The Human Genome Project (HGP), the name given to the scientific program to map and decode all of human genetic material, has been projected to revolutionize the conduct of biological science in the twenty-first century. For several years before its formation in 1990, a federally-funded, systematic study of the human genome was discussed first in the scientific arena and then in the public arena. The central thesis of this dissertation is that the arguments supporting or rejecting creation of the HGP and the rhetorical devices used to further those arguments had a major influence on the shape the HGP took in 1991. The argumentation used both for and against the creation of the HGP before the public as well as on the border between the public and scientific arenas is studied. The rhetorical devices such as metaphor, narrative, and selective word choices used to further these arguments are also examined. In particular, a rhetorical content analysis was performed on the 1986-1991 argumentation available to the most crucial audience for such persuasion: the members of Congress who ultimately voted for or against the program's funding and its establishment as a part of U.S. science policy. The proponents of the HGP, especially after the first year of public debate, presented their arguments in a wider arena of discussion and presented more and more varied arguments to advocate the project. The opposition raised questions that had for the most part been answered earlier in the debate. Often anti-HGP arguments focused on less effective audiences (scientists instead of members of Congress). Opposition to the project didn't become organized until near the end of the time frame studied, too late to have much of an impact on the outcome of the debate. The rhetorical devices studied served to magnify the impact of arguments used: in particular, the metaphor served as a boundary object to bridge discussions between the scientific and the public arenas. Ultimately the victory in the debate over the establishment of the HGP was awarded to the promulgators of the strongest underlying metaphor--the idealized excitement and profit of exploration of unknown territory--and the benefits to come from filling in and conquering the unknown areas of the human genetic map, territory the U.S. was eager to claim for its own. / Ph. D.

The ability to generate multiplexed genomic modifications using CRISPR-based gene editing has fundamentally changed the scope of possible reverse genetic screening approaches that can be executed in human cells. A diversity of Cas effector proteins lies at the center of pooled CRISPR screens. Working in unison with targeting gRNAs, CRISPR-Cas effector complexes can produce a range of alterations at user specified genomic sites. The type of alteration, ranging from double-strand break (DSB) formation to precise single nucleotide substitutions, is dictated by the Cas protein. Initially, pooled CRISPR screens were conducted using the Cas9 endonuclease to generate loss of function mutations in single genes through the formation of DSBs. As CRISPR technologies matured, the discovery and engineering of novel Cas proteins has allowed for increasingly complex sets of genomic alterations to be studied in a high-throughput manner. In Chapter 1, I introduce a variety of CRISPR-based functional genomic technologies that have been used in high-throughput screening approaches. Here, I also describe discoveries that have been made in the human DNA damage response (DDR) using these approaches. In Chapter 2, I present my work using Cas12a to interrogate the genetic interaction landscape of the DDR. This work leverages the ability of Cas12a to generate several DSBs from a single gRNA array to investigate ~27,000 genetic interactions between 233 DDR genes. In these screens, novel synthetic lethal interactions were identified, with three sets of synthetic lethal interactions between gene complexes being highlighted. In Chapter 3, I present a published manuscript that demonstrates the utility of precision base editing screens. This study uses BE3-dependent base editing to induce mutational tiling of 86 human DDR genes and analyze the effects of these mutations in response to DNA damaging agents. In total, the work presented here highlights the utility of novel CRISPR screening platforms through the interrogation of the human DDR.

The expression of retroviral genes depends on the establishment of the provirus - the DNA copy of retroviral genome integrated into the host genome. The transcriptional state of provirus is then influenced by the environment at the site of integration. The phenomenon of proviral silencing is an obstacle to the usage of retroviral vectors and a barrier to the eradication of human immunodeficiency virus type 1 (HIV-1) from infected individuals. Taking advantage of single cell clones bearing one provirus, this diploma thesis investigates the distribution of (epi)genomic features at the sites occupied by stably expressed proviruses. In total, long-term expression profiles of 245 and 255 clones carrying avian sarcoma-leucosis virus (ASLV) and HIV-1, respectively, were obtained. The database-based analysis of 42 integration sites of ASLV and three integration sites of HIV-1 proviruses shows that proviral stable expression highly correlates with the transcriptional start sites (TSS) at the sites of integration. Histone marks characteristic for the proximity of active TSSs and regulatory elements at the sites of integration of stably expressed proviruses confirm this finding. The results presented in this thesis could inspire other analyses investigating the relationship between the integration site and the...

Etiquetas de sequências expressas (ESTs) são fundamentais para a identificação de genes no genoma humano e para definir características de expressão gênica. Neste trabalho, descrevemos uma nova abordagem para a geração de bibliotecas de cDNA, utilizando iniciadores arbitrários para a produção, por PCR, de mini-bibliotecas a partir de mRNA derivado de câncer de mama. Clones destas bibliotecas foram sequenciadas para gerar 6029 ESTs. Utilizando esta abordagem, foi possível observar uma significante normalização das diferentes sub-populações de mRNA e amplificação preferencial de porções centrais dos genes. Análise bioinformática destas sequências mostra que 3.350 ESTs (56%) tem similaridade significante a sequências de DNA e/ou cDNA já conhecidas (sequências anotadas) descritas em diferentes organismos, e 1509 ESTs (25%) não possuem qualquer similaridade a diferentes bancos de dados. Dentre as sequências anotadas, identificamos algumas sequências com alta similaridade a genes conhecidos em diferentes organismos, indicando a descoberta de alguns genes homólogos possivelmente envolvidos com processos carcinogênicos. Como exemplo, isolamos e caracterizamos parcialmente (i) uma nova isoforma do gene NABC1 (novel amplified sequence in breast carcinoma 1), o qual é pouco expresso em tumores coloretais, (ii) um novo gene da família de semaforinas (moléculas de motilidade axonal) que apresenta uma baixa expressão em linhagens celulares de glioblastoma tratadas com ácido retinóico, um agente antitumoral e (iii) o gene ortólogo humano Notch 2, aparentemente superexpresso em tumores mamários com maior malignidade. / Expressed sequence tags (ESTs) are of fundamental importance for the identification of genes within the human genome and defining gene expression characteristics. In this work, we describe a new approach for generating cDNA libraries using essentially arbitrary primers to construct PCR-based minilibraries from breast tumor mRNA. Clones from these libraries were sequenced to generate 6,029 ESTs. Using this approach, we were able to observe a significant normalization of the different mRNA subpopulations and a preferential amplification of the central portions of the genes. Bioinformatic analysis of these sequences shows that 3,350 ESTs (56%) have significant similarity to known DNA and/or cDNA sequences (annotated sequences) from different organisms and 1,509 ESTs (25%) show no similarity to any sequences on different databases. From the annotated sequences, we have identified some sequences with high similarity to known genes from different organisms, indicating the discovery of some homologous genes possibly correlated with carcinogenic processes. For instance, we have isolated and partially characterized (i) a new NABC1 (novel amplified sequence in breast carcinoma 1) isoform which is downregulated in colorectal tumors, (ii) a novel semaphorin member of axon guidance molecules that is down-regulated in glioblastoma cell lines treated with all-trans-retinoic acid, an anti-tumor agent and (iii) the ortolog Notch 2 human gene, apparenty overexpressed in breast tumors with higher malignancy.

Partindo da concepção de que a ciência é, por definição, uma atividade coletiva, organizada localmente e através de instituições, esta dissertação realiza um estudo empírico do Centro de Estudos do Genoma Humano (CEGH), situado na Universidade de São Paulo. A pergunta mais geral do trabalho diz respeito à maneira pela qual se dá a organização social da produção de conhecimento e da produção de produtores de conhecimento em uma área específica de pesquisa a genética em um país periférico. Para isso, parte-se do processo de institucionalização da pesquisa em genética no Brasil, enfatizando os arranjos entre pesquisadores, universidade e agência de fomento em três aspectos considerados essenciais à atividade científica: padrão de financiamento, padrão disciplinar e padrão de circulação internacional de ideias e pesquisadores. A preocupação central é compreender a dinâmica da disciplina, pensada como um conjunto de processos sociais de produção de conhecimentos (e não como uma lista de descobrimentos, acumuladas por homens singulares), e demonstrar como a institucionalização da pesquisa em genética foi conformando uma tradição local de pesquisa. Essa tradição servirá como pano de fundo para compreender a incorporação das mudanças na pesquisa em genética humana passagem da genética clássica à molecular nos laboratórios que atualmente compõem o CEGH e as transformações no padrão de financiamento da pesquisa. Ao olhar para o CEGH, a partir dessa tradição científica local da qual ele é tributário, é possível descrever quais são os atuais arranjos organizacionais, as práticas de pesquisa e a divisão do trabalho que remodelam e atualizam essa tradição. Este trabalho considera o CEGH como um microcosmo social, que faz parte de um espaço disciplinar mais amplo que, por sua vez, insere-se no universo hierarquizado das áreas de conhecimento e disciplinas científicas. / Starting from the understanding that science is, by definition, a collective activity, organized locally and through institutions, this dissertation carries out an empirical study on the Human Genome Research Center (HGRC), situated in the University of São Paulo (USP). The broader question of this study regards the way through which the social organizing of production of knowledge occurs, and the production of the producers of knowledge in a specific field of research genetics in a peripheral country. For this, we begin from the process of institutionalisation of the genetics research in Brazil, emphasizing the arrangement between researchers, university and funding agencies in three aspects considered essentials in scientific activities: funding pattern, disciplinary pattern and the pattern of international circulation of ideas and researchers. The main concern is to understand the dynamics of the discipline, conceived as an ensemble of social processes in the production of knowledge (and not as a list of discoveries accumulated by singular men), and demonstrate how the institutionalization of research in genetics conformed to a local research tradition. This tradition will serve as a background to comprehend the incorporation of changes in human genetics research the passage from classical genetics to molecular biology in laboratories which nowadays integrate the HGRC and the transformations in the patterns of research funding. By observing the HGRC from the perspective of this local scientific tradition, from which this research center is tributary, it is possible to describe what are the recent organizational arrangements, such as the practices of research and the division of labor which reshaped and updated this tradition. This dissertation considers the HGRC a social microcosm, which integrates a disciplinary space which, in turn, is inserted in the hierarchical universe of the fields of knowledge and scientific disciplines.

Partindo da concepção de que a ciência é, por definição, uma atividade coletiva, organizada localmente e através de instituições, esta dissertação realiza um estudo empírico do Centro de Estudos do Genoma Humano (CEGH), situado na Universidade de São Paulo. A pergunta mais geral do trabalho diz respeito à maneira pela qual se dá a organização social da produção de conhecimento e da produção de produtores de conhecimento em uma área específica de pesquisa a genética em um país periférico. Para isso, parte-se do processo de institucionalização da pesquisa em genética no Brasil, enfatizando os arranjos entre pesquisadores, universidade e agência de fomento em três aspectos considerados essenciais à atividade científica: padrão de financiamento, padrão disciplinar e padrão de circulação internacional de ideias e pesquisadores. A preocupação central é compreender a dinâmica da disciplina, pensada como um conjunto de processos sociais de produção de conhecimentos (e não como uma lista de descobrimentos, acumuladas por homens singulares), e demonstrar como a institucionalização da pesquisa em genética foi conformando uma tradição local de pesquisa. Essa tradição servirá como pano de fundo para compreender a incorporação das mudanças na pesquisa em genética humana passagem da genética clássica à molecular nos laboratórios que atualmente compõem o CEGH e as transformações no padrão de financiamento da pesquisa. Ao olhar para o CEGH, a partir dessa tradição científica local da qual ele é tributário, é possível descrever quais são os atuais arranjos organizacionais, as práticas de pesquisa e a divisão do trabalho que remodelam e atualizam essa tradição. Este trabalho considera o CEGH como um microcosmo social, que faz parte de um espaço disciplinar mais amplo que, por sua vez, insere-se no universo hierarquizado das áreas de conhecimento e disciplinas científicas. / Starting from the understanding that science is, by definition, a collective activity, organized locally and through institutions, this dissertation carries out an empirical study on the Human Genome Research Center (HGRC), situated in the University of São Paulo (USP). The broader question of this study regards the way through which the social organizing of production of knowledge occurs, and the production of the producers of knowledge in a specific field of research genetics in a peripheral country. For this, we begin from the process of institutionalisation of the genetics research in Brazil, emphasizing the arrangement between researchers, university and funding agencies in three aspects considered essentials in scientific activities: funding pattern, disciplinary pattern and the pattern of international circulation of ideas and researchers. The main concern is to understand the dynamics of the discipline, conceived as an ensemble of social processes in the production of knowledge (and not as a list of discoveries accumulated by singular men), and demonstrate how the institutionalization of research in genetics conformed to a local research tradition. This tradition will serve as a background to comprehend the incorporation of changes in human genetics research the passage from classical genetics to molecular biology in laboratories which nowadays integrate the HGRC and the transformations in the patterns of research funding. By observing the HGRC from the perspective of this local scientific tradition, from which this research center is tributary, it is possible to describe what are the recent organizational arrangements, such as the practices of research and the division of labor which reshaped and updated this tradition. This dissertation considers the HGRC a social microcosm, which integrates a disciplinary space which, in turn, is inserted in the hierarchical universe of the fields of knowledge and scientific disciplines.

This thesis examines one of the premier "big science" projects of the contemporary era - the globalised genetic mapping and sequencing initiative known as the Human Genome Project (HGP), and how Australia has responded to it. The study focuses on the relationship between the HGP, the biomedical model of health, and globalisation. It seeks to examine the ways in which the HGP shapes ways of thinking about health; the influence globalisation has on this process; and the implications of this for smaller nations such as Australia. Adopting a critical perspective grounded in political economy, the study provides a largely structuralist analysis of the emergent health context of the HGP. This perspective, which embraces an insightful nexus drawn from the literature on biomedicine, globalisation and the HGP, offers much utility by which to explore the basis of biomedical dominance, in particular, whether it is biomedicine's links to the capitalist infrastructure, or its inherent efficacy and efficiency, that sustains the biomedical paradigm over "other" or non-biomedical health approaches. Additionally, the perspective allows for an assessment of whether there should be some broadening of the way health is conceptualised and delivered to better account for social, economic, and environmental factors that affect living standards and health outcomes, and also the capacity of globalisation to promote such change. These issues are at the core of the study and provide the theoretical frame to examine the processes by which Australian policy makers have given an increasing level of support to human genomic research over the past decade and also the implications of those discrete policy choices. Overall, the study found that globalisation is renewing and extending the dominance of the biomedical model, which will further marginalise other models of health while potentially consuming greater resources for fewer real health outcomes. While the emerging genomic revolution in health care may lead to some wondrous innovations in the coming decades, it is also highly likely to exacerbate the problems of escalating costs and diminishing returns that characterise health care systems in industrialised countries, and to lead to greater health inequities both within and between societies. The Australian Government has chosen to underwrite human genomic research and development. However, Australia's response to the HGP has involved both convergences and variations from the experiences of more powerful industrial nations. The most significant divergence has been in industry and science policy, where until the mid-1990s, the Australian Government displayed no significant interest in providing dedicated research funding, facilities, or enabling agencies to the emerging field. Driven by the threat of economic marginalisation and cultural irrelevance, however, a transformation occurred. Beginning with the Major National Research Facilities Program of the Department of Industry, Science and Technology, and then the landmark Health and Medical Research Strategic Review, support for human genomic research grew strongly. Comprehensive policy settings have recently been established to promote the innovation, commercialisation, promotion and uptake of the products of medical biotechnology and genomics. As such, local advocates of a broader model of health will be forced to compete on the political and economic stage with yet another powerful new area of biomedicine, and thus struggle to secure resources for perhaps more viable and sustainable approaches to health care in the 21st century.

Meiotic recombination is a prerequisite for exchange of genetic information in all Sexually reproducing organisms. This process is initiated by the formation of double stranded breaks (DSBs) in DNA followed by homology directed repair. The process is subjected to surveillance mechanisms that control DSB formation and allow for repair of DSBs by halting cell cycle progression. Interestingly, though generation of DSBs is an Essential event in meiosis they are nevertheless regarded as the most lethal forms of DNA damage. If left unrepaired a single DSB can lead to gene deletion, duplication, translocations and missegregation of large chromosome fragments leading to cell death. In Saccharomyces cerevisiae, genetic screens for mutants defective in meiotic recombination led to the identification of a group of genes called the RAD52 epistasis group which includes RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, MRE11 and XRS2. A subset of these genes, namely MRE11, RAD50 and XRS2, have been shown by genetic studies to be essential for several nuclear events including sensing DSBs, double strand break repair (DSBR) by homologous recombination (HR) and non homologous end joining (NHEJ), telomere length maintenance, cell cycle activation in response to DSBs, mitotic and meiotic recombination. In vitro, Mre11 displays Mn2+-dependent endonuclease activity on ssDNA, 3'-5' Exonuclease on single- and double-stranded DNA, strand annealing and weak hairpin Opening activities. Mutational analyses have revealed two functional domains in Mre11- Then terminal nuclease domain involved in telomere length maintenance and DSB Processing and the C terminal DNA binding domain involved in DSB formation during Meiosis. Rad50, a 153 kDa protein shares homology with the SMC (Structural Maintenance of Chromosome) family of proteins which are involved in chromosome Condensation and cohesion. It consists of a bipartite N- and C terminal Walker A and Walker B motifs separated by a heptad repeat sequence which folds into an antiparallel Coiled-coil structure. The heptad repeats are separated by a metal binding globular region the Zn hook. Rad50 is an ATP-dependent DNA-binding protein. hRad50 regulates the exonuclease activity of hMre11. Unlike Mre11 and Rad50, which are evolutionarily conserved, Xrs2 is found only in S. cerevisiae and Nbs1 in mammals. Xrs2 appears to be sequence non-specific DNA- binding protein. Xrs2 in yeast or Nbs1 is its counterpart in mammals target Mre11 and Rad50 to the sites of DNA damage and mediate S-phase cell cycle checkpoint activation. Mutations in either one of the MRX subunits results in defects in repair of DSBs, activation of cell cycle checkpoint and shortened telomeres leading to genomic instability. Hypomorphic mutations in MRE11 and NBS1 lead to genetic disorders- A-TLD (ataxia-telangiectasia-like disorder) and NBS (Nijmegen breakage syndrome) respectively, that are phenotypic ally related to AT (ataxia-telangiectasia) caused by mutations in ATM. Patients with AT, A-TLD or NBS syndromes are hypersensitive to radiomimetic agents and are predisposed to cancer. Several lines of evidence suggest that S. cerevisiae strains bearing mre11Δ, rad50Δ or xrs2Δ display shortening of telomeres. Telomeres are the nucleoprotein ends of all linear eukaryotic chromosomes that are important in maintaining the integrity of the genome.Telomeres are comprised of repetitive G rich sequence most of which is double stranded but the extreme 3' end protrudes to form 3' single stranded overhang called the G tail. elopers are essential in preventing end-end fusion of chromosome, are important for chromosome replication, segregation and genome stability. Genetic studies have implicated the MRX complex in both telomerase-dependent and independent telomere length maintenance. Studies have indicated a direct role for S. cerevisiae MRE11 in the proper establishment of telomere end-structure. However, the molecular mechanism of MRX at telomeres is poorly understood. To understand the role(s) of MRX complex at telomeres, it is important to elucidate the biochemical activities of MRX complex as well as its individual subunits on the telomere DNA structures. Since, Mre11 complex is known to function in several processes related to DNA metabolism it becomes imperative to study the function of Mre11 complex on DNA substrates in the context of a given nuclear process. The 3' single trended telomeric sequence is capable of acquiring folded conformation(s) as a mechanism of end protection which is mediated by several telomere-specific and nonspecific ending proteins. In mammals, the 3' ssDNA has been demonstrated to fold into tloop configuration mediated by some of the components of sheltrin protein complex, wherein the ssDNA invades the duplex DNA resulting in the formation of a displacement loop (D loop). Evidence for the formation of t-loop has been shown in vitro with human telomeres. However, the formation of t-loops has not been demonstrated in S. cerevisiae. Nevertheless, there is growing body of evidence which suggests the formation of alternative DNA structures such as G4 DNA at the yeast telomeres. G quadruplexes (G quartets or G4 DNA) are thermodynamically stable structures formed by Hoogsteen base pairing between guanine residues. In a G quartet the four guanine residues are paired, where each guanine residue is an electron acceptor and a donor and stabilized by a metal cation. The presence of G rich motifs at the promoter regions, rDNA, telomeres and recombination hot spots indicate that G4 DNA has important functions in vivo. Although the existence of G4 DNA has been the subject of much debate, the identification of several proteins that promote (Rap1, Hop1, Topo I, TEBPβ), modify and resolve (POT1, TERT, KEM1, GQN1, BLM, WRN, Rte1) G4 DNA, together with the direct visualization of G4 DNA using G4 DNA specific antibodies and RNA interference have provided compelling for the existence of G4 DNA in vivo. To elucidate the function of MRX complex or its individual subunits at telomeres, the biochemical activities of purified MRX complex and its individual subunits on G4 DNA, D loop, duplex DNA and G rich ssDNA has been analyzed in this study. G4 DNA was assembled from S. cerevisiae telomeric sequence. G4 DNA was isolated and its identity was ascertained by chemical probing and circular dichroism. S. cerevisiae MRE11 and XRS2 was cloned and expressed in E. coli BL21 (DE3)plysS. S. cerevisiae RAD50 in pPM231 vector in S. cerevisiae BJ5464 strain was a gift from Dr. Patrick Sung (Yale University). Mre11, Rad50 and Xrs2 were overexpressed and purified to >98% homogeneity. The identity of the proteins was ascertained by Western bloting using polyclonal antibodies. Using purified proteins heterotrimeric MRX and heterodimeric MR and MX protein complexes were formed in the absence of ATP, DNA or Mn2+. The ability of M/R/X to bind to telomeric DNA substrates was studied by electrophoretic mobility shift assays. Mre11, Rad50, Xrs2 and MRX displayed higher binding affinity for G4 DNA over D loop, ss- or dsDNA. MRX bound G4 DNA more efficiently compared to its individual subunits as 10-fold lower concentration of MRX was able to shift the DNA into the protein-DNA complex. The protein-G4 DNA complexes were stable as >0.8 M NaCl as required to dissociate 50% of protein-G4 DNA complexes. Efficient competition by poly(dG), which is known to fold into G4 DNA, suggested that the protein-G4 DNA complex was specific. Competition experiments with tetra-[N-methyl- pyridyl]-porphyrin suggested that M/R/X recognizes distinct determinants and makes specific interactions with G4 DNA. G4 DNA is highly polymorphic and can exist as intramolecular or intermolecular (parallel and antiparallel) structures. High affinity binding of Mre11 to G4 DNA (parallel) over G2' DNA (antiparallel), ss- and dsDNA suggests the existence of parallel G4 DNA structures at the telomeres and that G4 DNA may be the natural substrate for MRX complex in vivo. Telomeres are elongated by telomerase that requires access to the 3' G-tail for its activity. Formation of G4 DNA structures renders the 3' G-tail inaccessible to telomerase thereby inhibiting telomere elongation. To elucidate the functional relevance of high affinity of M/R/X for G4 DNA, the ability of the complex to generate the appropriate DNA structure for telomere elongation has been analyzed. In this study, I considered the possibility that MRX could act as: (a) a helicase that opens up the G4 DNA structures making it accessible to telomerase or (b) as a nuclease that cleaves the G4 DNA generating substrates for telomerase. Helicase assay with Mre11, Xrs2, MX and MRX on G4 DNA and duplex DNA showed no detectable DNA unwinding activity. Interestingly, nuclease assays with Mre11 on G4 DNA showed that Mre11 cleaved G4 DNA in Mn2+-dependent manner and the cleavage was mapped to the G residues at the stacks of G quartets. Mre11 cleaved telomeric duplex DNA in the center of TGTG repeat sequence, G rich ssDNA at 5' G residue in an array of 3 G residues and D loop structure preferentially at the 5' ends at TG residues. Significantly, the endonuclease activity of Mre11 was abrogated by Rad50. Xrs2 had no effect on the endonuclease activity of Mre11. Structural studies on Rad50 and Mre11 showed that binding of ATP by Rad50 positions the Rad50 catalytic domain in close proximity to the nuclease active site of Mre11. In yeast, disruption of ATP binding Walker motifs results in a null phenotype, suggesting that ATP is required for Rad50 functions in vivo. hRad50 is known to regulate the exonuclease activity of hMre11 in the presence of ATP. Therefore, can ATP modulate the effect of S. cerevisiae (Sc) Rad50 on ScMre11? To address this question, I monitored the ATPase activity of Rad50 in the absence or presence of DNA. Rad50 hydrolyzed ATP in a DNA-independent manner; however, ATPase activity was enhanced in the presence of Mre11 and Xrs2. However, Rad50 exhibited a low turnover indicating that ATP could function as a switch molecule. Based on these observations, the effect of ATP on the nuclease activity was examined. The binding of ATP and its hydrolysis by Rad50 attenuated the inhibition exerted by Rad50 on the Mre11 endonuclease activity. Cleavage of G4 DNA, D loop, duplex DNA and ssDNA required ATP hydrolysis, since no cleavage product was observed when ADP or ATPγS was substituted for ATP. This observation was corroborated using a hairpin DNA substrate that mimics a intermediate in VDJ recombination, thereby confirming the generality of regulation of Rad50 on the endonuclease activity of Mre11. Does Rad50 regulate the exonuclease activity of Mre11 as well? To address this question, exonuclease activity of Mre11, MR and MRX on 3' labeled duplex DNA and G4 DNA was assayed. Rad50 had no measurable effect on the exonuclease activity of Mre11. Based on previous studies and my observations, I propose a model for the role of MRX in telomere length maintenance and its regulation by the ATP-binding pocket of Rad50. MRX binds telomeric DNA substrates in a non-productive complex, which is converted to a catalytically active complex upon binding of ATP by Rad50. ATP induces conformational changes, repositioning the complex such that the catalytic site of Mre11 now has access to the substrate. Following cleavage of DNA by Mre11, the release of ADP and inorganic phosphate, generate the cleaved product. The cleaved DNA is now accessible to telomerase or telomere binding proteins. In summary, the data presented in my PhD thesis demonstrates that Mre11 is a structure- and sequence-specific endonuclease. The natural substrate for telomerase is the 3' ssDNA. G quartets at telomeres not only protect the ends from degradation but also make the ends inaccessible for telomerase activity. Genetic studies have shown that cells proficient for telomerase activity but lacking any one of the components of the MRX complex display shortening in telomere length. The ability of Mre11 to cleave G4 DNA at the stacks of G quartets therefore, suggests a mechanism by which the 3' ssDNA is rendered accessible to telomerase or other telomere binding proteins. Yeast telomeres are characterized by the presence of subtelomeric Y' elements proximal to the terminal TG1- 3 repeat sequences. The Y' element has been shown to be amplified by telomerase in a fraction of mutants with short telomeres. The mechanism by which Y' DNA is amplified is unclear. The ability of Mre11 to cleave telomere duplex DNA at the center of TGTG repeats could contribute to the generation of appropriate substrate for elongation by telomerase, thereby contributing to Y' DNA amplification. Telomere length is maintained by homeostasis between processes that contribute to telomere elongation and those that cause attrition in telomeric ends. Overelongated telomeres are brought to wild type telomere size by a unique recombinational single step deletion process termed telomere rapid deletion (TRD). TRD involves invasion of the elongated 3' G tail into the proximal telomeric tract resulting in the formation of the D loop structure. Following branch migration the D-loop is nicked and resolved into a deleted telomere and a circular liner product. Cells deleted for MRE11, RAD50 or XRS2 are deficient in TRD process. It has been hypothesized that Mre11 could be a candidate for cleaving the D-loop structure. The endonuclease activity of Mre11 on D-loop structure, preferentially at the 5' ends at TG residues demonstrated in this study, show that Mre11 could function as the nuclease required to generate the deleted telomere in TRD. MRX complex is involved in several processes involving DNA metabolism. It is important that the activities of the complex are regulated in the in vivo context. Complex formation and the interaction of the individual subunits with nucleotide cofactors and metal ions constitute a mode of regulation. This study shows that Rad50 regulates the endonuclease, but not exonuclease activity of Mre11. The binding of ATP and its hydrolysis by Rad50 brings in the regulatory factor necessary to keep the uncontrolled nuclease activity of MRX in check, thus preventing any deleterious effects on telomere length. Telomere maintenance by telomerase is activated in 80% of cancer cells. Inhibition of telomerase by G quartets provides a new drug targets for potential anti-cancer drugs. It is, therefore, likely that understanding the biological consequences of G quadruplex interactions would provide a better insight in development of therapeutics for cancer.

The Human Genome Project, initiated in 1990, creates an enormous amount of excitement in human genetics—a field of study that seeks answers to the understanding of human evolution, diseases and development, gene therapy, and preventive medicine. The first completion of a human genome in 2003 and the breakthroughs of sequencing technologies in the past few years deliver the promised benefits of genome studies, especially in the roles of genomic variability and human health. However, intensive resource requirements and the associated costs make it infeasible to experimentally verify the effect of every genetic variation. At this stage of genome studies, in silico predictions play an important role in identifying putative functional variants. The most common practice for genome variant evaluation is based on the evolutionary conservation at the mutation site. Nonetheless, sequence conservation is not the absolute predictor for deleteriousness since phylogenetic diversity of aligned sequences used to construct the prediction algorithm has substantial effects on the analysis. This dissertation aims at overcoming the weaknesses of the conservation-based assumption for predicting the variant effects. The dissertation describes three different integrative computational approaches to identify a subset of high-priority amino acid mutations, derived from human genome data. The methods investigate variant-function relationships in three aspects of genome studies—personal genomics, genomics of epilepsy disorders, and genomics of variable drug responses. For genetic variants found in genomes of healthy individuals, an eight-level variant classification scheme is implemented to rank variants that are important towards individualized health profiles. For candidate genetic variants of epilepsy disorders, a novel 3-dimensional structure-based assessment protocol for amino acid mutations is established to improve discrimination between neutral and causal variants at less conserved sites, and to facilitate variant prioritization for experimental validations. For genomic variants that may affect inter-individual variability in drug responses, an explicit structure-based predictor for structural disturbances is developed to efficiently evaluate unknown variants in pharmacogenes. Overall, the three integrative approaches provide an opportunity for examining the effects of genomic variants from multiple perspectives of genome studies. They also introduce an efficient way to catalog amino acid variants on a large scale genome data.

[Truncated abstract] Polymorphic Alu insertions (POALINs) are known to contribute to the variation and genetic diversity of the human genome. In this report specific POALINs of the Major Histocompatibility Complex (MHC) were studied. Previous population studies on the MHC POALINs have focused on individuals of African, European and Asian descent. In this study, we expand the research by studying a new and previously uncharacterised population, focusing on the Bedouin from the Middle East. Specifically we report on the individual insertion frequencies of four POALINs within the MHC class I region of this population. POALINs are members of a young Alu subfamily that have only recently been inserted into the human genome. POALINs are either present or absent at particular sites. Individuals that share the inserted (or deleted) polymorphism inherited the insertion (or deletion) from a common ancestor, making Alu alleles identical by decent. In population genetics a comparison of the resulting products from each population can then be done by comparing the lengths of the PCR products in a series of unrelated individuals and may also detect polymorphisms with regard to the presence or absence of the Alu repeats. As a direct result of their abundance and sequence identity, they promote genetic recombination events that are responsible for large-scale deletions, duplication and translocations. The deletions occur mostly in the A-T rich regions and have found to be unlikely to have been created independently of the insertions of the Alu elements (Callinan et al, 2005) The easy genotyping of the POALINs has proven to be very valuable as lineage markers for the study of human population genetics, pedigree and forensics as well as genomic diversity and evolution. POALINs have been used in a range of applications, primarily focusing on anthropological analysis of human populations. As a result of its ease of use and its utility as a marker in human evolutions studies, combining the POALINs along with other markers used in forensics could lead to improved identity testing in forensic science. More specifically, in combination with more traditional markers, race specific genotypes and haplotypes could be used for profiling crime scene samples. ... This is supported by previously reported molecular data using various types of genetic markers. In a study using six separate Alu genes, Antunez-de-Mayolo et al were able to generate a phylogenetic tree, in which the biogeographical groups followed a pattern. The biogeographical groups started with African populations that were found to relate closely to the hypothetical ancestral African population. The African populations were then followed in order by Southwest Asian populations, European populations which include Middle Eastern groups (Antunez-de-Mayolo et al, 2002). This study shows the similarities and differences between the frequencies of the Middle Eastern Bedouin and the rest of the compared populations. Though no clear results were determined, the information from the POALINs along with information provided from other genetic markers can lead to further research on the Bedouin population and the improvement of the forensic population database in order to accurately test individual ethnic background of samples to be analysed.