Thesis (Ph. D.)--Institute of Paper Chemistry, 1942. / Includes bibliographical references (p. 106-107).

No description available.

abstract: V(D)J recombination is responsible for generating an enormous repertoire of immunoglobulins and T cell receptors, therefore it is a centerpiece to the formation of the adaptive immune system. The V(D)J recombination process proceeds through two steps, site-specific cleavage at RSS (Recombination Signal Sequence) site mediated by the RAG recombinase (RAG1/2) and the subsequent imprecise resolution of the DNA ends, which is carried out by the ubiquitous non-homologous end joining pathway (NHEJ). The V(D)J recombination reaction is obliged to be tightly controlled under all circumstances, as it involves generations of DNA double strand breaks, which are considered the most dangerous lesion to a cell. Multifaceted regulatory mechanisms have been evolved to create great diversity of the antigen receptor repertoire while ensuring genome stability. The RAG-mediated cleavage reaction is stringently regulated at both the pre-cleavage stage and the post-cleavage stage. Specifically, RAG1/2 first forms a pre-cleavage complex assembled at the boarder of RSS and coding flank, which ensures the appropriate DNA targeting. Subsequently, this complex initiates site-specific cleavage, generating two types of double stranded DNA breaks, hairpin-ended coding ends (HP-CEs) and blunt signal ends (SEs). After the cleavage, RAG1/2 proteins bind and retain the recombination ends to form post-cleavage complexes (PCC), which collaborates with the NHEJ machinery for appropriate transfer of recombination ends to NHEJ for proper end resolution. However, little is known about the molecular basis of this collaboration, partly attributed to the lack of sensitive assays to reveal the interaction of PCC with HP-CEs. Here, for the first time, by using two complementary fluorescence-based techniques, fluorescence anisotropy and fluorescence resonance energy transfer (FRET), I managed to monitor the RAG1/2-catalyzed cleavage reaction in real time, from the pre-cleavage to the post-cleavage stages. By examining the dynamic fluorescence changes during the RAG-mediated cleavage reactions, and by manipulating the reaction conditions, I was able to characterize some fundamental properties of RAG-DNA interactions before and after cleavage. Firstly, Mg2+, known as a physiological cofactor at the excision step, also promotes the HP-CEs retention in the RAG complex after cleavage. Secondly, the structure of pre-cleavage complex may affect the subsequent collaborations with NHEJ for end resolution. Thirdly, the non-core region of RAG2 may have differential influences on the PCC retention of HP-CEs and SEs. Furthermore, I also provide the first evidence of RAG1-mediated regulation of RAG2. Our study provides important insights into the multilayered regulatory mechanisms, in modulating recombination events in developing lymphocytes and paves the way for possible development of detection and diagnotic markers for defective recombination events that are often associated immunodeficiency and/or lymphoid malignancy. / Dissertation/Thesis / Ph.D. Molecular and Cellular Biology 2012

RAGs (Recombination Activating Genes) are responsible for generation of antigen receptor diversity in case of B-cells and T-cells, through the process of combinatorial joining of different V (variable), D (diversity) and J (joining) gene segments. Each of these segments are flanked by recombination signal sequences (RSS), which consist of a conserved heptamer and nonamer separated by a less conserved spacer of 12 or 23 bp. RAGs recognize and cleave at the 5’ end of heptamer, leading to the formation of hairpin coding ends and blunt signal ends. The coding ends are joined through the process of no homologous DNA end joining (NHEJ), leading to the rearrangement of variable region of antigen receptors. Apart from its physiological property, RAGs can also act as a structure-specific nuclease. Previously, it has been shown that inadvertent action of RAGs on cryptic RSS and non B-DNA structures can lead to the generation of genomic instability and cancer. A very coordinated expression of RAGs has been observed in pro- and pre-B cells of the lymphoid system, which overlaps with the window of productive rearrangement during V(D)J recombination. Besides, studies by us and others have shown that RAG cleavage at altered DNA structures and cryptic RSS leads to chromosomal translocations resulting into cancer. However, several questions related to regulation of RAG expression and its activity in lymphoid cells remains to be answered. Previous studies have suggested regulation of RAG expression at different levels, such as methylation, ubiquitination, phosphorylation and by coordinate action of various transcription factors. In the present study, we evaluate the potential role of miRNAs in the regulation of RAG expression and its function in lymphoid cells. miRNAs are small, single-stranded non-coding RNAs, which play an important role in the regulation of gene expression. They play a critical role in the regulation of different cellular functions. Although there are miRNAs identified to play critical role during development of immune system, several key questions such as its role in the regulation of RAGs is yet to be addressed. In the current study, we have used bioinformatics approach to extract potential miRNAs that bind to 3’UTR of RAG1 and RAG2. miRNA expression datasets were downloaded from NCBI SRA database and extensive evaluation was done using various bioinformatics tools such as Bowtie, Sam tools, Bam tools, Bed tools and R package. We screened the miRNA expression profile across different stages of B-cell development (pro, pre, immature and mature B-cells), which overlap with the narrow window of RAG expression. The shortlisted miRNAs were further analyzed using miRNA databases such as miRBase, Targetscan and EMBL. Results showed that 33 miRNAs were specific to RAG1, among that one (miRNA1) followed RAG expression profile in B-cells. Besides miRNA2, which is a novel miRNA, was selected only on the basis of RAGs expression profile in a stage specific manner and the complementarity of the seed sequence of miRNA2 to the 3’UTR of RAG1 was checked manually. Interestingly, we observed that RAG1 expression was significantly down regulated in the presence of these miRNAs. However, there was no significant difference in the levels of other genes analysed. Further, semi-quantitative RT-PCR analysis confirmed the endogenous processing of pre-miRNA into mature miRNA using the cellular machinery. Besides, enrichment of 3’UTR of seed region of these miRNAs, enhanced the expression level of RAG1. Importantly, the enhancement in RAG1 expression level was limited in case of mature B-cells, where RAG expression is normally not observed. Further, transfection of lymphoid cells with miRNA inhibitors, specific to the miRNAs under study, showed the enhancement in RAG1 expression in lymphoid cells. In addition to this, specificity of selected miRNAs was confirmed by performing 3’UTR reporter assays, where enhanced luciferase expression was observed in case of mutant 3’UTR, while it was minimal in case of wild type constructs. Endogenous expression levels of selected miRNAs were evaluated in both lymphoid and nonlymphoid normal tissues and cancer cells using RT-PCR. Interestingly, we observed inverse correlation of expression levels of miRNA and RAG expression in all the cells tested. Besides, miRNA expression levels were less in pre-B cells and T-cells, owing to the increased expression of RAGs. Apart from this, recombinogenic potential of candidate miRNAs was assessed using episomal based V(D)J recombination assays. Interestingly we observed significant decrease (2-4 fold) in the V(D)J recombination efficiency when miRNA1 or 2 constructs were transfected in Nalm6 cells, as compared to that of controls, where no miRNAs were used. However, in case of Reh cells upon transfection with miRNA1construct, the decrease in recombination potential was upto 9 fold. Hence, we identify two miRNAs that can play an important role in the regulation of RAG1 expression and its physiological activity. Further, studies are being carried out to confirm their role in the regulation of RAG1 during different developmental stages of lymphoid cells in mice. As stated above, in addition to the sequence-specific activity, RAG possesses structure-specific nuclease activity as well. It has been shown that RAGs can cleave different types of altered DNA structures. Studies from our laboratory showed that even when RAGs act as a structure-specific nuclease there is a sequence bias. Presence of cytosine and thymine at the single-stranded region of heteroduplex DNA is important for RAG nicking and double-strand break (DSB) formation. In addition, proximity of a nonamer to bubble structures can enhance RAG cleavage. However, the role of immediate flanking sequences in the RAG mediated cleavage at heteroduplex regions is not understood. We investigated the role of flanking double-stranded DNA sequences in the regulation of RAG cleavage on non-B DNA structures. We found that RAG binding and cleavage on heteroduplex DNA is dependent on the length of double-stranded flanking region. Besides, immediate flanking regions of the heteroduplex DNA affected the RAG binding and cleavage in a sequence dependent manner. Interestingly, we also observed that the cleavage efficiency of RAGs at heteroduplex region was influenced by the phasing of DNA. Thus, our results suggest that sequence, length and phase positions of the DNA can affect the efficiency of RAG cleavage when it acts as a structure-specific nuclease. These findings provide novel insights into regulation of the pathological action of RAGs. Previous studies have shown that in addition to formation of coding and signal joints during V(D)J recombination, nonstandard V(D)J recombination products known as hybrid joints and open-shut joints may be formed, particularly in certain aberrant conditions such as defective NHEJ machinery. Interestingly, the hybrid and open-shut joints closely resemble the transposition mechanisms associated with transposons oretroviruses. Studies have also shown that RAGs possess structural similarity with integrases in domain organization. Both the proteins have Zinc Finger Binding domain (ZFB) which helps in multimerization of the protein, a central catalytic core domain comprising three acidic amino acids D, D and E essential for enzymatic activity and C-terminal domain (CTD) responsible for nonspecific binding to the DNA. Previous studies from our laboratory showed that, Elvitegravir, an inhibitor of integrase could interfere with the biochemical functions of the RAGs in vitro. Specifically, it inhibited the RAG binding and cleavage at RSS, hairpin formation, post-cleavage complex formation involving 12RSS and 23RSS. Using the episomal assay system that mimics signal joints (pGG49) and coding joints (pGG51), we show that Elvitegravir can inhibit V(D)J recombination inside cells. Interestingly we observed 3-6 fold decrease in the recombination frequency in signal ends joining, when treated with increasing concentrations (100, 500 and 1000 nM) of Elvitegravir. A 5-8 fold decrease in coding joints formation was also observed upon treatment with the inhibitor. The presence of recombination was confirmed by restriction digestion followed by sequencing analysis. Further analysis of recombination junctions revealed extensive deletion before joining in the case of Elvitegravir treated samples. Insertions or substitutions near to the recombination junctions were also prominent in treated samples. In depth analysis of sequenced junctions showed the presence of sequence having the features to form hairpins both upstream and downstream to the RSS sequences and was the site of cleavage in cases were higher deletion was observed. The analyzed recombinants did not show any signal joints or coding joints formation in treated samples. This suggests that Elvitegravir affects the physiological function, the V(D)J recombination of RAGs inside the cells. Thus, in the present study, we show that RAGs can be regulated by specific miRNAs. We have identified two potential miRNAs, which can regulate the RAG expression as well as its function in different stages of B- and T-cell development. Further, we also identify a novel regulatory mechanism for the structure-specific activity of the RAG complex. In addition to this, we find that integrase inhibitor, Elvitegravir, affects V(D)J recombination within B-cells, indicating its potential deleterious impact in HIV patients, which needs to be further evaluated.

No description available.

Characterizing the uncertainty in rainfall using stochastic models has been a challenging area of research in the field of operational hydrology for about half a century. Simulated sequences drawn from such models find use in a variety of hydrological applications. Traditionally, parametric models are used for simulating rainfall. But the parametric models are not parsimonious and have uncertainties associated with identification of model form, normalizing transformation, and parameter estimation. None of the models in vogue have gained universal acceptability among practising engineers. This may either be due to lack of confidence in the existing models, or the inability to adopt models proposed in literature because of their complexity or both. In the present study, a new nonparametric Matched Block Bootstrap (MABB) model is proposed for stochastic simulation of rainfall at daily time scale. It is based on conditional matching of blocks formed from the historical rainfall data using a set of predictors (conditioning variables) proposed for matching the blocks. The efficiency of the developed model is demonstrated through application to rainfall data from India, Australia, and USA. The performance of MABB is compared with two non-parametric rainfall simulation models, k-NN and ROG-RAG, for a site in Melbourne, Australia. The results showed that MABB model is a feasible alternative to ROG-RAG and k-NN models for simulating daily rainfall sequences for hydrologic applications. Further it is found that MABB and ROG-RAG models outperform k-NN model. The proposed MABB model preserved the summary statistics of rainfall and fraction of wet days at daily, monthly, seasonal and annual scales. It could also provide reasonable performance in simulating spell statistics. The MABB is parsimonious and requires less computational effort than ROG-RAG model. It reproduces probability density function (marginal distribution) fairly well due to its data driven nature. Results obtained for sites in India and U.S.A. show that the model is robust and promising.

Human herpesvirus 6A (HHV-6A) has yet to be definitively linked to a specific disease. This is due in part to the ubiquitous nature of the virus. Humanized Rag2-/-γc-/- (Rag-hu) mice were tested to determine if these were a suitable animal model to study the virus. Both cell-free and cell-associated virus was used for infection and both were found to be efficient at infecting the mice. Viral DNA was found in the plasma and cellular blood fractions, bone marrow, lymph node, and thymus, indicating successful infection and propagation of the virus in vivo. The CD3+CD4- population was depleted, while the CD3-CD4+ was increased in infected animals. The CD3-CD4+CD8- and CD3+CD4+CD8- populations were depleted and the CD3+CD4+CD8+ population increased when analysis was gated upon CD4+ cells. The CD3-CD4+CD8+ population expanded and the CD3-CD4+CD8- population was reduced when analysis was gated on the CD3- population. Additional flow cytometry analysis revealed increases in CD4+CD8+ double positive cells in the peripheral blood of cell-free infected mice, which could indicate improper T cell selection and a premature departure of these cells from the thymus, possibly contributing to autoimmunity. Previous research has shown that HIV and HHV-6A may have a synergistic effect on one another and that HHV-6A may act as a cofactor in the progression to AIDS. After determining the Rag-hu mouse model was suitable for studying HHV-6A infection, a coinfection of HHV-6A and HIV-1 was performed. Coinfected mice had fewer thymocytes when compared with the HIV-1 only, mock-infected, and to a lesser extent HHV-6A only groups which could indicate increased cell death in the coinfected group as well as possible disruptions in migration of cells, either causing cells to be sequestered in the bone marrow and unable to migrate to the thymus, or causing premature egress of the cells in the thymus due in part to premature upregulation of CCR7, both of which would explain the smaller cellular populations found in the coinfected mouse thymi. Additional studies were performed to determine if a preferential targeting existed between HHV-6A and HIV-1 as these viruses are found simultaneously coinfecting the same cell. Preferential targeting was not observed by cell-associated migration assay, but increased migration of HHV-6A-infected cells was observed in a CCL21 dependent manner. These studies have provided useful information about HHV-6A and its relevance to HIV/AIDS as well as a possible mechanism of the involvement of HHV-6A in multiple sclerosis (MS) and other autoimmune diseases.

Vascular stiffening is defined as the reduced ability of the blood vessels to expand in response to an increase in blood pressure. Vascular stiffening is often not appreciated as a disease in and of itself but is important to recognize because it is considered a predictor of many cardiovascular disease states. Mechanisms of vascular stiffening remain largely unknown; however the immune system has been found to play major roles in cardiovascular disease and arterial remodeling. This dissertation therefore seeks to illustrate the role of the adaptive immune system in vascular stiffening. This has been done by modeling vascular stiffness in transgenic mice lacking an adaptive immune system as well as immunosuppression in normal mice using a novel method to stimulate regulatory T cells with a cytokine immune complex. We have found that inhibition of the immune system by the use of a genetic knockout (RAG 1 ⁻/⁻ mice) or suppression of an existing immune system with an IL-2/anti-IL-2 complex reduces the development of angiotensin II-induced vascular stiffening. This dissertation supports the role of the adaptive immune system, and particularly CD4⁺T cells, in the development of vascular stiffening as well as the protective roles of Tregs in the disease. It also highlights the use of the IL-2/anti-IL-2 complex as a new potential therapy for vascular stiffness. Therapeutics that suppress adaptive immune function may be beneficial in the treatment of vascular stiffening.

The lymphoid tissues consist of distinct cell subpopulations of B and T cell lineages and possess complex signaling pathways that are controlled by a myriad of molecular interactions. During the fine-tuned developmental process of the lymphoid system, inappropriate activation of oncogenes and loss of tumor suppressor gene activity can push lymphocytes into uncontrolled clonal expansion, causing several lymphoid malignancies. V(D)J recombination is one such essential process, important for the proper development of the mammalian immune system. However, mistakes in normal V(D)J recombination can lead to deletion of tumor suppressor genes or activation of proto-oncogenes. In the first part of the study, the physiological and pathological roles of DNA binding domain of RAG1 have been characterized. RAG (Recombination Activating Gene) complex consisting of RAG1 and RAG2, is a site specific endonuclease responsible for the generation of antigen receptor diversity. It cleaves a specific DNA sequence termed as recombination signal sequence (RSS), comprising of a conserved heptamer and nonamer. Recent studies have shown that RAGs can also act as a structure-specific nuclease by cleaving flaps, heterologous loops, bubbles, hairpins etc. Nonamer binding domain (NBD) of RAG1 plays a central role in the recognition of RSS during its sequence specific activity. To investigate its DNA binding properties, NBD of murine RAG1 was cloned, overexpressed and purified from E. coli. Electrophoretic mobility shift assays showed that NBD binds with high affinity to nonamer in the context of 12/23 RSS. However, it did not bind to heteroduplex DNA, irrespective of the sequence of the single-stranded region. Interestingly, when a nonamer was present next to a heteroduplex DNA, NBD exhibited robust binding. NBD binding was specific to thymines when single stranded DNA containing poly A, C, G and T were used. Biolayer interferometry studies showed that the observed poly T binding to NBD was robust with a binding constant of 0.45±0.16 µM. >23 nt was essential for NBD binding at homothymidine stretches. On a double-stranded DNA, NBD could bind to A:T stretches, but not G:C stretches or random sequences. Although NBD is indispensable for sequence-specific activity of RAGs, external supplementation of purified nonamer binding domain to NBD deleted cRAG1/cRAG2 did not restore the sequence specific activity, suggesting that the overall domain architecture of RAG1 is important for maintaining its properties. Therefore, we define the sequence requirements of NBD binding to double- and single-stranded DNA, which will have implications in generation of chromosomal rearrangement and genomic instability in lymphoid cells. Genetic alterations are one of the hallmarks of lymphoid malignancies. Many genes involved in chromosomal abnormalities are known to play central roles in the development of normal lymphocytes. In the second part of the study, molecular mechanism associated with fragility of the transcription factor, B cell leukemia 11B (BCL11B) that drives malignant transformation of T-cells has been studied. BCL11B is a zinc finger protein transcription factor with multiple functions. It plays a key role in both development and subsequent maintenance of T-cells. BCL11B gene alterations are implicated in a number of diseases including T-cell malignancies. It acts as a haplo-insufficient tumor suppressor and loss of BCL11B allele leads to susceptibility to mouse thymic lymphoma and human T-ALL. Recent studies reveal heterozygous BCL11B mutations and deletions across each of the major molecular subtypes of T-ALL (15% of patients). Most of the BCL11B missense mutations identified so far affected the residues within BCL11B zinc finger domains of the exon 4. However, mechanism of generation of such specific mutations leading to altered functions of BCL11B remains to be explored. In the present study, we address the potential mechanism of fragility of BCL11B gene during leukemia genesis. Firstly, we have evaluated different regions of BCL11B gene for presence of non-B DNA sequence motifs. Studies using non-B DB database reveal clustering of several non-B DNA forming motifs at the region spanning exon 4 of BCL11B gene. In order to biochemically evaluate the potential of non-B DNA structure formation, two different regions of exon 4 were PCR amplified and cloned. Using bisulfite modification assay we demonstrate that, single strandedness exists at both region I and II of BCL11B exon 4, when the region is present on a plasmid DNA. Bisulfite reactivity on chromosomal DNA confirmed existence of such altered DNA structures in the context of human genome. In vitro gel shift assays showed formation of both intra and intermolecular G-quadruplexes. Primer extension studies revealed that non-B DNA structures could block polymerization during replication on a plasmid, leading to DNA replication arrest. Extrachromosomal assays showed that non-B DNA structure motifs, in contrast to its mutants, blocked transcription leading to reduced expression of green fluorescent protein (GFP) within cells. Many non-B DNA-forming sequences have been mapped to regions of common chromosomal breakpoints in human tumors, known as “hotspots”, which are associated with leukemia, lymphomas and genomic disorders. Thus, alternative DNA conformations are believed to contribute to mutations, deletions and other genetic instability, leading to the deregulation of cancer-related genes in malignant diseases such as leukemia and lymphoma. Activation induced cytidine deaminase (AID), is an essential enzyme involved in antibody diversification of immunoglobulin genes. However, aberrant AID expression in B- cell and non-B cell background is reported in various cancers including leukemia and lymphoma. AID activity requires single stranded DNA (ssDNA) as a substrate. Since activation induced cytidine deaminase (AID) deaminates cytosines when present on a single stranded DNA and its expression is deregulated in many cancers, we investigated the role of AID in BCL11B gene mutagenesis. We observed substantial AID expression in many T-cell leukemic cell lines. Thus, we hypothesize that AID might be targeted to single stranded DNA present at BCL11B exon 4 due to formation of non-B DNA structures such as G-quadruplexes causing AID mediated deamination, further leading to nucleotide alterations and the mutational signature observed at BCL11B exon 4 resulting in T-ALL. Based on our findings, we propose that single strandedness resulted due to formation of non-B DNA structures such as G-quadruplex DNA, triplex DNA or cruciform DNA during physiological processes like DNA replication and transcription at exon 4 of BCL11B, can act as the target for AID. Thus, our findings uncover a new possible link between non-B DNA structure motifs and AID expression in causing mutations at BCL11B exon 4 which could lead to T cell leukemia genesis. BCL11B is a bifunctional transcriptional regulator that can act as a repressor and transactivator, and is known to differentially control the expression of specific genes in a context-dependent manner. In order to understand the transcriptional network involving BCL11B, it was cloned, overexpressed and purified from E. coli. To investigate the DNA binding properties of BCL11B protein, electrophoretic mobility shift assays were performed. Our results lead to identification of a specific sequence motif that is responsible for DNA binding. Competition experiments in presence of specific and nonspecific oligomers further confirmed the binding specificity. Thus, in the present study, we have characterized the binding properties of nonamer binding domain of RAG1, emphasizing its pathological relevance in causing genomic instability in lymphoid cells. The study may help in better understanding of RAG induced genomic instability in lymphoid tissues and role of aberrant AID expression in inducing mutations at BCL11B Zinc finger domain, leading to its deregulation and culminating into T-cell leukemia

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.