Prostate cancer is the most frequently diagnosed cancer in American men and the second leading cause of cancer deaths. Supplementation with Se has reduced the incidence of prostate cancer and Se status is inversely correlated with prostate cancer risk. One molecular mechanism by which high Se concentrations may affect cancer risk is by catalyzing disulfide bond formation or otherwise complexing with reactive sulfhydryl groups in cellular proteins. The estrogen receptor (ER) contains cysteines in zinc (Zn) fingers that are susceptible to oxidation and internal disulfide formation, which can prevent DNA binding. We examined ER binding to its DNA response element and gene expression levels for estrogen-regulated genes in human prostate cancer cells (LNCaP) treated with control (50 nM) or high (5 ìM) concentrations of Se. High Se treatment resulted in a non-significant 16 % decrease in ER binding to the estrogen response element (ERE), and no significant changes were found in expression levels of estrogen-regulated genes for either run-on nuclear transcripts or total mRNA. The well documented reaction of Se with reactive sulfhydryl groups, if it occurs in the ER in vivo, has a minimal effect on the binding of ER to DNA and its regulation of gene expression.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 72-79).

A side effect of generating an immune system for defense against invading pathogens is the potential to develop destructive cells that recognize self-tissues. Typically, through the "education" of developing immune cells, the organism inactivates potentially self-destructive cells, resulting in what is called self-tolerance. I proposed to explore the molecular mechanisms responsible for the induction and maintenance of tolerance. Our lab has developed a model of induced immune tolerance to skin and islet allografts utilizing a donor-specific transfusion of spleen cells and a brief course of anti-CD40L antibody. Because the difficulty in isolation of tolerant T cells from this system is prohibitive to performing large screens on these cells directly, I have chosen to study an in vitro CD4+Th1 cell line, A.E7, which can be made anergic via stimulation through the T cell receptor in the absence of costimulation. I hypothesized that anergized T cells upregulate genes that are responsible for the induction and maintenance of anergy and therefore exhibit a unique RNA expression profile. I have screened anergic cells using Affymetrix GeneChips and identified a small number of genes that are differentially expressed long-term in the anergic population compared to mock-stimulated and productively activated controls. The results have been confirmed by quantitative RT-PCR for each of the candidates. One of the most promising, the zinc-finger transcription factor Egr-2, was verified to be expressed long-term by western blotting, demonstrating perfect correlation between Egr-2 protein expression and the anergic phenotype. Silencing Egr-2 gene expression by siRNA in A.E7 T cells prior to anergy induction rescues the cells from the inability to phosphorylate ERK-1 and ERK-2 and also results in increased proliferation in response to antigen rechallenge. In this study I report that Egr-2 is specifically expressed long-term in anergic cells, protein expression correlates inversely with responsiveness to antigen rechallenge, and that Egr-2 is required for the full induction of anergy in T cell clones.

Très répandues dans le monde vivant, les protéines à doigt de zinc constituent une super-famille dont les membres possèdent un site à zinc de formule générale [ZnII(Cys)4-X(His)X] (x=0, 1 or 2). Tandis que la majorité de ces sites joue un rôle purement structural, certains présentent une fonction réactive, comme la détection de stress oxydant par exemple. En effet, les sites doigt de zinc de Hsp33 et de RsrA ont été décrits comme des interrupteurs rédox[1,2] : transmettant l'information « stress oxydant » sous forme d'un signal structural, via l'oxydation/réduction des cystéines coordonnées au zinc, détruisant/reformant le domaine doigt de zinc. Cependant, certains aspects de l'étape d'oxydation restent mal compris.Étant donné le grand nombre des protéines à doigt de zinc et leurs rôles clés, il est de tout intérêt d'identifier les facteurs contrôlant leur réactivité afin de comprendre pourquoi certaines espèces réactives de l'oxygène (ERO) sont capable d'oxyder des doigts de zinc in vivo, contrairement à H2O2. Durant ce projet, nous avons décidé de nous focaliser sur deux ERO très puissantes et jouant un rôle important en biologie : l'acide hypochloreux (HOCl) et l'oxygène singulet (1O2). Nous étudierons la réactivité des doigts de zinc vis-à-vis de ces deux oxydant en utilisant des modèles peptidiques, reproduisant parfaitement la structure de doigts de zinc courants. / Widely spread in the living world, zinc finger proteins constitute a large superfamily, with a zinc site of general formula [ZnII(Cys)4-X(His)X] (x=0, 1 or 2) as a common feature. Whereas the majority of such sites plays a purely structural role, a few of them exhibit a reactive function (e.g. oxidative stress detection). Indeed, zinc finger sites of Hsp33 and RsrA have been shown to act as redox switches[1,2]: transmitting the information “oxidative stress” as a structural signal, by means of oxidation/reduction of its ZnII-coordinating cysteines. However, the precise mechanism of the oxidation step remains poorly understood.Given the occurrence of zinc finger proteins and their key roles, it is of high biological interest to identify factors controlling their reactivity and to understand why some ROS are able to oxidize them in vivo, on the contrary to H2O2. In this project, we decided to focus on two major ROS: hypochlorous acid (HOCl), a key player of the immune response, and singlet oxygen (1O2), produced in significant amount by photosynthetic organisms. By use of peptide model complexes, reproducing perfectly the structure of some archetypal zinc fingers, we investigated the reactivity of zinc fingers toward those ROS.

The utility of a model organism for studying biological processes is closely tied to its amenability to genome manipulation. Although tools for targeted genome engineering in mice have been available since 1987, most organisms including zebrafish have lacked efficient reverse genetic tools, which has stymied their broad implementation as a model system to study biological processes. The development of zinc finger nucleases (ZFNs) that can create double-strand breaks at desired sites in a genome has provided a universal platform for targeted genome modification. ZFNs are artificial restriction endonucleases that comprise of an array of 3- to 6-C2H2-zinc finger DNA-binding domains fused with the dimeric cleavage domain of the type IIs endonuclease FokI. C2H2-zinc fingers are the most common, naturally occurring DNA-binding domain, and their specificity can be engineered to recognize a variety of DNA sequences providing a strategy for targeting the appended nuclease domain to desired sites in a genome. The utility of ZFNs for gene editing relies on their activity and precision in vivo both of which depend on the generation of ZFPs that bind desired target sites high specificity and affinity. Although various methods are available that allow construction of ZFPs with novel specificities, ZFNs assembled using existing approaches often display negligible in vivo activity, presumably resulting from ZFPs with either low affinity or suboptimal specificity. A root cause of this deficiency is the presence of interfering interactions at the finger-finger interface upon assembly of multiple fingers. In this study we have employed bacterial-one-hybrid (B1H)-based selections to identify two-finger zinc finger units (2F-modules) containing optimized interface residues that can be combined with published finger archives to rapidly yield ZFNs that can target more than 95% of the zebrafish and human protein-coding genes while maintaining a success rate higher than that of ZFNs constructed using available methods. In addition to genome engineering in model organisms, this advancement in ZFN design will aid in the development of ZFN-based therapeutics. In the process of creating this archive, we have undertaken a broader study of zinc finger specificity to better understand fundamental aspects of DNA recognition. In the process we have created the largest protein-DNA interaction dataset for zinc fingers to be described that will facilitate the development of better predictive models of recognition. Ultimately, these predictive models would enable the rational design of synthetic zinc finger proteins for targeted gene regulation or genomic modification, and the prediction of genomic binding sites for naturally occurring zinc finger proteins for the construction of more accurate gene regulatory networks.

A idade normal para início da puberdade em meninas varia bastante, de 8 a 13 anos, e os genes envolvidos nesse controle são parcialmente conhecidos. Fatores ambientais, como alimentação e exposição a disruptores endócrinos, contribuem para essa variabilidade, de modo que genes modulados epigeneticamente podem justificar parte da complexidade desse processo. O termo epigenética se refere às modificações na expressão gênica que não são causadas por alterações na sequência do DNA. A metilação do DNA é o mecanismo epigenético mais bem estudado. Na última década surgiram evidências demonstrando a relação entre metilação do DNA e desenvolvimento puberal. Em fêmeas de roedores, a hipermetilação do DNA levou à puberdade precoce. Em humanos, a puberdade precoce central (PPC) familial causada por mutações nos genes MKRN3 e DLK1 é considerada um defeito do imprinting, fenômeno epigenético no qual apenas um dos alelos parentais é expresso, estando o outro metilado e inativo. Além disso, um conceito atual propõe que o início da puberdade requer a repressão epigenética de fatores inibidores do eixo gonadotrófico. Recentemente, genes zinc finger (ZNF) foram relacionados ao processo puberal, e muitos deles codificam repressores transcricionais. Neste trabalho, estudamos a metilação do DNA do sangue periférico de 10 pacientes do sexo feminino com PPC familial (casos índices) e 33 meninas com desenvolvimento puberal normal (15 pré-púberes e 18 púberes), usando a plataforma Human Methylation 450 BeadChip. Duas pacientes tinham PPC de causa genética (uma com mutação no MKRN3 e outra com deleção no DLK1) e oito tinham PPC idiopática, sem mutações identificadas pelo sequenciamento exômico global. Cento e vinte regiões diferencialmente metiladas foram identificadas entre as meninas saudáveis pré-púberes e púberes, estando 74% delas no cromossomo X. Apenas uma região mostrou-se hipometilada no grupo púbere e, de maneira importante, contém a região promotora do ZFP57, fator necessário para manutenção do imprinting. Uma vez que a hipermetilação nas regiões promotoras dos genes é relacionada à inibição transcricional, o achado de hipermetilação global do DNA na puberdade sugere que haja inibição de fatores inibidores do eixo gonadotrófico, o que resultaria no início do processo puberal. O receptor estrogênico destacou-se como um fator transcricional que se liga a sete genes diferencialmente metilados entre os controles pré-púberes e púberes. As pacientes com PPC apresentaram mais sítios CpG hipermetilados tanto na comparação com as meninas pré-púberes (81%) quanto púberes (89%). Há doze genes ZNF contendo sítios CpG hipermetilados na PPC. Não foram encontradas anormalidades de metilação nos genes MKRN3 e DLK1 nem em suas regiões regulatórias. Em conclusão, este estudo evidenciou hipermetilação global do DNA em meninas com puberdade normal e precoce, sugerindo que esse padrão é uma marca epigenética da puberdade. Pela primeira vez, mudanças no metiloma de pacientes com PPC foram descritas. Modificações na metilação de vários genes ZNF parecem compor a complexa rede de mecanismos que leva ao início da puberdade humana / Normal puberty initiation varies greatly among girls, from 8 to 13 years, and the genetic basis for its control is partially known. Environmental factors, such as nutrition and exposure to endocrine disruptors, contribute to this variance, and epigenetically modulated genes may justify some of the complexity observed in this process. Epigenetics refers to alterations in gene expression that are not caused by changes in DNA sequence itself. DNA methylation is the best studied epigenetic mechanism. In the last decade, evidence has emerged showing the relationship between DNA methylation and pubertal development. In female mice, DNA hypermethylation led to precocious puberty. In humans, familial central precocious puberty (CPP) caused by mutations in the MKRN3 and DLK1 genes is considered a disorder of imprinting, an epigenetic phenomenon in which only one parental allele is expressed, and the other allele is methylated and inactive. In addition, animal studies indicated that pubertal timing requires epigenetic repression of inhibitory factors of the gonadotrophic axis. Recently, zinc finger genes (ZNF) were related to pubertal development, many of which encode transcriptional repressors. In the present study, we analyzed the DNA methylation of peripheral blood samples from 10 female patients with familial CPP (index cases) and 33 girls with normal pubertal development (15 pre-pubertal and 18 pubertal), using the Human Methylation 450 BeadChip assay. Genetic CPP was diagnosed in two patients (one with a MKRN3 mutation and the other with a DLK1 deletion). The remaining eight cases with idiopathic CPP were previously evaluated by whole exome sequencing and no causative mutations were identified so far. We evidenced 120 differentially methylated regions between pre-pubertal and pubertal healthy girls, and 74% of them were located at the X chromosome. Only one genomic region was hypomethylated in the pubertal group. Of note, it contains the promoter region of ZFP57, an important factor for imprinting maintenance. As DNA hypermethylation in gene promoters is related to gene silencing, the finding of global DNA hypermethylation in puberty suggests inhibition of inhibitory factors of the hypothalamic-pituitary-gonadal axis that results in puberty onset. Importantly, the estrogen receptor was identified as a transcriptional factor that binds to seven differentially methylated genes associated with pubertal process. Patients with CPP exhibited more hypermethylated CpG sites compared to both pre-pubertal (81%) and pubertal (89%) controls. Twelve ZNF genes were recognized as having hypermethylated CpG sites in CPP. The methylation analyses of MKRN3 and DLK1 genes showed no abnormalities. In conclusion, this study revealed a widespread DNA hypermethylation in girls with normal and precocious puberty, suggesting that this pattern can be an epigenetic signature of puberty. For the first time, changes in the methylome of patients with CPP were described. We highlight that alterations in methylation levels of several ZNF genes may impact the onset of human puberty

By means of reverse-transcription polymerase chain reaction, overexpression of ZNF146 was detected in two human HCC cell lines HepG2 and Hep3B and a clear relationship between HCC and overexpression of ZNF146 has been established. Subcellular localization of ZNF146 protein in liver cells was studied by generation and expression of a green fluorescent protein (GFP) fusion protein. The nuclear localization and the reported DNA binding ability of ZNF146 protein provided a hint that ZNF146 may carry out its function in the cell system by interacting with specific genomic DNA sequences. Recombinant ZNF146 protein was expressed using bacterial and yeast system for the genomic DNA pull down assay in the identification of potential interacting genomic DNA sequences. Several potential genomic DNA sequences that interact with ZNF146 were identified and the gene MDM2 is the one of the candidates that is directly related to human carcinogenesis. MDM2 is a negative regulator of the tumor suppresser protein p53. Deregulation of MDM2 will impair the cell's ability in cell cycle arrest, DNA repair and apoptosis upon induced DNA damage. / Hepatocellular carcinoma (HCC) is a type of primary malignant liver tumor. And is one of the most frequent malignancies worldwide. The focus of this research project is the characterization of a Kruppel zinc finger protein, zinc Finger Protein 146 (ZNF146) using HCC as a disease model. The aim of this project is to understand the functional role ZNF146 and try to explore the mechanism of how ZNF146 might be involved in the carcinogenesis of HCC. / In order to have a better understanding with the protein ZNF146, SUMOylation properties of this protein has been studied. SUMO1 modification on ZNF146 has already been reported. And in our study, experimental result demonstrated that ZNF146 is also modified by SUMO2 and SUMO3 in liver cells. Other than the SUMOylation sites for SUMO1 protein which has been reported, modification sites for SUMO2 at the K247 and K275 positions were mapped, while K191R, K219R, K247R, K256R and K275R, five positions were mapped for SUMO3 modification. A more complete picture of the SUMOylation properties of ZNF146 has been revealed. Since we hypothesized that ZNF146 is related to the p53 tumor suppressor, cell cycle control and DNA repair pathway, a cell cycle study using flow cytometry was performed for the investigation of the effect on cell cycle regulation by ZNF146 overexpression. In our study, ZNF146 overexpression promoted the G1/S transition in the cell division cycle, which indicated that liver cells were more active for the progression of cell cycle. / On the other hand, using cDNA microarray technology expression profiles of ZNF146 overexpressing and non-overexpressing liver cell lines were compared and with real-time polymerase chain reaction, six candidate genes CRLF1, IFI44, ST6GAL1, LOC441601, IL18 and RAD17 were confirmed with their deregulation induced by the overexpression of ZNF146. Four of the candidates, IFI44, LOC441601, IL18 and RAD17 were found to be related to the p53 tumor suppressor activity or DNA damage, repair response and control. This observation, together with the result of genomic DNA pull down assay, gives us a hint that ZNF146 is possibly involved in liver carcinogenesis by affecting DNA repair and cell cycle control upon induced DNA damage. / The gene ZNF146 codes for a member of the Kruppel zinc finger proteins, however ZNF146 protein is different from most members of the Kruppel zinc finger proteins subfamily. It encodes a 33 kDa protein solely composed of 10 zinc finger motifs and is devoid of any non-zinc finger regulatory domain for interactions with other proteins. ZNF146 overexpression has been reported in a number of cancers including colon cancer and pancreatic carcinoma. However, the functional role of ZNF146 overexpression in tumorigenesis is yet to be solved and not much research on how ZNF146 might be invovled in the establishment of HCC was published. / To conclude, the experimental results of this study support the hypothesis that ZNF146 overexpression may deregulating the cell division cycle and some genes differentially regulated upon over-expression of ZNF146 are related to the regulations of DNA damage response. Future research on ZNF146 can be focused on the detail regulatory pathway of ZNF146 overexpression and its interaction between the p53 tumor suppressor, DNA damage response and cell cycle regulation, and a fuller picture of how ZNF146 overexpression might induce hepatocarcinogenesis can be revealed. / Yeung, Tsz Lun. / Adviser: Miu Yee (Mary) Waye. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3329. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 287-304). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Os projetos genoma têm fornecido uma grande quantidade de informação sobre a arquitetura gênica e sobre a configuração física de suas respectivas regiões flanqueadoras (RF). Estas RF contêm informações com o potencial de auxiliar na elucidação de vários processos biológicos, como os mecanismos de expressão gênica e de sua regulação. Estes mecanismos são de extrema importância para a compreensão do correto funcionamento dos organismos e das patologias que os afetam. Uma parte significativa dos mecanismos de controle de expressão gênica atuam na fase transcricional. Na base destes mecanismos está o recrutamento de proteínas que se ligam às regiões promotoras da transcrição, as quais são segmentos específicos de DNA que podem estar localizados tanto próximos à região de início da transcrição (TSS) quanto a centenas ou até a milhares de pares de bases dela. Essas proteínas compõem a maquinaria transcricional e podem ativar ou inibir o processo de transcrição. Experimentalmente, os segmentos regulatórios podem ser identificadas utilizando métodos complexos de biologia molecular, tais como SELEX, ChiP-ChiP, ChIP-Seq, dentre outros. Uma estratégia alternativa aos métodos experimentais é a utilização de metodologias computacionais. Análises computacionais tendem a ser mais rápidas, baratas e flexíveis do que protocolos experimentais, além de poderem ser utilizadas em larga escala. Atualmente, os métodos computacionais disponíveis necessitam de informações experimentais para a definição de padrões globais de preferências de sequências de DNA para a ligação de fatores de transcrição (TFBS, em inglês transcription factor binding sites). Entretanto, esses métodos apresentam uma elevada taxa de falso positivos e, por vezes, apresentam também taxas significativas de falso negativos, além de serem limitados ao estudo de fatores de transcrição de espécies bem conhecidas, o que diminui a área de aplicação dos mesmos. Diante deste cenário, o uso de métodos computacionais que não necessitem da informação referente aos sítios de ligação, bem como os que utilizem parâmetros mais robustos de detecção dos resultados, em detrimento dos escores de pontuação provindos de alinhamentos, podem acrescentar uma sensível melhoria ao processos de predição de regiões regulatórias. Neste projeto, foi desenvolvido um novo modelo computacional (TFBSAnalyzer) para análise e identificação de TFBS em elementos regulatórios, que utiliza técnicas de modelagem molecular para a construção de complexos entre um fator de transcrição ancorado a estruturas de DNA com sequências variáveis de bases e, através de cálculos termodinâmicos de entalpia de ligação, determina uma função de pontuação baseada na energia de ligação e realiza a predição de sítios de ligação ao DNA para o fator de transcrição em análise. Esta abordagem foi testada com três fatores de transcrição como sistemas-modelo, pertencentes à família dos receptores nucleares, a saber: o receptor de estrógeno ER-alfa (Estrogen Receptor Alpha), o receptor de ácido retinoico RAR-beta (Retinoid Acid Receptor Beta) e o receptor X retinóico RXR (Retinoid X Receptor). Os modelos previstos computacionalmente foram comparados aos dados experimentais disponíveis para estes receptores nucleares, os quais apresentaram as seguintes taxas de FP/FN: 10%/0 para RAR-beta e RXR, 21%/6% para ER-alfa. Também simulamos um experimento de ChIP-seq do ER-alfa no genoma humano, cujos genes selecionados foram submetidos a uma análise de enriquecimento de fatores de transcrição curados experimentalmente, que fazem sua regulação, revelando que o receptor de estrógeno está realmente envolvido no processo. Para mostrar a aplicabilidade geral de nosso método, nós modelamos a distribuição de energia de ligação para o receptor NHR-28 isoforma a de Caenorhabditis elegans com DNA . Obtivemos distribuições de energia semelhantes àquelas encontradas para os NRs modelos, portanto seria possível aplicar o método para buscar possíveis TFBSs para este receptor no genoma de C. elegans. Os dados gerados e as metodologias desenvolvidas neste projeto devem acrescentar uma sensível melhoria aos processos de predição de regiões regulatórias e consequentemente auxiliar no entendimento dos mecanismos envolvidos no processo de expressão gênica e de sua regulação. / The genome projects have provided a lot of information about the genetic architecture, as well as on the physical configuration of their flanking regions (FR). These FR have the potential to aid in the elucidation of many biological processes, such as the mechanisms involved in gene expression and its regulation. These mechanisms are extremely important for undeerstanfind the correct functioning of organisms as well as the pathologies that affect them. A significant part of the control mechanisms of gene expression act during transcription. On the basis of this mechanisms is the recruitment of proteins that bind to promoter regions of transcription, which are specific segments of DNA that can be located either near the transcription start site or at hundreds or even thousands of base pairs away. These proteins form the transcription machinery, which can activate or inhibit the transcription process. The regulatory segments can be identified experimentally using complex methods of molecular biology, such as SELEX, ChIP-chip, ChIP-seq, among others. An alternative strategy to these experimental methods is the use of computational methodologies for predicting regulatory regions. Computational analysis tend to be faster, cheaper and more flexible than the experimental protocols, and can be used on a larger scale. Currently, the available computational methods require information previously obtained from experiments in order to define global standards of preference of DNA-Binding sequences for transcription factors (TFBS - Transcription Factor Binding Sites). However, these methods have a high rate of false positives and sometimes also have significant rates of false negatives, besides being limited to the study of transcription factors of well-known species, which decreases their application area. In this scenario, the use of computational methods that do not require previous information concerning the binding sites and use more robust parameters of results detection, instead of alignment scores, may add significant improvement to the processes of predicting regulatory regions. In this project, we developed a new computational model TFBSAnalyzer) for analysis and identification of regulatory elements using molecular modeling techniques for the construction of complexes between a transcription factor bound to specific DNA structures with variable sequences of bases and, by means of thermodynamic calculations of bond enthalpy, provides a scoring function based on the binding energy and predicts the DNA binding sites for the transcription factor in analysis. This approach was tested initially with three transcription factors as models, belonging to the nuclear receptor family, namely estrogen receptor ER-alpha (Estrogen Receptor Alpha), the retinoic acid receptor RAR-beta (Retinoid Acid Receptor Beta) and the retinoic X receptor RXR (Retinoid X Receptor). The computationally predicted models were compared to experimental data available for these nuclear receptors, and presented the following rates of FP/FN: 10%/0 for RAR-beta and RXR, 21%/6% for ER-alpha. We also simulated an experiment of ChIP-seq with ER-alpha with the human genome, where the selected genes were subjected to a transcription factor enrichment analysis, with curated information, revealing that the estrogen receptor is indeed involved in their regulation. To show that our method has a general applicability, we modeled the binding energy distribution for the NHR-28 receptor, isoform a, from Caenorhabditis elegans. The energy distributions obtained were similar to the ones obtained for the model NR, so it would be possible to use the method and search for possible TFBS in the C. elegans genome. The data generated and the methodologies developed in this project should add a significant improvement to the prediction processes of regulatory regions and, consequently, help to understand the mechanisms involved in the gene expression process and its regulation.

Saccharomyces cerevisiae HOP1, which encodes a component of the synaptonemal complex, plays an important role in both gene conversion and crossing over between homologs, as well as enforces the meiotic recombination checkpoint control over the progression of recombination intermediates. The zinc-finger motif (Znf) 348CX2CX19CX2C374) of Hop1 is crucial for its function in meiosis, since mutation of conserved Cys371 to Ser in this motif results in a temperature-sensitive phenotype, which is defective in sporulation and meiosis. The direct role for Hop1 or its ZnF in the formation of joint molecules and checkpoint control over the progression of meiotic recombination intermediates is unknown. To understand the underlying biochemical mechanism, we constructed a series of recombination intermediates. Hop1 or its ZnF were able to bind different recombination intermediates. Interestingly, the binding affinity of Hop1 and its ZnF was much higher for the Holliday junction as compared to other recombination intermediates. The complexes of Hop1 or its ZnF with the Holliday junction were stable and specific as shown by NaCl titration and competition experiment. Hop1 and its ZnF blocked BLM helicase-induced unwinding of the Holliday junction, indicating that the interaction between Hop1 and its ZnF with the Holliday junction is specific. DNase I footprinting experiment showed that Hop1 or its ZnF bind to the center of the Holliday junction. 2-aminopurine fluorescence and KMnO4 experiments showed that Hop1 or its ZnF can distort the Holliday junction in a 2-fold symmetrical manner. The molecular modeling study showed that Hop1 ZnF folded into unique helix-loop-helix motif and bound to center of the Holliday junction. In summary, this study shows that Hop1 protein or its ZnF interact specifically with the Holliday junction and distort its structure. Taken together, these results implicate that Hop1 protein might coordinate the physical monitoring of meiotic recombination intermediates during the process of branch migration and that Hop1 ZnF acts as a structural determinant of Hop1 protein functions.

Poly(ADP-ribosyl)ation, a covalent modification of proteins catalyzed by poly(ADP-ribose) polymerases (PARPs), plays a crucial role in regulating DNA repair, DNA replication, and cell death. Poly(ADP-ribose) Polymerase-1 (PARP-1) is a nuclear zinc-finger DNA-binding protein that is the most extensively studied member of the PARP family. The activation of PARP-1 depends on the N-terminal DNA-binding domain, which consists of two unusually long zinc finger-like motifs (termed FI and FII) of the form CX₂CX₂₈/₃₀HX₂C and a newly discovered zinc-ribbon motif (FIII). Though zinc is indispensible for PARP-1 activity, the metal binding affinities of the unusual zinc fingers of PARP-1 is not yet known. In this dissertation, the second zinc finger of PARP-1 was used as a model peptide to study the binding properties of several divalent metal ions (Co²⁺, Cd²⁺, Zn²⁺, and Pb²⁺). Metal-induced protein folding was investigated by circular dichroism, and the effects of the metal ions on PARP-1 activity were investigated by poly(ADP-ribosyl)ation activity assays. This study represents the first detailed biochemical characterization of the PARP zinc fingers. The functional role of each zinc finger in DNA damage recognition is critical for understanding how PARP-1 is involved in DNA repair. Thus, we constructed a series of PARP-1 zinc finger variant proteins and investigated their DNA binding properties and their effects on PARP activity. Using a combination of southwestern blotting and activity assays, we demonstrated that FII is more important for DNA binding, while FI and FIII seem to facilitate PARP activity. The DNA sequence-independent binding properties of PARP-1 were further characterized using DNA probes bearing defined secondary structures. Together, our results indicate that the zinc fingers help position the enzyme at specific DNA damage sites, and also help to activate the catalytic domain upon DNA binding. PARP-1 is involved in caspase-independent apoptosis, and the translocation of apoptosis inducing factor (AIF) out of the mitochondrial matrix has been shown to require PARP-1 activity. However, it is not readily apparent how the catalytic activity of PARP-1 (a nuclear protein) triggers the release of AIF from the mitochondrial matrix. In an attempt to understand the relationship between PARP-1 activity and caspase-independent apoptosis, we demonstrate here that AIF is an in vitro protein substrate for PARP-1. The possible implications of this finding will be discussed. / text