Homologous recombination represents a DNA repair pathway. Its role in a plant cell is not limited to double strand break repair. It also extends to genome evolution via rearranging of DNA sequences, and has an important application in foreign DNA integration in the plant genome. Our study demonstrated that effects exerted by stress on homologous recombination and genome stability are not restricted to the exposed generation. The progeny of plants exposed to stress exhibited elevated spontaneous homologous recombination, changes in DNA methylation and higher tolerance to stress. These heritable changes are mediated by an unknown stress-inducible epigenetic signal. Furthermore, we demonstrated that using factors that enhance homologous recombination can improve the efficiency of genetic transformation by Agrobacterium. We have developed and patented a plant growth medium enhancing homologous recombination and significantly increasing the transformation frequency. The role of several other chemicals for the improvement of transformation was also evaluated. / xxi, 246 leaves : ill. ; 29 cm. --

Downstream purification and analysis of a model biopharmaceutical protein (recombinant human myelin basic protein) is described. The recombinant protein was expressed in the milk of transgenic cows and was found exclusively associated with the casein micellar phase. Binding of milk calcium to the active sites of a cation exchanger resin was used beneficially in this study in order to gently disrupt the casein micelles and liberate the recombinant protein. This approach was found superior to the conventional micelle disruption procedures with respect to product recovery, resin fouling due to milk components and column hydrodynamic properties. Further purification was carried out using Ni2+ affinity chromatography and resulted in purity more than 90% and a total recovery of 78%. A capillary electrophoresis total protein assay employing large volume sample stacking and a microsphere-based, sandwich-type immunoassay were developed and validated. Both methods were successfully integrated with the downstream purification protocol in order to evaluate various quality attributes of the recombinant protein. A onestep capillary isoelectric focusing protocol was developed in order to monitor the recombinant protein in milk samples. The results showed extra protein bands in the transgenic milk that had isoelectric points significantly lower than the theoretically calculated one which indicated that the protein had been modified during expression. The association between the recombinant protein and bovine milk caseins was explored at the molecular level using the surface plasmon resonance technique. Results showed a calciummediated interaction between the recombinant protein and the phosphorylated caseins. This selective interaction was not noted between the human myelin basic protein and milk caseins which indicated mammary gland-related posttranslational modifications, most likely phosphorylation. The co-expression of the recombinant protein and caseins in the mammary gland, along with the ability of the recombinant protein to form calcium bridges with caseins explained its association with the casein micellar phase in the transgenic milk. Despite this and owing to the low expression levels of the recombinant protein in milk, light scattering investigations using diffusing wave spectroscopy showed no significant differences between the transgenic and the non-transgenic milk samples with respect to the average micelle size and the micelle surface charges.

Homologous recombination (HR) is conserved across all three domains of life and is associated with a number of key biological processes. Over the years, numerous genetic, biochemical and structural studies have uncovered important mechanistic details and established a role for HR in DNA damage repair, control of DNA replication fidelity and suppression of various types of cancer. Much of our current understanding of the mechanistic aspects of HR is gained from the study of Escherichia coli paradigm. E. coli RecA is the founding member of a nearly ubiquitous family of multifunctional proteins and is substantially conserved among eubacterial species. During HR, RecA protein promotes homologous pairing followed by strand exchange reaction leading to heteroduplex formation. In addition to HR, RecA is a central component of SOS response, recombinational DNA repair and rescue of collapsed replications forks. Moreover, recent work has suggested that DNA recombination/repair mechanisms might contribute to genome evolution and consequently to the generation of multidrug-resistant strains of the pathogen. The disease caused by Mycobacterium tuberculosis, endemic in certain regions of the world, is a leading cause of disability and death. A thorough knowledge of the function and interaction of specific HR proteins/enzymes involved in the maintenance of genome integrity is essential in order to elucidate the impact of genome perturbation effects on M. tuberculosis. Toward this end, modulation of RecA protein activity, a central component of HR, represents a potential novel target for design of new drugs because of its involvement in various processes of DNA metabolism. Additionally, small molecule modulators of RecA activity may offer novel insights into the regulation and its role in cellular physiology and pathology. Traditionally, antibiotics have been used to treat infections caused by bacteria. Despite their importance, the development of new antibiotics against M. tuberculosis has considerably decreased over the past several years due to disappointing results in clinical trials. These failures may be due the fact that they suffer from low potency or low cell permeability. Therefore, one of the aims of studies described in this thesis was to test the effect of suramin, a known inhibitor of E. coli RecA, on various biochemical activities of mycobacterial RecA proteins and determine its mechanism of action. Furthermore, the most crucial step in the HR pathway and rescue of collapsed DNA replication forks is the resolution of Holliday junctions and other branched intermediates. Because Holliday junction resolvases are essential for the resolution of different types of DNA recombination/repair intermediates, therefore, we considered it worthwhile to study the genomic expression and biochemical properties of HJRs in M. tuberculosis. Suramin is a commonly used antitrypanosomal and antifiliarial drug, and a novel experimental agent for the treatment of several cancers. A forward chemical screen assay identified several small molecule inhibitors of E. coli RecA. In this screen, suramin (also called germanin), a polysulfonated naphthylurea, and suramin-like agents were found to inhibit EcRecA catalyzed ATPase and DNA strand exchange activity. However, the mechanism underlying such inhibitory action of suramin and whether it can exert antibacterial activity under in vivo conditions remains largely unknown. In an attempt to delineate the range of suramin action, we reasoned that it might be useful to test its effect on mycobacterium RecA proteins. We found that suramin is a potent inhibitor of all known biochemical activities of mycobacterial RecA proteins with IC50 values in the low μM range. The mechanism of action involves, in part, its ability to disassemble the nucleoprotein filaments of RecA-ssDNA. To validate the above results and to obtain quantitative data, a pull-down assay was developed to assess the effect of suramin on RecA–ssDNA filaments. The data indicated that suramin was able to dissociate >80% of RecA bound to ssDNA. Altogether, these results indicated the effectiveness of suramin in the disassembly of RecA nucleoprotein filament. Next, we sought to test whether suramin binds to RecA by using a CD spectropolarimeter. Significant spectral changes were observed upon addition of increasing concentrations of suramin, indicating alterations in the secondary structure of RecA protein. Additional evidence revealed that suramin impaired RecA catalyzed proteolytic cleavage of LexA repressor and blocked ciprofloxacin-inducible recA gene expression and SOS response. More importantly, suramin potentiated the cidal action of ciprofloxacin and reduced the growth of Mycobacterium smegmatis recA+ strain but not its isogenic recA∆ mutant, consistent with the idea that it acts directly on RecA protein. This approach, which appears as an appealing concept, opens up new possibilities to chemically disrupt the pathways controlled by RecA and treat drug-sensitive as well as drug-resistant strains of M. tuberculosis for better infection control and the development of new therapies. The annotated genome sequence of M. tuberculosis revealed the presence of putative homologues of E. coli DNA recombination/repair genes. However, it is unknown whether these putative genes have the ability to encode catalytically active proteins or participate in biochemical reactions intrinsic to the process of HR or DNA repair. Studies in the second half of the thesis originated from an in silico analysis for genes that encode functional equivalents of E. coli RuvC HJ resolvase(s) in M. tuberculosis. The central intermediate formed during mitotic and meiotic recombination is a four-way DNA junction, also known as the Holliday junction (HJ), and its efficient resolution is essential for proper segregation of chromosomes. The resolution of HJ is mediated by a group of structure specific endonucleases known as the Holliday junction resolvases (HJR) which have been identified in a wide variety of organisms based on their shared biochemical characteristics. Bioinformatics analyses of the evolutionary relationships among HJ resolvases suggests that HJR function has arisen independently from four distinct structural folds, namely RNase H, endonuclease VII-colicin E, endonuclease and RusA. Furthermore, similar analyses of HJRs identified another family within the RNaseH fold, along with previously characterized RuvC family of junction resolvases. This new family of putative HJRs is typified by E. coli Yqgf protein. The yqgf gene is highly conserved among bacterial genomes. Nuclear magnetic resonance structural studies have disclosed notable structural similarities between E. coli RuvC and YqgF proteins. Utilizing homology-based molecular modelling, YqgF is predicted to function as a nuclease in various aspects of nucleic acid metabolism. Sequence analysis of M. tuberculosis genome has revealed the presence of two putative HJ resolvases, ruvC (Rv2594c) and ruvX (Rv2554c, yqgF homolog). Previous studies have demonstrated that M. tuberculosis ruvC is induced following DNA damage and ruvX is expressed during active growth phase of M. tuberculosis. More importantly, the absence of ruvC increased the potency of moxifloxacin in M. smegmatis. Although, these results imply that the ruv genes play crucial roles in DNA recombination and repair in M. tuberculosis, the biochemical properties of their gene products have not been characterized. In this study, we have isolated M. tuberculosis ruvC and yqgF genes and purified their encoded proteins, M. tuberculosis RuvC (MtRuvC) and M. tuberculosis RuvX (MtRuvX), respectively, to near homogeneity. Protein-DNA interaction assays conducted with purified MtRuvC and MtRuvX revealed that both can bind HJ, albeit with different affinities. However, in contrast to MtRuvC, MtRuvX showed robust HJ resolvase activity. The endonuclease activity of MtRuvX was completely dependent on Mg2+and Mn2+ partially substituted for Mg2+. Additional experiments showed that RuvX exhibits >2-fold higher binding affinity for HJ over other recombination/ replication intermediates. As demonstrated for other HJRs, MtRuvX failed to cleave static HJ and linear duplex DNA. The cleavage sites were mapped within the homologous core of a branch-migratable HJ. To identify catalytic residues in RuvX, we conducted mutational analysis of an acidic amino acid residue guided by the bioinformatics data. The product of MtRuvXD28N retained full HJ-binding activity, but showed extremely reduced HJ-specific endonuclease activity. Further biochemical characterization revealed that MtRuvX exists as a homodimer in solution. Notably, we found that disulfide-bond mediated intermolecular homodimerization is crucial for the ability of MtRuvX to cleave Holliday junctions, implicating that stable junction binding is necessary to promote branch migration and to create cleavable sites. Analysis of qPCR data suggested that the pattern of yqgF gene expression was similar to those of ruvC and recA genes following DNA damage. Together, these data indicate that ruvX expression is induced by DNA-damaging agents and that RuvX might be functionally involved in recombinational DNA repair in M. tuberculosis. These findings are all consistent with the idea that RuvX might be the bona fide HJ resolvase in M. tuberculosis analogous to that of E. coli RuvC. More importantly, we provide the first detailed characterization of RuvX and present important insights into the mechanism of HJ resolution, which could be directly linked to the regulation of different DNA metabolic processes, including HR, DNA replication and DNA repair. Overall, this study opens a new avenue in the understanding of HR in this human pathogen, together with elucidation of the function of some of the uncharacterized genes may represent a novel set of recombination enzymes.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / RESUMO A confirmaÃÃo diagnÃstica de leishmaniose visceral ou calazar à realizada atravÃs da detecÃÃo do parasito por mÃtodos diretos, atravÃs do encontro de formas amastigotas no interior de macrÃfagos ou monÃcitos, em aspirado esplÃnico, hepÃtico, medula Ãssea, histopatolÃgico de linfonodo e esfregaÃo do sangue perifÃrico, com ampla variaÃÃo de sensibilidade de acordo com o sÃtio pesquisado, constituindo-se o padrÃo-ouro a visualizaÃÃo de Leishmania sp. A pesquisa de anticorpos especÃficos para triagem diagnÃstica da leismaniose visceral atravÃs do ELISA, reaÃÃo de imunofluorescÃncia indireta e os testes de aglutinaÃÃo direta possuem sensibilidade alta e especificidade limitada pelas reaÃÃes cruzadas com tripanosomatÃdeos e micobactÃrias, incluindo-se tuberculose, hansenÃase, doenÃa de Chagas, e ainda, leishmaniose tegumentar e histoplasmose. O teste intradÃrmico de Montenegro à sempre negativo durante a fase ativa da doenÃa, e baseia-se na memÃria imunolÃgica dependente de linfÃcitos Th1. O ojetivo deste trabalho foi evidenciar a relevÃncia do teste rÃpido para detecÃÃo de anticorpo anti-leishmania chagasi utilizando o antÃgeno recombinante rK39 que apresentou sensibilidade de 99,2%. Foram realizados testes sorolÃgicos pelo ELISA que apresentou sensibilidade de 80%, e a reaÃÃo de imunofluorescÃncia indireta para calazar com sensibilidade de 82%. O gold-standard diagnÃstico empregado para todos exames foi a pesquisa microscÃpica direta do parasito no aspirado de medula Ãssea e a positividade foi de 71%, em uma pesquisa realizada em hospitais terciÃrios de Fortaleza-CE. A reaÃÃo imunoenzimÃtica para teste rÃpido em fita contendo antÃgeno rK39 apresentou elevada sensibilidade e especificidade permitindo diagnÃstico imediato e inÃcio precoce do tratamento, alÃm de confirmar alta densidade de epÃtopos exclusivamente para o gÃnero Leishmania, pois nÃo houve reaÃÃes cruzadas dentre vÃrias doenÃas testadas. Detectamos tÃtulos de anticorpos IgG anti-leishmania chagasi persistentemente positivos em pacientes tratados adequadamente para calazar, corroborando com evidÃncias de que o rK39 nÃo define doenÃa em atividade e nem estabelece critÃrios de cura, fortalecendo que outras possibilidades diagnÃsticas sejam investigadas mediante um resultado positivo em indivÃduo procedente de zona endÃmica para calazar, em paciente portador de hepatoesplenomegalia febril e pancitopenia. O teste rÃpido rk39 deverà ter sua leitura imediatamente relizada e interpretada em atà no mÃximo 10 minutos, objetivando tomada de decisÃes clÃnicas, e que sua implantaÃÃo como mÃtodo auxiliar de diagnÃstico deveria fazer parte da rotina laboratorial, particularmente de serviÃos secundÃrios e terciÃrios de saÃde em regiÃes endÃmicas para leshmaniose visceral. / ABSTRACT The visceral leishmaniasis diagnostic confirmation or Kala-azar is performed by detection of the parasite through direct methods of amastigotes within macrophages or monocytes, spleen aspirated, hepatic, bone marrow, lymph node histopathological and peripheral blood smear, with wide variation in sensitivity in accordance with the site searched, being the gold standard of Leishmaniasis sp . The specific antibodies for diagnostic of visceral leishmaniasis by ELISA, indirect immunofluorescence reaction and direct agglutination tests have high sensitivity and specificity cross reactions with limited by trypanosomatids and mycobacterium, including tuberculosis, leprosy, Chagas disease , leishmaniasis cutaneous and histoplasmosis. The Montenegro intradermal test is always negative during the active phase of the disease, and is based on immune memory bound on lymphocyte Th1. The purpose of this work was to highlight the importance of quick test for the detection of antibody anti-leishmania chagasi using recombinant rK39 antigen that presented sensitivity of 99.2%. There were serological by ELISA tests that presented sensitivity of 80%, and the indirect immunofluorescence reaction to Kala-azar with sensitivity of 82%. The gold-standard diagnosis made for all examinations was the direct microscopic search on parasite bone marrow aspirated and positivity was 71%, in a survey in tertiary hospitals in Fortaleza-CE. The immunoenzymatic reaction to quick test in tape containing rK39 Antigen has high sensitivity and specificity allowing immediate diagnosis and in the early beginning of treatment, in addition to confirm high-density epitopes exclusively for the Leishmaniasis genus, because there was no cross reactions among several diseases tested. We found evidence of IgG anti-leishmaniasis chagasi antibodies persistently -positive in patients treated appropriately for kala-azar, supporting with evidence that rK39 does not define disease activity and establishes criteria for cure, strengthening that other homogenizing are investigated by a positive result in individual from endemic area of Kala-azar, in disease carrier of febrile hepatosplenomegaly and pancytopenia . The quick test Rk39 must be read and interpreted immediately up to a maximum of 10 minutes driving clinical decision-making, and that its deployment as diagnostic helper method should be part of the laboratorial routine services, particularly in secondary and tertiary health endemic regions of visceral leshmaniasis . Keywords: American visceral leishmaniasis. Kala-azar. Recombinant K39 Antigen. Kala-azar through rK39 antigen diagnostic.

Watson-Crick paired B-form DNA is the genetic material in most of the biological systems. Integrity of DNA is of utmost importance for the normal functioning of any organism. Various environmental factors, chemicals and endogenous agents constantly challenge integrity of the genome resulting in mutagenesis. Over the past few decades multiple reports suggest that DNA can adopt alternative conformations other than the right handed double helix. Such structures occur within the context of B-DNA as sequence dependent structural variations and are facilitated by free energy derived from negative supercoiling, which may be generated during physiological processes like transcription, replication, etc. or binding of proteins. Multiple groups have shown that these structures render fragility to the genome owing to single-strandedness (presence of unpaired bases). This conformational polymorphism of the DNA is due to the presence of several repetitive elements across the genome. Some of the common non-B DNA structures include Z-DNA, H-DNA (triplex DNA), cruciform DNA, G-quadruplexes and RNA: DNA hybrid (R-loops). Over the past few decades G-quadruplex structures have gained tremendous importance owing to its role in physiology and pathology. Recently it has been shown that novel sequence motifs, called GNG or bulges can fold into G-quadruplexes, thus increasing the propensity of such structures genome-wide. Neurological diseases, psychiatric diseases and genomic disorders (due to deletions, translocations, duplications and inversions) are some of the consequences of non-B DNA structures in the human genome. Inadvertent genomic rearrangements in human can lead to different diseases including cancer. Immediate consequence of genomic rearrangement includes structural alteration of genome through joining of distant sequences. t(8;14) translocation is the hallmark of Burkitt’s lymphoma, which results in deregulation of c-MYC gene that may contribute to oncogenic transformation. In the present study, we delineate the causes of fragility within the c-MYC gene. In order to do this, breakpoints at the c-MYC locus from Burkitt’s lymphoma patient sequences reported in database were plotted and analysed. Interestingly, unlike many other translocations, breakpoints at c-MYC locus were widespread, except for a cluster of breakpoints downstream to promoter 2 (P2). Previous studies indicate that the translocation breakpoint clusters often correlate with formation of non-B DNA structures. The entire breakpoint cluster downstream of P2 was divided into Region 1, Region 2 and Region 3. Interestingly, in silico analysis of the breakpoint clusters revealed no evidence for predictive classic non-B DNA motifs in Region 2; whereas Region 1 harboured a G-quadruplex motif on the template strand and Region 3 had two short inverted repeats. Intriguingly, as the nontemplate strand of Region 2 was G skewed with a good number of AID binding motifs, we tested the MYC breakpoint Region 2 for its potential to form R-loop due to binding of nascent RNA to template DNA. Our results showed that MYC Region 2 can form RNA-DNA hybrid in a transcription dependent manner in physiological orientation. Observed structure was sensitive to RNase H. We showed Region 2 hindered action of Dpn I upon transcription confirming formation of R-loop structure. Owing to single strandedness, Region 2 R-loop was shown to be sensitive to P1 nuclease as opposed to the untranscribed control. The single strandedness of the Region 2 R-loop was characterized at a single molecule level through bisulfite modification assay. The assay corroborated formation of R-loop along with providing snapshots of various length R-loops formed upon Region 2 transcription. Besides, various biophysical and biochemical assays showed the complementary region (template strand) to be single-stranded in stretches, upon transcription. Length of RNA within the R-loop was within a range of 75 to 250 nt. To delineate the mechanism of R-loop formation we tested the sensitivity of R-loop formation to RNase A during and post transcription; and found that R-loop formation was abrogated in presence of RNase A during transcription suggesting that R-loop formation followed a “thread back model”. Intriguingly we observed that two short regions of the template strand exhibited high degree of single strandedness. To investigate the reason for such unusual single strandedness, oligonucleotides spanning the region was designed and subjected for CD and EMSA studies. EMSA showed robust intramolecular G-quadruplex structure formation in presence of KCl, whereas CD confirmed that both regions formed parallel G-quadruplexes. We also showed the precise involvement of guanines in structure formation through DMS protection assay. Further, the region of interest was cloned into appropriate vectors and primer extension assays were performed in presence of G-quadruplex stabilizing agents like TMPyP4 and KCl. Increasing concentration of these stabilizing agents enhanced the formation of G-quadruplexes in a double stranded context, which hindered polymerase progression. Since these G-quadruplex structures utilized sequences which are deviant to the consensus of G-quadruplex motifs, non-B DNA predicting tools were unable to score them. On closer analysis of the sequences we found that, these G-quadruplexes involve duplex hairpin and GNG motifs during structure formation. Besides, both the G-quadruplexes were highly thermostable and were able to fold back upon renaturation. Till recently, it has been believed that G-quadruplex structures are formed using a minimum of four, 3 guanine tracts, with connecting loops ranging from one to seven. Recent studies have reported deviation from this general convention. One such deviation is the involvement of bulges in the guanine tracts. In the present study, guanines along with GNG motifs have been extensively studied using recently reported HOX11 breakpoint fragile region I as a model template. By strategic mutagenesis approach we show that the core elements of a G-quadruplex are not equally important in structure formation when flanked by GNG motifs. Importantly, the positioning and number of GNG/GNGNG can dictate the formation of G-quadruplexes. In addition to HOX11 fragile region, GNG motifs of HIF1-alpha can fold into intramolecular G-quartet. However, GNG motifs in mutant VEGF sequence could not participate in structure formation, suggesting that the usage of GNG is context dependent. Importantly, we show that when two stretches of guanines are flanked by two independent GNG motifs in a naturally occurring sequence (SHOX), it can fold into an intramolecular G-quadruplex. Interestingly, intra molecular GNG G-quadruplexes were able to fold back after complete denaturation of the oligonucleotides. Besides one of the intra molecular GNG G-quadruplexes was purified and confirmed for parallel conformation. Finally, we show the specific binding of G-quadruplex binding protein, Nucleolin and G-quadruplex antibody BG4 to SHOX G-quadruplex through EMSA studies. Thus, the study provides novel insights into the role of GNG motifs in G-quadruplex structure formation, which may have both physiological and pathological implications. In conclusion, we show formation of transcription dependent R-loop and G-quadruplex structures at the c-MYC gene locus in a mutually exclusive manner. The data presented here, in conjunction with studies from other laboratories suggests that these structures could impart fragility within the c-MYC gene locus during t(8;14) translocation. Besides, we characterised unusual G-quadruplexes harbouring GNG motifs. We find that positioning and number of GNG can dictate the formation of G-quadruplexes and is context dependent.

A hierarchy of different chromosome conformations plays a role in many biological systems. These conformations contribute to the regulation of gene expression, cellular development, chromosome transmission, and defects can lead to human disease. The highest functional level of this hierarchy is the partitioning of the genome into compartments of active and inactive chromatin domains (1’s -10’s Mb). These compartments are further partitioned into Topologically Associating Domains (TADs) that spatially cluster co-regulated genes (100’s kb – 1’s Mb). The final level that has been observed is long range loops formed between regulatory elements and promoters (10’s kb – 100’s Mb). At all of these levels, mechanisms that establish these conformations remain poorly understood. To gain new insights into processes that determine chromosome folding I used the mating type switching system in budding yeast to study the chromosome conformation at length scales analogous to looping interaction. I specifically examined the role in chromosome conformation in the mating type switching system. Budding yeast cells can have two sexes: MATa and MATα. The mating types are determined by allele-specific expression of the MAT locus on chromosome III. The MATa allele encodes for transcription factors responsible for the MATa mating type and the MATα allele encodes transcription factors responsible for the MATα mating type. Yeast cells can switch their mating type by a process that repairs a break at MAT using one of two silent loci, HML or HMR, as a donor to convert the allele at the MAT locus. When MATa cells switch they prefer to use HML, which contains the MATα allele, located at the end of the left arm. MATα cells prefer to use HMR, which contains the MATa allele, located on the end of the right arm of chromosome III. The sequences of the HM loci are not important for donor preference. Instead the cell chooses the donor on the left arm in MATa cells and chooses the donor on the right arm in MATα cells. This lack of sequence specificity has led to the hypothesis that the conformation of the chromosome may play a role in donor preference. I found that the conformation of chromosome III is, indeed, different between the two mating types. In MATa cells the chromosomes displays a more crumpled conformation in which the left arm of the chromosome interacts with a large region of the right arm which includes the centromere and the MAT locus. In MATα cells, on the other hand, the left arm of the chromosomes displays a more extend conformation. I found that the Recombination Enhancer (RE), which enhances recombination along the left arm of the chromosome in MATa cells, is responsible for these mating type-specific conformations. Deleting the RE affects the conformation of the chromosomes in both MATa and MATα cells. The left portion of the RE, which is essential for donor preference during the switching reaction in MATa cells, does not contribute to the conformation in MATa. This region does have a minor effect on the conformation in MATα cells. However, I found that the right portion of the RE is responsible for the conformation of chromosome III in both mating types prior to initiation of switching. This work demonstrates that chromosome conformation is determined by specific cis regulatory elements that drive cell-type specific chromosome conformation.

Indiana University-Purdue University Indianapolis (IUPUI) / The Cre/loxP system is a tool for targeted recombination of DNA. For applying Cre recombinase-mediated genome modifications, there is a requirement for reliable, high-fidelity, and specific transgenic expression of the Cre recombinase. This study focuses on the reliability of different bone cell specific Cre models in the Cre/loxP system. In this study, DMP1-Cre transgenic mouse which has a transgene driven by DMP1 promotor that allows Cre-expression only in late stage osteoblasts and osteocytes was used. Ctsk-Cre mouse with a driven by Ctsk promoter was used so that only osteoclasts would undergo Cre-mediated recombination. E2A-Cre mouse where the Cre recombinase is driven by a global promoter E2A was also included in this study as a control line to test the Cre reporter line Ai9. Dmp1-Cre, Ctsk-Cre and E2A-Cre mice were crossed to the fluorescent Cre-reporter line—Ai9, which harbors a floxed stop codon, followed by the fluorophoremTomato, inserted into the Rosa26 locus. This construct is expected to give red fluorescence when it recombines with Cre-expressing mouse cells and no fluorescence in non-recombinant mouse cells. Double positive (Ai9+/Cre+) offspring selected by PCR were perfused, and 5mu-m thick section of bone and soft tissues were examined for red fluorescent expression. Cre positive cells were quantitated using ‘ImageJ’ software program. The DMP1-vi Cre mouse results showed significant expression in the targeted osteocytes and osteoblasts. In addition, skeletal muscle tissue also showed significant Cre- expression. Ctsk-Cre mice showed significant expression in targeted osteoclasts. But brain tissue was positive in Cre-expression. Bone-Cre mouse models are expected to express Cre recombinase only in their respective bone cells and they have been used for gene deletion studies in bone cells. However, this study has revealed that the bone cell specific Cre mouse models DMP1-Cre and Ctsk-Cre have unexpected expression in muscle and brain respectively. In order to use these models for targeted gene deletion in bone cells, further testing and studies have to be conducted.

La maladie pied-main-bouche (PMB) et l’herpangine sont deux maladies pédiatriques bénignes causées par les entérovirus (EV), en particulier les sérotypes de l’espèce A (EV-A). Le sérotype EV-A71 fait l’objet d’une surveillance dans les pays du Sud Est de l’Asie car il est associé à des atteintes neurologiques sévères chez les très jeunes enfants, parfois mortelles (défaillance cardio-pulmonaire). Les infections causées par les autres EV-A tel que le coxsackievirus A16 (CV-A16) provoquent rarement des atteintes sévères. En Europe, les cas de maladie PMB causés par l’EV-A71 ne font pas l’objet d’une déclaration obligatoire, car ce virus ne cause pas d’épidémies de grande ampleur. L’objectif général de la thèse était d’étudier l’épidémiologie des EV-A en Europe et nous avons utilisé une approche phylogénétique bayésienne pour analyser un échantillon de 500 souches. Nous montrons la circulation discontinue de l’EV-A71 de deux populations virales principales (sous génogroupes C1 et C2), ce qui explique la rareté des épidémies en Europe. L’épidémiologie de ce virus est aussi caractérisée par des transports de souches entre les pays Européens et sporadiquement entre l’Europe et l’Asie (sous génogroupes B5 et C4). La recombinaison génétique intertypique survient rarement parmi les populations d’EV-A71 en circulation et ne contribue pas significativement à leur diversité génétique. Cependant, ce mécanisme génétique est relié à l’émergence d’un sous génogroupe CV-A16 qui circule en France depuis 2011. Comparés à l’EV-A71, les sérotypes CV-A2, CV-A4, CV-A6 sont plus fréquemment sujets à des événements de recombinaison intertypiques. L’analyse de la sélection à l’échelle moléculaire indique que la fixation des mutations dans les protéines de capside de l’EV-A71 est lente, probablement à cause des contraintes structurales et fonctionnelles. La surveillance des infections à EV-A71 en Europe devrait être renforcée à cause de la neurovirulence de ce virus, de l’introduction récente et répétée de souches variantes « asiatiques » et de l’existence d’une grande diversité de génogroupes en Afrique et en Inde encore peu explorée. / Hand-Foot and Mouth Disease (HFMD) and Herpangina are two benign pediatric diseases caused by Enteroviruses (EV), especially enterovirus A species serotypes (EV-A). Infections caused by the EV-A71 serotype are monitored in countries of South East Asia because they are associated with severe neurological symptoms in young children and may be fatal (cardiopulmonary failure). Infections caused by the other EV-A serotypes, e.g. coxsackievirus A16 (CV-A16), rarely induce severe symptoms. In Europe, EV-A71 HFMD cases are not notifiable because this virus does not cause large-scale epidemics. The overall objective of this thesis was to study the EV-A epidemiology in Europe and we used a Bayesian phylogenetic approach to analyze 500 viral strains. We show a discontinued circulation of two EV-A71 populations (C1 and C2 subgenogroups), which explains the rare outbreaks in Europe. The epidemiology of this virus is characterized by transportation events of viral strains between European countries and sporadically between Europe and Asia (C4 and B5 subgenogroups). Intertypic genetic recombination occur rarely among circulating EV-A71 populations and does not contribute significantly to their genetic diversity. We found that genetic mechanism was related to the emergence of a new CV-A16 subgenogroup, which is circulating in France since 2011. In comparison with EV-A71, a number of serotypes (CV-A2, CV-A4, and CV-A6) are more frequently involved in intertypic recombination events. The structural and functional constraints are possible factors involved in the slow mutation fixation in the EV-A71 capsid proteins as determined by analyses of molecular selection. Neurovirulence, the recent and repeated introductions of variants “Asian” strains, and the diversity of genogroups in Africa and India call for strengthened surveillance of EV-A71 infections among European countries.

Indiana University-Purdue University Indianapolis (IUPUI) / DNA double strand break (DSB) is one of the most threatening of all types of DNA damages as it leads to a complete breakage of the chromosome. The cell has evolved several mechanisms to repair DSBs, one of which is break-induced replication (BIR). BIR repair of DSBs occurs through invasion of one end of the broken chromosome into a homologous template followed by processive replication of DNA from the donor molecule. BIR is a key cellular process and is implicated in the restart of collapsed replication forks and several chromosomal instabilities. Recently, our lab demonstrated that the fidelity of DNA synthesis associated with BIR in yeast Saccharomyces Cerevisiae is extremely low. The level of frameshift mutations associated with BIR is 1000-fold higher as compared to normal DNA replication. This work demonstrates that BIR stimulates base substitution mutations, which comprise 90% of all point mutations, making them 400-1400 times more frequent than during S-phase DNA replication. We show that DNA Polymerase δ proofreading corrects many of the base substitutions in BIR. Further, we demonstrate that Pif1, a 5’-3’ DNA helicase, is responsible for making BIR efficient and also highly mutagenic. Pif1p is responsible for the majority of BIR mutagenesis not only close to the DSB site, where BIR is less stable but also at chromosomal regions far away from the DSB break site, where BIR is fast, processive and stable. This work further reveals that, at positions close to the DSB, BIR mutagenesis in the absence of Pif1 depends on Rev3, the catalytic subunit of translesion DNA Polymerase ζ. We observe that mutations promoted by Pol ζ are often complex and propose that they are generated by a Pol ζ- led template switching mechanism. These complex mutations were also found to be frequently associated with gross chromosomal rearrangements. Finally we demonstrate that BIR is carried out by unusual conservative mode of DNA synthesis. Based on this study, we speculate that the unusual mode of DNA synthesis associated with BIR leads to various kinds of genomic instability including mutations and chromosomal rearrangements.

Indiana University-Purdue University Indianapolis (IUPUI) / Break-induced replication (BIR) is a mechanism to repair double-strand breaks (DSBs) that possess only a single end that can find homology in the genome. This situation can result from the collapse of replication forks or telomere erosion. BIR frequently produces various genetic instabilities including mutations, loss of heterozygosity, deletions, duplications, and template switching that can result in copy-number variations (CNVs). An important type of genomic rearrangement specifically linked to BIR is half crossovers (HCs), which result from fusions between parts of recombining chromosomes. Because HC formation produces a fused molecule as well as a broken chromosome fragment, these events could be highly destabilizing. Here I demonstrate that HC formation results from the interruption of BIR caused by a defective replisome or premature onset of mitosis. Additionally, I document the existence of half crossover instability cascades (HCC) that resemble cycles of non-reciprocal translocations (NRTs) previously described in human tumors. I postulate that HCs represent a potent source of genetic destabilization with significant consequences that mimic those observed in human diseases, including cancer.