protect the extremities of linear chromosomes, avoiding end-to-end fusions and nucleolytic degradation of chromosome ends. The failure of cells to properly maintain telomeres can be an important source of chromosome instability involved in cancer progression and aging.A major role of telomeres is to prevent chromosome ends from being recognized as damage-induced double-strand DNA breaks (DSBs). However, many proteins involved in recognition and processing of DSBs are also involved in telomeres maintenance, like Tel1 and MRX. Based on this analogy, I have studied the role at telomeres of the role of the endonuclease Sae2 and the helicase Sgs1, two proteins that have a key function in the processing of DSBs through nucleolytic degradation of their 5' end.The role of protein Sae2 and Sgs1 has been studied at native and eroded telomeres. My results showed that eroded telomeres, in telomerase deficient cells, are recognized and resected as a double-strand break DNA by a set of nucleases and helicases including Sae2 and sgs1. In contrast, the main role of Sae2 and Sgs1 at native telomeres would be to protect telomeres against illegitimate recombination during replication.