Master's paper (M.S.I.S.)--University of North Carolina at Chapel Hill, 2003. / "January 2003." Bibliography: p. 32-33. Also available in PDF via the World Wide Web.

A recombinação durante a transcrição reversa é um fator importante no aumento da diversidade genética e adaptação do HIV-1, permitindo que mutações vantajosas presentes em diferentes linhagens sejam combinadas em um mesmo genoma. No Brasil, vários recombinantes foram descritos e seis formas recombinantes circulantes (CRFs) já foram identificados, demonstrando a relevância destes recombinantes na epidemia brasileira. Portanto, um dos objetivos desta tese foi analisar os dados gerados pela Rede de Diversidade Genética Viral (VGDN) (sequências parciais de gag, pol e env), a fim de identificar recombinantes inter-subtipos de HIV-1 e avaliar a frequência e distribuição geográfica destes vírus. Utilizando diferentes técnicas foram identificados 152/1083 pacientes portadores de recombinantes BF. A frequência destes recombinantes foi maior em cidades como São Vicente (30%) e Sorocaba (22,6%), sendo que os recombinantes circulantes em São Vicente foram geralmente relacionados às CRF28 e CRF29, enquanto que os vírus presentes na região de Sorocaba comumente apresentam um envelope subtipo F1, independente do subtipo nos demais genes. Além disso, o gene da integrase de 159 pacientes foi amplificado e sequenciado. A análise deste gene revelou mais 10 pacientes infectados com recombinantes BF e nenhuma mutação de resistência primária aos inibidores da integrase foi encontrada. O segundo objetivo foi isolar e caracterizar recombinantes BF in vitro. O isolamento viral foi realizado por co-cultivo e ao final foram obtidos 10 isolados primários. O sequenciamento do genoma quase completo desses dez isolados primários revelou que três isolados primários pertencem ao grupo da CRF28_BF, três ao grupo da CRF29_BF e quatro foram classificados como formas recombinantes únicas (URFs). Ainda, o uso de correceptores desses isolados foi avaliado in vitro em ensaios com as células GHOST(3), e revelou três duplo-trópicos (X4/R5) vírus, quatro CXCR4 (X4) e três isolados utilizaram apenas CCR5 (R5). Em suma, uma alta frequência de URFs foi encontrada em algumas cidades do Estado de São Paulo, e também foi desenvolvido e caracterizado um painel de isolados primários representando as CRF28_BF, CRF29_BF e algumas URFs. / Recombination during reverse transcription is an important factor promoting HIV-1 diversity and adaptive change, allowing advantageous mutations arising on different genomes to undergo linkage in the same progeny recombinant genome more frequently than what would be expected under random mutation alone. In Brazil, several recombinant viruses were reported, and six circulating recombinant forms (CRFs) have already been identified. Therefore, the first objective of this Thesis was to analyze the data generated by the Viral Genetic Diversity Network (VGDN) (gag, pol and env partial sequences), in order to identify HIV-1 intersubtype recombinants and evaluate the frequency and geographical distribution of these viruses. Using different techniques we identified 152/1083 patients harboring BF recombinants. The frequency of these recombinants was higher in cities like São Vicente (30%) and Sorocaba (22.6 %). The recombinant viruses circulating in São Vicente were generally related to CRF28 and CRF29, while those viruses circulating in Sorocaba commonly presented an envelope region of subtype F1, irrespective the subtype composition on the remaining genes. Additionally, the integrase gene of HIV-1 from 159 patients was further amplified and sequenced. The analysis of this viral gene revealed ten more patients infected with BF recombinants and no primary mutations related to integrase inhibitor resistance were found. The second objective was to isolate and characterize BF recombinants in vitro, which resulted in ten primary HIV-1 isolates. The near full-length genomes of these ten primary isolates revealed that three were related to CRF28_BF, three to CRF29_BF and four were unique recombinant forms (URFs), according to their breakpoints profile determined with the jpHMM program. Additionally, the coreceptor usage of these isolate was investigated in vitro using GHOST assays, which revealed three dual-tropic (X4/R5) viruses, four CXCR4 (X4) viruses and three CCR5 (R5) viruses. In sum, we report a high frequency of URFs in some cities of São Paulo State, and also developed a well-characterized panel of viruses representing CRF28_BF, CRF29_BF and URFs.

Orientador: Maria Beatriz Machado Bonacelli / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Geociências / Made available in DSpace on 2018-08-07T03:16:33Z (GMT). No. of bitstreams: 1 Dias_ElianeLaranja_M.pdf: 1534782 bytes, checksum: 198c16ef5c088bd64b1e73aa1de5da87 (MD5) Previous issue date: 2006 / Resumo: A revolução nas áreas científicas e tecnológicas nos últimos anos, assim como a complexidade que envolve, atualmente, o desenvolvimento da pesquisa vêm ensejando a organização de novos arranjos e formas de cooperação entre distintos atores que compõem o processo de inovação. As redes de pesquisa em biotecnologia e genômica, como programas de investigação e seus nexos com a geração de novas tecnologias, vêm se apresentando como exemplos recentes das novas formas de articulação, sendo um importante instrumento de desenvolvimento científico e tecnológico e de busca de maior competitividade. O objetivo dessa dissertação é o de analisar e avaliar as motivações para a formação de duas redes de pesquisa genômica no país: a do fungo Crinipellis perniciosa, causador da doença vassoura de bruxa em cacau e a do Genolyptus, que trabalha com o eucalipto. A primeira, com forte viés de política pública, procura enfrentar um dos principais problemas fitopatológicos do país - o ataque de fungos nos cacaueiros - e resgatar uma atividade de grande importância econômica e social, notadamente na Bahia. A segunda, formada por uma rede nacional de pesquisa pré-competitiva em eucalipto, visa aumentar a produtividade e a competitividade de um dos setores de destaque na geração de divisas para o Brasil - o de celulose e papel. Procura-se verificar a influência de diferentes elementos na sua estruturação, configuração e desenvolvimento, incluindo a dinâmica técnico-concorrencial dos mercados em questão (cacau e chocolate e papel e celulose), a organização da pesquisa e do processo inovativo e o papel estratégico dos diferentes atores envolvidos. O trabalho está estruturado em três capítulos, sendo que os dois primeiros discutem as principais ferramentas da abordagem evolucionista integrada aos conceitos de cadeias produtivas e inovativas e de redes de pesquisa. O terceiro capítulo discute a formação dessas duas redes genômicas, com base nos conceitos apresentados e em dados primários levantados, visando apontar os elementos mais relevantes para a compreensão da constituição e do desenvolvimento das atividades relacionadas às redes, assim como suas principais particularidades. A realização da dissertação permitiu identificar um referencial importante para o estudo de arranjos cooperativos que congrega elementos das pesquisas sobre redes e sobre cadeias produtivas e inovativas, aplicando-o ao estudo das redes genômicas em questão. A contribuição deste estudo reside na proposição de que a articulação de uma rede de pesquisa é fortemente influenciada pelas características das cadeias produtivas e inovativas dos setores industriais envolvidos / Abstract: In recent years, the revolutions in science and technology and the complexity inherent to research development have led to the organization of nove I cooperation arrangements between the different parties involved in the process of innovation. Research networks in the areas of biotechnology and genomics, as well as research programs aimed at the production of new technologies are recent examples of these novel associations, constituting an important element in scientific and technological development and in the search for higher levels of competitive ability. The goal of this dissertation was to analyze and evaluate the motivations for the creation of two genomic research networks in Brazil: the Crinipellis perniciosa Genome Project, a fungus that causes. Witches' Broom disease of cocoa, and the Genolyptus Project, aimed at deciphering the transcriptome of Eucalyptus crops. The first network has a strong orientation towards public policies and aims at solving one of the main phytopathological problems in the country - fungal diseases of cacao trees - and to restore an agricultural activity of great economical and social importance to the State of Bahia. The latter network consists of a nation-wide association of pre-competitive research in Eucalyptus that aims at increasing the productivity and the competitive ability of one of the main production sectors that contributes to the generation of monetary assets in Brazil: the business of pulp and paper. We intend to verify the influence of the different elements to network structure, configuration, and development, also including the technical-competitive dynamics of the markets under consideration (cocoa and chocolate, as well as pulp and paper), the organization ofthe research and innovation process, and the strategic role of the different parties involved. This work is organized into three chapters, with the first two discussing the main tools used for the evolutionary approach integrated to the concepts of productive and innovative chains and research networks. The third chapter discusses the creation of these two genomic networks, based on the concepts presented previously and on the primary data obtained, with the intention of pointing out the most important elements necessary to understand the constitution and development of the activities related to these networks, as well as their main particularities. The realization of this dissertation allowed the identification of an important referential for the study of cooperative arrangements that conjugates elements of the research concerning networks and productive and innovative chains, and finally applying this referential to the study of the genomic networks under consideration. The contribution of this study resides in the proposition that the organization of a research network is strongly influenced by the particular characteristics of the productive and innovative chains ofthe industrial sectors involved. / Mestrado / Mestre em Política Científica e Tecnológica

O presente estudo teve como objetivo realizar a triagem de PHA sintases numa biblioteca metagenômica de solo de Mata Atlântica, empregando duas técnicas de busca: o método de detecção fenotípica e a técnica de PCR. Resultados positivos foram obtidos com este último, empregando iniciadores descritos na literatura e iniciadores descritos neste trabalho. Em 10.67% dos clones da biblioteca foram obtidos amplicons, dos quais 7 foram seqüenciados, apresentando similaridade para os genes phaC dos tipos II e IV. Adicionalmente, 67 clones positivos para a classe III foram obtidos e 4 destes foram seqüenciados. Duas das seqüências obtidas mostraram alta similaridade com o gene codificador da enzima glutamina sintetase tipo I, outro deles para a proteína hipotética conservada pertencente à família de enzimas oxidoredutases e a outra para o componente D do gene hidrogenase-4. A partir da análise dos resultados, iniciadores mais específicos são propostos. Assim, a técnica de PCR foi mais eficiente na detecção de genes da biossíntese de PHA na biblioteca metagenômica estudada. / To perform a PHA synthase screening in a metagenomic library from Atlantic forest soil two search methods were applied: phenotypic detection and PCR. Positive results with PCR were obtained by using primers described in the literature and proposed in this study. Amplicons were obtained in 10.67% of the library, 7 of them were sequenced showing similarity with class II and IV phaC genes. In addition, 67 positive clones for class III were obtained and 4 of them were sequenced. Two of these sequences showed high similarity to the glutamine synthase gene type I, the third one showed similarity to the conserved hypothetical protein of the reductase family, and the forth presented similarity to the component D of the hidrogenase-4. According to the results, more specific primers are suggested. Therefore, PCR was more efficient in the detection of PHA biosynthesis genes in the studied metagenomic library.

Caulobacter crescentus pertence ao grupo das proteobactérias e apresenta a característica distinta de diferenciação celular a cada divisão. Este trabalho visou desvendar os mecanismos de reparo de DNA em C. crescentus. Identificamos 44 genes pertecentes ao regulon SOS através da construção de um mutante para o repressor deste, LexA. Caracterizamos funcionalmente alguns dos genes do regulon, como CC_2272 (que codifica uma proteína da família das endonucleases III) e CC_2433. A cepa deficiente em LexA apresentou morfologia filamentosa, e por esse motivo, buscamos também desvendar quais seriam os fatores genéticos responsáveis por esta morfologia. Investigamos também os processos de controle do ciclo celular após a introdução de danos na molécula de DNA pela luz UVC, em mutantes deficientes para diferentes vias de reparo. Estes experimentos nos mostraram que as células procariontes possuem mecanismos para acoplar a progressão do ciclo celular a integridade do material genético. Este trabalho abre novas e excitantes possibilidades no campo da biologia bacteriana. / Caulobacter crescentus belongs to the proteobacteria group and exhibts the distinctive feature of cellular differentiation after each division. This work aimed to reveal the DNA repair mechanisms in C. crescentus. We have identified 44 genes belonging to the SOS regulon through the construction of a mutant strain to its repressor. We have functionally characterized some of its genes, like CC_2272 (that encodes an endonuclease III family protein) and CC_2433. The lexA strain showed filamentous morphology, e because of that, we have tried to discover which the genetic factors responsible for this morphology were. We have also investigated the cell cycle control processes after the introduction of damages in the DNA by the UVC light, in mutant strains deficient in different repair pathways. These experiments showed us that prokaryotic cells possess mechanisms to couple the cell cycle progression to the integrity of the genetic material. This work opens new and exciting possibilities in the field of bacterial biology.

Orientador: Zanoni Dias / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Computação / Made available in DSpace on 2018-08-21T22:58:04Z (GMT). No. of bitstreams: 1 Iizuka_VictordeAbreu_M.pdf: 1453681 bytes, checksum: 1fec01321d56a93084d2597366b44422 (MD5) Previous issue date: 2012 / Resumo: A teoria da seleção natural de Darwin afirma que os seres vivos atuais descendem de ancestrais, e ao longo da evolução, mutações genéticas propiciaram o aparecimento de diferentes espécies de seres vivos. Muitas mutações são pontuais, alterando a cadeia de DNA, o que pode impedir que a informação seja expressa, ou pode expressá-la de um modo diferente. A comparação de sequências é o método mais usual de se identificar a ocorrência de mutações pontuais, sendo um dos problemas mais abordados em Biologia Computacional. Rearranjo de Genomas tem como objetivo encontrar o menor número de operações que transformam um genoma em outro. Essas operações podem ser, por exemplo, reversões, transposições, fissões e fusões. O conceito de distância pode ser definido para estes eventos, por exemplo, a distância de reversão é o número mínimo de reversões que transformam um genoma em outro [9] e a distância de transposição é o número mínimo de transposições que transformam um genoma em outro [10]. Nós trataremos os casos em que os eventos de reversão e transposição ocorrem de forma isolada e os casos quando os dois eventos ocorrem simultaneamente, com o objetivo de encontrar o valor exato para a distância. Nós criamos modelos de Programação por Restrições para ordenação por reversões e ordenação por reversões e transposições, seguindo a linha de pesquisa utilizada por Dias e Dias [16]. Nós apresentaremos os modelos de Programação por Restrições para ordenação por reversões, ordenação por transposições e ordenação por reversões e transposições, baseados na teoria do Problema de Satisfação de Restrições e na teoria do Problema de Otimização com Restrições. Nós fizemos comparações com os modelos de Programação por Restrições para ordenação por transposições, descrito por Dias e Dias [16], e com as formulações de Programação Linear Inteira para ordenação por reversões, ordenação por transposições e ordenação por reversões e transposições, descritas por Dias e Souza [17] / Abstract: The Darwin's natural selection theory states that living beings of nowadays are descended from ancestors, and through evolution, genetic mutations led to the appearance of different kinds of living beings. Many mutations are point mutations, modifying the DNA sequence, which may prevent the information from being expressed, or may express it in another way. The sequence comparison is the most common method to identify the occurrence of point mutations, and is one of the most discussed problems in Computational Biology. Genome Rearrangement aims to find the minimum number of operations required to change one sequence into another. These operations may be, for example, reversals, transpositions, fissions and fusions. The concept of distance may be defined for these events, for example, the reversal distance is the minimum number of reversals required to change one sequence into another [9] and the transposition distance is the minimum number of transpositions required to change one sequence into another [10]. We will deal with the cases in which reversals and transpositions events occur separately and the cases in which both events occur simultaneously, aiming to find the exact value for the distance. We have created Constraint Programming models for sorting by reversals and sorting by reversals and transpositions, following the research line used by Dias and Dias [16]. We will present Constraint Logic Programming models for sorting by reversals, sorting by transpositions and sorting by reversals and transpositions, based on Constraint Satisfaction Problems theory and Constraint Optimization Problems theory. We made a comparison between the Constraint Logic Programming models for sorting by transpositions, described in Dias and Dias [16], and with the Integer Linear Programming formulations for sorting by reversals, sorting by transpositions and sorting by reversals and transpositions, described in Dias and Souza [17] / Mestrado / Ciência da Computação / Mestre em Ciência da Computação

Les systèmes toxine-antitoxine sont divisés en trois classes suivant la nature et le mode d’action de l’antitoxine. Ils sont fortement représentés au sein du règne bactérien et se trouvent sur des éléments génétiques mobiles qu’ils stabilisent dans la population bactérienne, mais aussi sur les chromosomes bactériens où leur fonction n’a pas encore été établie avec certitude. Au cours de ce travail, nous avons étudié les systèmes toxine-antitoxine bactériens de classe II, qui sont généralement composés de deux gènes organisés en opéron. Le premier gène code pour une antitoxine qui antagonise l’activité de la toxine, le produit du second gène. L’antitoxine, en complexe avec la toxine, est également capable de réguler l’expression de l’opéron en se fixant au promoteur de l’opéron. Lors du commencement de cette étude, les systèmes toxine-antitoxine étaient divisés en 10 familles sur base des similarités de séquences partagées par les toxines. A chaque famille de toxine était associée une famille d’antitoxine. <p>\ / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Le travail effectué dans cette thèse présente une nouvelle approche de la théorie du code circulaire dans les gènes qui a été initiée en 1996. Cette approche consiste à analyser les motifs construits à partir de ce code circulaire, ces motifs particuliers sont appelés motifs de code circulaire. Ainsi, nous avons développé des algorithmes de recherche pour localiser les motifs de code circulaire dans les séquences d'acides nucléiques afin de leur trouver une signification bioinformatique. En effet, le code circulaire X identifie dans les gènes est un ensemble de trinucleotides qui a la propriété de retrouver, synchroniser et maintenir la phase de lecture. Nous avons commencé notre analyse avec le centre de décodage du ribosome (ARNr) qui est une région majeure dans le processus de traduction des gènes aux protéines. Puis, nous avons étendu les résultats obtenus avec le ribosome aux ARN de transfert (ARNt) pour étudier les interactions ARNr-ARNt. Enfin, nous avons généralisé la recherche de motifs de code circulaire X dans l'ADN aux chromosomes d'eucaryotes complets. / The work done in this thesis presents a new direction for circular code identified in 1996 by analysing the motifs constructed from circular code. These particular motifs are called circular code motifs. We applied search algorithms to locate circular code motifs in nucleic acid sequences in order to find biological significance. In fact, the circular code X, which was found in gene sequences, is a set of trinucleotides that have the property of reading frame retrieval, synchronization and maintenance. We started our study in the ribosomal decoding centre (rRNA), an important region involved in the process of translating genes into proteins. Afterwards, we expanded our scope to study the interaction of rRNA through the X circular code. Finally, we search for the X circular code motifs in the complete DNA sequences of chromosomes of the eukaryotic genomes. This study introduced new properties to the circular code theory.

La compréhension de l'évolution des génomes est un des enjeux clé de la biologie actuelle. Nous avons rédigé une revue littéraire consacrée à la contribution de la génomique dans la compréhension de la diversité, de l'évolution et des phénotypes d'un super-phylum bactérien, le super-phylum PVC (Planctomycetes, Verrucomicrobiae et Chlamydiae). Ces bactéries proviennent d'environnements variés et présentent des caractéristiques phénotypiques intéressantes. Les analyses génomiques ont révélé la grande diversité de ces espèces, mais ont aussi permis de reconstruire l'évolution de leurs génomes et d'expliquer l'apparition de certains phénotypes particuliers. Une partie de notre travail était consacré à l'étude de l'évolution et de l'impact de la présence d'un plan cellulaire particulier chez les bactéries PVC. Ce plan cellulaire est sujet a différentes interprétations et induirait la compartimentation des cellules en deux régions distinctes. Les résultats obtenus semblent indiquer que cette caractéristique n'induit pas une protection des génomes bactériens vis à vis des transferts de gènes horizontaux, comme on pourrait le supposer. En revanche les observations microscopiques réalisées sur deux espèces ont permis de mieux appréhender l'évolution de ce plan cellulaire. Nous avons, de plus, détecté une contribution de l'environnement concernant la sélection des gènes transférés. Il semblerait que les gènes transférés soient en effet sélectionnés selon leurs fonctions par les différents environnements.Nos travaux ont donc permis d'améliorer la compréhension des relations entre l'évolution, les phénotypes et l'environnement, en particulier chez les bactéries du super-phylum PVC. / The comprehension of genomes evolution is a key issue of modern biology.We wrote a review dedicated to the genomic contribution in comprehension of diversity, evolution and phenotypes, in a bacterial super-phylum named PVC (for Planctomycetes, Verrucomicrobiae and Chlamydiae). These bacteria are distributed in varied environments and present specific phenotypic characteristics. Genomic analyzes revealed the important diversity of these species and allow also to reconstruct the genomes evolution and, in some cases, to explain the presence of specific phenotypes. One part of our work was dedicated to the study of evolution and impact of one of this phenotype, the special cell plan detected in PVC bacteria. This original cell plan is subject to different interpretations and induces the compartmentalization of cells in two different regions, whom one containing the nucleoid. Our results indicate that this feature has probably no role in the protection of bacterial genomes against horizontal genes transfers, so, its function is still unknown. Microscopic observations of two species from PVC super-phylum permit to better understand the evolution of the special cell plan. The environment seems to contribute in the genomes evolution, by selection of genes transferred. Genes transferred are probably selected according to their functions by the different environments.Our works allowed to improve the knowledge about relations between evolution, genomes, phenotypes and environment, especially in bacteria from PVC super-phylum.

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.