Dissertations / Theses on the topic 'Synaptonemal Complex'

The Synaptonemal Complex (SC) is a proteinaceous structure that connects homologous chromosomes lengthwise during meiotic prophase. In budding yeast, the SC consists of two parallel axes that become connected by the central element protein, Zip1 that extends along the chromosome axes (Sym, Engebrecht et al. 1993). Extension of the SC is coordinated to crossover formation by a group of proteins known as the ‘ZMM's (Zip1, Zip2, Zip3, Zip4, Msh4, Msh5 and Mer3) (Borner, Kleckner et al. 2004). Work outlined here demonstrates a role for the mismatch repair paralogue, Msh4 in preventing SC extension from being de-coupled from crossover formation. Furthermore, increased temperature serves as a positive effector for this decoupling. These findings suggest that SC extension is highly regulated to ensure that it is coupled with crossing over. As well as its role in crossover formation (Storlazzi, Xu et al. 1996), the work outlined here demonstrates an independent role for Zip1 in promoting the segregation of non-exchange chromosome pairs (NECs). Zip1 pairs the centromeres of NECs in pachytene through to metaphase I, where it aids their segregation at the first meiotic division. The localisation and function of Zip1 at the centromeres of non-exchange chromosomes depends on Zip3 and Zip2, respectively. Zip1 is observed at the centromeres of all chromosomes following SC disassembly through to the first meiotic division, where it promotes the segregation of exchange pairs also. A model is suggested whereby Zip1 promotes the segregation of both non-exchange and exchange chromosome pairs by tethering homologous centromeres throughout meiotic prophase. Finally, a parallel pathway for NEC segregation is also described that depends upon the spindle checkpoint component, Mad3. When both ZIP1 and MAD3 are deleted, NECs segregate at random.

Meiosis is a highly conserved specialized cell division that occurs in many organisms, including budding yeast and mammals. Meiosis divides the chromosome number of the cell in half to create gametes for sexual reproduction. A single round of chromosome duplication is followed by two rounds of chromosome segregation, Meiosis I (homologs segregate) and Meiosis II (sister chromatids segregate). Proper segregation at Meiosis I requires that homologs are connected by both crossovers and sister chromatid cohesion. Crossovers are formed by the repair of double strand breaks (DSBs) preferentially by the homolog. The choice of repair template is determined at the time of strand invasion, which is mediated by two recombinases, Rad51 and the meiosis-specific Dmc1. Rad51 is necessary for Dmc1 to function properly but its strand exchange activity is inhibited both by Dmc1 and Mek1, a meiosis-specific kinase, which is activated by DSBs. Mek1 suppresses interaction between Rad51 and its accessory factor Rad54 in two ways. First, phosphorylation of Rad54 lowers its affinity for Rad51. Second, phosphorylation stabilizes Hed1, a meiosis-specific protein that binds to Rad51 and excludes Rad54. Although RAD54 is not required for wild-type levels of interhomolog recombination, rad54Δ diploids exhibit decreased sporulation and spore viability, indicating the presence of unrepaired DSBs. My thesis tested the idea that Mek1 kinase activity is down-regulated after interhomolog recombination to allow Rad51-mediated repair of any remaining DSBs.

Meiotic recombination occurs in the context of a proteinaecous structure called the synaptonemal complex (SC). The SC is formed when sister chromatids condense along protein cores called axial elements (AEs) comprised of the meiosis-specific proteins, Hop1, Red1 and Rec8. AEs are brought together by interhomolog recombination, which creates stable connections and the gluing together of the AEs by the insertion of the transverse filament protein, Zip1, in a process called synapsis. Pachynema is the stage of meiotic prophase in which chromosomes are fully synapsed and where interhomolog recombination has proceeded to the double Holliday junction (dHJ) stage.

Meiotic progression requires transcription factor NDT80, a middle meiosis transcription factor required to express >200 genes, including the polo-like kinase, CDC5 (required for Holliday junction resolution and SC disassembly) and CLB1 (required for meiotic progression). Diploids deleted for NDT80 arrest in pachynema with unresolved dHJs. I used an inducible version of NDT80 (NDT80-IN ) to separate prophase into two phases: pre-NDT80, when interhomolog recombination occurs and post-NDT80, when it is proposed that inactivation of Mek1 allows intersister recombination to repair residual DSBs. RAD54 is sufficient to function after interhomolog recombination, as inducing both RAD54 and NDT80 simultaneously rescues the spore inviability defects observed in NDT80-IN rad54Δ diploids. Using an antibody specific for phosphorylated Hed1 as an indicator of Mek1 kinase activity, I showed that Mek1 is constitutively active in ndt80-arrested cells and that induction of NDT80 is sufficient to abolish Mek1 activity. Furthermore, inactivation of Mek1 by Ndt80 can occur in the absence of interhomolog strand invasion and synapsis. Mek1 inactivation correlates with the appearance of CDC5 and the degradation of Red1. My work demonstrates that the sole target of NDT80 responsible for inactivating Mek1 is CDC5.

Unrepaired DSBs trigger the meiotic recombination checkpoint resulting in prophase arrest, which requires Mek1 and works by sequestering Ndt80 in the cytoplasm. Mek1 also delays meiotic progression in wild-type cells, likely through inactivation of Ndt80. My work shows that Ndt80 in turn negatively regulates Mek1. Based on my observations, as well as published work showing that synapsis results in the removal of Mek1 from chromosomes, I propose that recombination and meiotic progression are coordinated by regulation of Mek1.

Defects in meiotic prophase I events, resulting in aneuploidy, are a leading cause of birth defects in humans; however, these are difficult to study in mammalian systems due to their occurrence very early in development. The nematode, Caenorhabditis elegans, is an excellent model for prophase I studies as its gonad is temporally and spatially organized around these meiotic events. Homolog pairing, synapsis, meiotic recombination and crossover formation are essential to the proper segregation of chromosomes into the respective gametes, either the egg or sperm. Disturbances in these events leads to missegregation of chromosomes in the gametes in the meiotic divisions. Synapsis is especially critical in meiosis as it precedes and is required for meiotic recombination in C. elegans. The formation of the synaptonemal complex (SC) is fundamental to chromosomal synapsis, yet the molecular mechanisms of synaptonemal complex morphogenesis are largely unknown. The investigations described in this thesis were undertaken to better understand the molecular contributions to synaptonemal complex morphogenesis. Chapter One reviews knowledge of morphogenesis and its relationship to the events of meiotic prophase I. Recent studies in our laboratory have implicated AKIRIN, a nuclear protein with multiple biological functions, as having a role in synaptonemal complex disassembly, specifically preventing the aggregation of synaptonemal proteins (Clemons et al., 2013). As a result of our efforts to discern the mechanism by which AKIRIN regulates disassembly, we found that the highly conserved CSN/COP9 signalosome has a role in SC assembly, leading to defects in prophase I events and in MAPK signaling , leading to the arrest of nuclei in the later stages of meiosis. While the CSN/COP9 signalosome has been implicated in general fertility in C. elegans (Pintard et al., 2003), no role had been defined in earlier meiotic stages until this study. Chapter Two describes an RNAi enhancer/suppressor screen undertaken in the akir-1 mutant background. Several RNAi clones were selected for future study based on a reduction in brood size; one of which, csn-5/, is the focus of the analysis presented in Chapter 3. Chapter Three describes the phenotypic characterization of two CSN/COP9 signalosome subunits, csn-2 and csn-5. Alleles of both genes display synaptonemal complex protein aggregation and defects in mitotic cell proliferation, homologous chromosome pairing, meiotic recombination and crossover formation, leading to an increase in apoptosis. Oocyte maturation is also disrupted by a lack of MAPK signaling, resulting in a lack of viable oocytes, which renders the csnmutant homozygotes sterile. These findings support a model suggesting the CSN/COP9 signalosome has an essential role in regulating meiotic prophase I events and oocyte maturation. Chapter 4 describes the methodology used in this study. Chapter 5 provides a summary of the thesis findings and examines the future directions to extend this work.

Meiosis is a specialized cellular division occurring in organisms capable of sexual reproduction that leads to the formation of gametes containing half of the original chromosome number. Meiosis involves two cell divisions, the first of which segregates homologous chromosomes to opposite poles, reducing ploidy by half. In most organisms, this segregation requires crossovers, the exchange of DNA sequences between homologous chromosomes, which in turn, is dependent upon stable associations of homologs. In early meiotic prophase I, chromosomes form pairing interactions that bring chromosomes into close physical associations. The process of synapsis then stabilizes these pairing interactions throughout the homolog pair, and is mediated by the synaptonemal complex (SC), a meiosis specific protein complex. Absent or misregulated assembly of the SC prevents the stabilization of pairing interactions that are essential for meiosis, leading to chromosome missegregation. Divided into two main projects, my work aimed to further our understanding of the regulation of synaptonemal complex assembly. One project examined meiotic chromosomal movement by characterizing a relatively unstudied protein in C. elegans, FKB-6. We showed that FKB-6 is important for creating pauses between chromosome movements. These pauses are needed for allowing chromosomes to properly pair and thus allowing for proper SC assembly. In the absence of FKB-6, a decrease in pausing occurs which perturbs chromosome pairing and causes SC assembly defects. A second project examined the role of CUL-4, an E3 ubiquitin ligase, in meiotic prophase I. We show that CUL-4 plays a role in both SC assembly and meiotic recombination. This work exemplifies the multiple levels of control of SC assembly which still require further study.

Meiotic recombination is initiated by self-inflicted DNA breaks and primarily involves homologous chromosomes, whereas mitotic recombination involves sister chromatids. Whilst the mitotic recombinase Rad51 exists during meiosis, its activity is suppressed in favour of the meiosis-specific recombinase, Dmc1, thus establishing a meiosis-specific mode of homologous recombination (HR). A key contributor to the suppression of Rad51 activity is the synaptonemal complex (SC), a meiosis-specific chromosomal structure that adheres homologous chromosomes along their entire lengths. Here, in budding yeast, we show that two major cell cycle kinases, Dbf4-dependent Cdc7 kinase (DDK) and Polo-kinase (Cdc5), collaborate to link the mode change of HR to the meiotic cell cycle by. This regulation of HR is through the SC. During prophase I, DDK is shown to maintain SC integrity and thus inhibition of Rad51. Cdc5, which is produced during the prophase I/metaphase I transition, interacts with DDK to cooperatively destroy the SC and remove Rad51 inhibition. By enhancing the interaction between DDK and Cdc5 or depleting DDK at late prophase I, meiotic DNA breaks are repaired even in the absence of Dmc1 by utilising Rad51. We propose that the interplay between DDK and Polo-kinase reactivates mitotic HR mechanisms to ensure complete repair of DNA breaks before meiotic chromosomem segregation.

The synaptonemal complex is a complex of proteins that connect homologous chromosomes, allowing genetic recombination to occur during meiosis in C. elegans. It is made up of lateral element proteins that assemble along each pair of sister chromatids and central region proteins that extend from, and join, each homolog. Here we aimed at understanding how synaptonemal complex activity is regulated through the study of nuclear import and isoform of its structural protein SYP-2. AKIR-1 is a protein with a previously identified meiotic role in the unloading of the Synaptonemal Complex (SC) Central Region (CR) proteins from the short arms of bivalent chromosomes exiting diakinesis. A Yeast-2-hybrid (Y2H) screen for additional proteins that interact with AKIR-1 found a potential interaction with the α-importin IMA-2. The efficient transfer of proteins from the cytoplasm into a meiotic nucleus in C. elegans is dependent on the activity of α- and β-importins. The α-importins associate to both cargo proteins and β-importins forming a complex that transits into the nucleus. Once inside the nucleus, the complex is disassembled and the importin complex is cycled back into the cytoplasm for reuse. Based on this, a double AKIR-1 and IMA-2 mutant was created to determine what role this interaction has in C. elegans meiosis. We found the first evidence of a role for AKIR-1 in the meiotic nuclear import and chromatin loading of meiotic cohesin complex proteins. We identified a meiotic phenotype in the double mutant akir-1(gk528);ima-2(ok256) that was not apparent in either of the akir-1(gk528) or ima-2(ok256) single mutants in which neither the cohesin REC-8 or the lateral element protein HTP-3 did not associate with chromatin normally, instead forming aggregates with which the central region SYP proteins co-localized. Additionally, we found that the pairing center protein HIM-8 was not being efficiently transported into the nucleus, localizing to the nuclear envelope instead of chromatin, as well as in cytoplasmic poly complexes (PCs). This loss of efficient import and localization resulted in morphological changes in the gonad as well. The gonad of akir-1;ima-2 contained significantly fewer nuclei and did not have a transition zone. We saw an increase in recombination intermediaries represented by RAD-51 foci, an ssDNA associating protein, in late pachytene. This increase indicated a lack of synapsis and crossover formation. We also examined the effect of a hypomorph of syp-2 on the proper assembly of the synaptonemal complex. The hypomorph displayed similar embryonic lethality to that of the syp-2 null mutant. However, unlike the null mutant, the syp-2 hypomorph showed a partial assembly of CR proteins. We also found an increased incidence of recombination intermediaries compared to the syp-2 null mutant, indicating hat DSB repair pathway is altered in this mutant. Autophagy is a regulator of aging in many organisms, including C. elegans, as well as functioning in the immune response pathway and muscle protein degradation. AKIRIN has been shown to have myriad roles in the soma in other model animals, and can be found in nearly every tissue. It has been shown to coordinate neural development and skeletal muscle differentiation, migration, and repair across multiple species. AKIRIN also acts as a cofactor in chromatin and cytoskeleton remodeling, gene transcription, and mediates interactions between the Twist transcription factor and Brahma. My research found the first evidence for a role of AKIR-1 in autophagosome inhibition after finding a significant increase in autophagosome puncta in the transgenic akir-1(gk529)::mCherry;LGG-1 mutants. We also found increased muscle deterioration and decreased motility in the akir-1(gk528) mutant, suggesting a role for AKIR-1 in actin filament maintenance. To test that association, we created a double mutant with akir-1(gk528);sma-1(ru18), a SMAD protein found in the apical membrane cytoskeleton, and that is required for body elongation in C. elegans. Our research provided evidence of AKIR-1 operating in a parallel pathway to SMA-1 as the double mutant displayed an additive reduction in body length.

La méiose est une étape essentielle de la reproduction chez tous les organismes sexués. En effet, celle-ci permet l’obtention de quatre gamètes haploïdes à partir d’une seule cellule diploïde grâce à la réalisation deux divisions successives suivant une seule étape de réplication. Un des éléments essentiels permettant une bonne ségrégation en première division méiotique est la création d’un échange physique entre les chromosomes homologues parentaux. Ce lien physique, plus communément appelé crossing-over (CO), est produit par un mécanisme de recombinaison entre les chromosomes homologues au cours de la prophase I méiotique. La recombinaison homologue est initiée par la formation simultanée de nombreuses cassures double-brin au sein du génome. Chez la levure de boulanger, la formation des COs est dépendante de la famille protéique ZMM (un acronyme pour Zip1/2/3/4-Msh4/5-Mer3-Spo16) composée de huit protéines hautement conservées, et impliquées dans la reconnaissance et la stabilisation des intermédiaires d’ADN formés au cours de la recombinaison homologue. Nous avons montré que la protéine Zip4 forme un complexe stable avec deux autres protéines ZMM, Zip2 et Spo16. Le complexe Zip2-Zip4-Spo16 (ZZS), de type XPF-ERCC1, serait capable de reconnaitre et de stabiliser les intermédiaires de recombinaison afin de promouvoir leur réparation en tant que CO. Chez les mammifères, Zip2 et Zip4 possèdent des homologues décrits, SHOC1 et TEX11 respectivement, mais aucun homologue n’a été découvert pour Spo16. Nous avons réalisé une analyse in silico et pu déterminer un homologue de Spo16 chez les mammifères, MmSPO16. Par la suite, j’ai pu co-purifier MmSPO16 avec le domaine XPF de SHOC1, ce qui suggère la conservation du complexe ZZS chez les mammifères. De plus, le processus de formation des COs est corrélé́ à la mise en place d’un complexe protéique formé entre les deux chromosomes homologues, appelé complexe synaptonémal (CS). Le CS est composé de deux éléments axiaux, accolés entre eux à une distance précise de 100 nm par la région centrale. La région centrale comprend un élément central, composé de l’hétérodimère Ecm11-Gmc2, et d’un élément transversal formé par la protéine Zip1. Les éléments transversaux partant des axes opposés se lient tête-bêche au niveau de l’élément central. Malgré des liens fonctionnels évidents entre la formation des COs et l’assemblage du CS entre les chromosomes homologues, aucun lien physique direct n’a été établi à ce jour. Au cours de mon doctorat, j’ai pu démontrer l’existence d’une interaction physique entre la protéine du CS Ecm11 et la protéine ZMM Zip4. Cette interaction est nécessaire pour la localisation et la polymérisation d’Ecm11 sur les chromosomes, l’assemblage correct du CS et la ségrégation des chromosomes homologues en première division méiotique
Meiosis is a highly conserved mechanism among organisms with sexual development. This process consists in producing four haploid gametes from one diploid cell by executing two successive rounds of cell division. During the first meiotic division, reciprocal exchanges of parental DNA strands, also known as crossing-overs (COs), ensure the faithful segregation of homologous chromosomes. COs arise from a specific type of DNA repair, homologous recombination. This pathway is initiated by the simultaneous induction of hundreds of double strand breaks (DSBs) in the genome. In budding yeast, the major CO pathway is promoted by a family of eight conserved proteins, named ZMMs (acronym for Zip1/2/3/4-Msh4/5-Mer3-Spo16), involved in recognizing and stabilizing DNA intermediates formed during homologous recombination. We showed that the Zip4 protein forms a stable tripartite complex with two other ZMM proteins, Zip2 and Spo16. Our data suggests that the Zip2-Zip4-Spo16 (ZZS) complex binds recombination intermediates through its XPF-ERCC1-like domain and drives them towards a CO fate. The homologs of Zip2 and Zip4 in mammals, SHOC1 and TEX11 respectively, have been described, but no Spo16 homolog has been found so far. We could identify the homolog of Spo16 in mammals by an in silico screen, MmSPO16. In addition, I could co-purify MmSPO16 with the XPF domain of SHOC1, thus revealing the potential conservation of the entire ZZS complex in mammals. ZMM-dependent COs are formed within the context of a meiosis-specific structure, named synaptonemal complex (SC). The SC is a proteinaceous structure composed of two axial elements physically maintained together at a precise distance of 100 nm by a central region. The central region encompasses a central element, composed of the two proteins Ecm11 and Gmc2, and the transverse filaments composed of Zip1. The transverse filaments from opposing axial elements overlap and bind head-to-head in the central element. However, despite evidence of a close relationship between SC assembly and CO formation, nothing is known about a direct link that could coordinate these two events spatially and temporally. During my PhD, I found a new interaction between the SC protein Ecm11 and the ZMM protein Zip4. This newly discovered interaction is necessary for Ecm11 association and polymerization on chromosomes, the SC assembly and the homolog disjunction in meiosis I. Our results suggest a direct connection that ensures SC assembly from CO sites through the Zip4-Ecm11 interaction. This way, ensuring SC polymerization from emerging CO sites could be a way of fine-tuning CO distribution, by participating to CO interference and/or by regulating nearby DSB formation. Moreover, I could identify an interaction between the mammalian ortholog of Zip4, TEX11, and one of the five members composing the SC central element, TEX12, raising the possibility that this mechanism synchronizing CO formation and SC polymerization could be conserved

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Resumo: Dentre todas as ordens de aracnideos conhecidas taxonomicamente, Araneae e a segunda mais diversa, com numero de especies menor somente em relacao a Acari. Atualmente, 39.725 especies ja foram descritas, sendo que centenas de novas descricoes sao feitas a cada ano em diversas familias de aranhas. O conhecimento citogenetico sobre a ordem restringe-se a analise de 638 especies (ca 2%) do total descrito do ponto de vista taxonomico. Este trabalho tem como objetivos fornecer uma compilacao dos dados citogeneticos existentes para a ordem na literatura ate a presente data, bem como caracterizar e estabelecer as estrategias de diferenciacao cromossomica em 13 especies de aranhas pertencentes ao grupo das haploginas, clado que corresponde a somente 3.257 especies (ca 8%) do total da ordem e a apenas 41 especies (ca 6%) do total cariotipado ate os dias atuais. Aliado a baixa representatividade dos dados cariologicos, outros pontos que fazem das haploginas um grupo interessante para estudos sao a predominancia de cromossomos meta/submetacentricos e de sistemas cromossomicos de determinacao sexual simples e multiplos, muitas vezes incluindo um cromossomo Y, ambas caracteristicas raras entre os outros clados de Araneae. As especies analisadas pertencem a tres familias de haploginas, Pholcidae (Mesabolivar luteus e Micropholcus fauroti), Sicariidae (Loxosceles amazonica, Loxosceles gaucho, Loxosceles hirsuta, Loxosceles intermedia, Loxosceles laeta, Loxosceles puortoi, Loxosceles similis e Sicarius tropicus) e Scytodidae (Scytodes fusca, Scytodes globula e Scytodes itapevi). Em Pholcidae, os resultados ineditos para os dois generos mostraram ... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Mesabolivar luteus (Keyserling 1891) and Micropholcus fauroti (Simon 1887) specimens were collected in Ubatuba and Rio Claro, both in the state of São Paulo, Brazil. Mesabolivar luteus showed 2n(.) = 15 = 14 + X and 2n(.) = 16 = 14 + XX in mitotic metaphases and 7II + X in diplotenic cells. During late prophase I, all bivalents presented a ring shape, evidencing two chiasmata per bivalent. In this species, some diplotenic cells appear in pairs, maybe due to specific characteristics of the intercellular bridges. The metaphases II showed n = 7 or n = 8 = 7 + X chromosomes. Micropholcus fauroti evidenced 2n(.) = 17 = 16 + X in spermatogonial metaphases and 8II+X in diplotenic cells, with only one chiasma per bivalent, contrasting with M. luteus. In both species, all chromosomes were metacentrics. The X sexual chromosome was the largest element and appeared as a univalent during meiosis I. These are the first cytogenetical data for the genera Mesabolivar and Micropholcus. Additionally, M. luteus is the first chromosomally analyzed species of the New World clade and the observed diploid number for M. fauroti had not yet been recorded in Pholcidae.
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Dentre todas as ordens de aracnideos conhecidas taxonomicamente, Araneae e a segunda mais diversa, com numero de especies menor somente em relacao a Acari. Atualmente, 39.725 especies ja foram descritas, sendo que centenas de novas descricoes sao feitas a cada ano em diversas familias de aranhas. O conhecimento citogenetico sobre a ordem restringe-se a analise de 638 especies (ca 2%) do total descrito do ponto de vista taxonomico. Este trabalho tem como objetivos fornecer uma compilacao dos dados citogeneticos existentes para a ordem na literatura ate a presente data, bem como caracterizar e estabelecer as estrategias de diferenciacao cromossomica em 13 especies de aranhas pertencentes ao grupo das haploginas, clado que corresponde a somente 3.257 especies (ca 8%) do total da ordem e a apenas 41 especies (ca 6%) do total cariotipado ate os dias atuais. Aliado a baixa representatividade dos dados cariologicos, outros pontos que fazem das haploginas um grupo interessante para estudos sao a predominancia de cromossomos meta/submetacentricos e de sistemas cromossomicos de determinacao sexual simples e multiplos, muitas vezes incluindo um cromossomo Y, ambas caracteristicas raras entre os outros clados de Araneae. As especies analisadas pertencem a tres familias de haploginas, Pholcidae (Mesabolivar luteus e Micropholcus fauroti), Sicariidae (Loxosceles amazonica, Loxosceles gaucho, Loxosceles hirsuta, Loxosceles intermedia, Loxosceles laeta, Loxosceles puortoi, Loxosceles similis e Sicarius tropicus) e Scytodidae (Scytodes fusca, Scytodes globula e Scytodes itapevi). Em Pholcidae, os resultados ineditos para os dois generos mostraram...
Mesabolivar luteus (Keyserling 1891) and Micropholcus fauroti (Simon 1887) specimens were collected in Ubatuba and Rio Claro, both in the state of São Paulo, Brazil. Mesabolivar luteus showed 2n(.) = 15 = 14 + X and 2n(.) = 16 = 14 + XX in mitotic metaphases and 7II + X in diplotenic cells. During late prophase I, all bivalents presented a ring shape, evidencing two chiasmata per bivalent. In this species, some diplotenic cells appear in pairs, maybe due to specific characteristics of the intercellular bridges. The metaphases II showed n = 7 or n = 8 = 7 + X chromosomes. Micropholcus fauroti evidenced 2n(.) = 17 = 16 + X in spermatogonial metaphases and 8II+X in diplotenic cells, with only one chiasma per bivalent, contrasting with M. luteus. In both species, all chromosomes were metacentrics. The X sexual chromosome was the largest element and appeared as a univalent during meiosis I. These are the first cytogenetical data for the genera Mesabolivar and Micropholcus. Additionally, M. luteus is the first chromosomally analyzed species of the New World clade and the observed diploid number for M. fauroti had not yet been recorded in Pholcidae.

L'etude de la spermatogenese d'hybrides de lemuriens a mis en evidence le role d'anomalies de l'appariement chromosomique, et de facteurs geniques sur la reduction de fertilite. L'etude reproductive et meiotique qui a ete menee sur 7 hybrides a montre que le bivalent sexuel s'associe dans un pourcentage eleve de spermatocytes (23%) chez 3 hybrides steriles, alors que les associations sont tres rares (<3%) chez les hybrides fertiles. D'autre facteurs, tels que le background genique herite de geniteur appartenant a des clades plus eloignes interviennent egalement dans la reduction de fertilite des hybrides. On observe ainsi une reduction de la fertilite chez des males hybrides porteurs de multivalents sans aucune association avec le bivalent sexuel. En particulier chez un hybride ou le multivalent n'est associe avec le bivalent sexuel que dans 3% des spermatocytes, on observe un nombre tres reduit de spermatozoides, une oligospermie importante, et une reduction de la spermatogenese pendant les deux saisons. La sterilite des hybrides issus du croisement de e. Collaris et e. Albocollaris montre, tout comme les donnees cytogenetiques et l'etude des complexes synaptonemaux, que ces 2 variete sont bien a considerer comme deux especes bien separees. L'etude ultrastructurale de la spermatogenese des hybrides steriles a montre une augmentation des phenomenes de degenerescence des cellules de la lignee germinale avec une presence accrue de cellules apoptotiques et une augmentation des inclusions lysosomales. L'etude ultrastructurale de la spermiogenese n'a mis en evidence aucune difference qualitative chez les hybrides steriles par rapport aux animaux feconds. La teratospermie augmentee chez les hybrides en periode de repos ne concerne pas la maturation de la chromatine evaluee par la coloration a l'acridine orange. Il existe une variation dans le taux de testosterone entre la periode de repos et l'activite sexuelle.

Der Synaptonemalkomplex (SC) ist eine hochkonservierte Proteinstruktur. Er weist eine dreiteili-ge, leiterähnliche Organisation auf und ist für die stabile Paarung der homologen Chromosomen während der Prophase der ersten meiotischen Teilung verantwortlich, die auch als Synpase be-zeichnet wird. Fehler während der Synpase führen zu Aneuploidie oder Apoptose der sich entwi-ckelnden Keimzellen. Seit 1956 ist der SC Gegenstand intensiver Forschung. Seine Existenz wurde in zahlreichen Orga-nismen von der Hefe bis zum Menschen beschrieben. Seine Struktur aus zwei parallel verlaufen-den Lateralelementen (LE), die durch eine Vielzahl von sogenannten Transversalfilamenten (TF) verbunden werden und dem Zentralen Element (CE) in der Mitte des SC ist dabei offensichtlich über die Millionen von Jahren der Evolution erhalten geblieben. Einzelne Proteinkomponenten des SC wurden jedoch nur in wenigen Modelorganismen charakterisiert, darunter Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster, Ceanorhabditis elegans und Mus mus-culus. Unerwarteter Weise gelang es bei dieser Charakterisierung nicht, eine evolutionäre Ver-wandtschaft, d.h. eine Homologie zwischen den Proteinsequenzen der verschiedenen SCs nach-zuweisen. Diese Tatsache sprach gegen die grundsätzliche Annahme, dass der SC in der Evolution nur einmal entstanden sei. Diese Arbeit hat sich nun der Aufgabe gewidmet, die Diskrepanz zwischen der hochkonservierten Struktur des SC und seiner augenscheinlich nicht-homologen Proteinzusammensetzung zu lösen. Dabei beschränkt sie sich auf die Analyse des Tierreichs. Es ist die erste Studie zur Evolution des SC in Metazoa und demonstriert die Monophylie der Säuger SC Proteinkomponenten im Tierreich. Die Arbeit zeigt, dass mindestens vier von sieben SC Proteinen der Maus spätestens im letzten gemeinsamen Vorfahren der Gewebetiere (Eumetazoa) enstanden sind und auch damals Teil ei-nes ursprünglichen SC waren, wie er heute in dem Nesseltier Hydra zu finden ist. Dieser SC weist die typische Struktur auf und besitzt bereits alle notwendigen Komponenten, um die drei Domä-nen – LE, TF und CE – zu assemblieren. Darüber hinaus ergaben die einzelnen Phylogenien der verschiedenen SC Proteine der Maus, dass der SC eine sehr dynamische Evolutionsgeschichte durchlaufen hat. Zusätzliche Proteine wurden während der Entstehung der Bilateria und der Wir-beltiere in den SC integriert, während andere ursprüngliche Komponenten möglicherweise Gen-Duplikationen erfuhren bzw. besonders in der Linie der Häutungstiere verloren gingen oder sich stark veränderten. Es wird die These aufgestellt, dass die auf den ersten Blick nicht-homologen SC Proteine der Fruchtfliege und des Fadenwurms tatsächlich doch von den ursprünglichen Prote-inenkomponenten abstammen, sich aber aufgrund der rasanten Evolution der Arthropoden und der Nematoden bis zu deren Unkenntlichkeit diversifizierten. Zusätzlich stellt die Arbeit Hydra als alternatives wirbelloses Modellsystem für die Meiose- und SC-Forschung zu den üblichen Modellen D. melanogaster und C. elegans vor. Die kürzlich gewon-nenen Erkenntnisse über den Hydra SC sowie der Einsatz der Standard-Methoden in diesem Orga-nismus werden in dem abschließenden Kapitel zusammengefasst und diskutiert
The synaptonemal complex (SC) is a highly conserved structure in sexually reproducing organism. It has a tripartite, ladder-like organization and mediates the stable pairing, called synapsis, of the homologous chromosomes during prophase of meiosis I. Failure in homolog synapsis result in aneuploidy and/or apoptosis of the developing germ cells. Since 1956, the SC is subject of intense research and its presence was described in various species from yeast to human. Its structure was maintained during millions of years of evolution consist-ing of two parallel lateral elements (LEs), joined by numerous transverse filaments (TFs) which run perpendicular to the LEs and an electron dense central element (CE) in the middle of the SC. Individual protein components, however, were characterized only in few available model organ-isms, as for example Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster, Ceanorhabditis elegans and Mus musculus. Rather unexpectedly, these characterizations failed to detect an evolutionary homology between the protein components of the different SCs. This fact challenged the general idea of a single origin of the SC in the evolution of meiosis and sexual reproduction. This thesis now addressed itself to the task to unravel the discrepancy between the high conser-vation of the SC structure and its diverse and apparently non-homologous protein composition, focusing on the animal kingdom. It is the first study dealing with the evolution of the SC in Meta-zoa and demonstrates the monophyly of the mammalian SC components in metazoan species. The thesis demonstrates that at least four out of seven murine SC proteins emerged in Eumeta-zoa at the latest and have been likewise part of an ancient SC as it can be found in the present-day cnidarian species Hydra. This SC displays the common organization and already possesses the minimal protein kit corresponding to the three different structural domains: LEs, TFs and the CE. Additionally, the individual phylogenies of the murine SC proteins revealed the dynamic evolu-tionary history of the ancient SC. Further components were added during the diversification of Bilateria and vertebrates while ancestral proteins likely duplicated in the vertebrate lineage and diversified or got lost in the branch leading to ecdysozoan species. It is hypothesized that the apparently non-homologous SC proteins in D. melanogaster and C. elegans actually do derive from the ancient SC proteins but diversified beyond recognition during the fast evolution of Ar-thropoda and Nematoda. The study proposes Hydra as an alternative invertebrate model system for meiosis and SC re-search to the standard organisms D. melanogaster and C. elegans. Recent results about the cni-darian SC as well as the possible application of standard methods is discussed and summarized in the concluding section

Thesis (M.Sc.)--York University, 2004. Graduate Programme in Biology.
Typescript. Includes bibliographical references (leaves 48-64). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pMQ99355

Thesis (Ph. D.)--York University, 1999. Graduate Programme in Biology.
Typescript. Includes bibliographical references (leaves 116-143). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pNQ39313.

Meiosis is a specialized type of cell division where two rounds of chromosome segregation follow a single round of DNA duplication resulting in the formation of four haploid daughter cells. Once the DNA replication is complete, the homologous chromosomes pair and recombine during the meiotic prophase I, giving rise to genetic diversity in the gametes. The process of homology search during meiosis is broadly divided into recombination-dependent (involves the formation of double-strand breaks) and recombination-independent mechanisms. In most eukaryotic organisms, pairing of homologs, recombination and chromosome segregation occurs in the context of a meiosis-specific proteinaceous structure, known as the synaptonemal complex (SC). The electron microscopic visualization of SC has revealed that the structure is tripartite with an electron-dense central element and two lateral elements that run longitudinally along the entire length of paired chromosomes. Transverse filaments are protein structures that connect the central region to the lateral elements. Genetic analyses in budding yeast indicate that mutations in SC components or defects in SC formation are associated with chromosome missegregation, aneuploidy and spore inviability. In humans, defects in SC assembly are linked to miscarriages, birth defects such as Down syndrome and development of certain types of cancer. In Saccharomyces cerevisiae, genetic screens have identified several mutants that exhibit defects in SC formation culminate in a decrease in the frequency of meiotic recombination, spore viability and improper chromosome segregation. Ten meiosis-specific proteins, viz. Hop1, Red1, Mek1, Hop2, Pch2, Zip1, Zip2, Zip3, Zip4 and Rec8, have been shown to be the bona fide components of SC and/or associated with SC function. S. cerevisiae HOP1 (HOmolog Pairing) gene was isolated in a genetic screen for mutants that showed defects in homolog pairing and, consequently, reduced levels of interhomolog recombination (10% of wild-type). Amino acid sequence alignment together with genetic and biochemical analyses revealed that Hop1 is a 70 kDa protein with a centrally embedded essential zinc-finger motif (Cys2/Cys2) and functions in polymeric form. Previous biochemical studies have also shown that Hop1 is a structure-specific DNA binding protein, which exhibits high affinity for the Holliday junction (HJ) suggesting a role of this protein in branch migration of the HJ. Furthermore, Hop1 displays high affinity for G-quadruplex structures (herein after referred to as GQ) and also promotes the formation of GQ from unfolded G-rich oligonucleotides. Strikingly, Hop1 promotes pairing between two double-stranded DNA molecules via G/C-rich sequence as well as intra- and inter-molecular pairing of duplex DNA molecules. Structure-function analysis suggested that Hop1 has a modular organization consisting of a protease-sensitive N-terminal, HORMA domain (characterized in Hop1, Rev7, Mad2 proteins) and protease-resistant C-terminal domain, called Hop1CTD. Advances in the field of DNA quadruplex structures suggest a significant role for these structures in a variety of biological functions such as signal transduction, DNA replication, recombination, gene expression, sister chromatid alignment etc. GQs and i-motif structures that arise within the G/C-rich regions of the genome of different organisms have been extensively characterized using biophysical, biochemical and cell biological approaches. Emerging studies with guanine- and cytosine-rich sequences of several promoters, telomeres and centromeres have revealed the formation of GQs and i-motif, respectively. Although the presence of GQs within cells has been demonstrated using G4-specific antibodies, in general, the in vivo existence of DNA quadruplex structures is the subject of an ongoing debate. However, the identification and isolation of proteins that bind and process these structures support the idea of their in vivo existence. In S. cerevisiae, genome-wide survey to identify conserved GQs has revealed the presence of ~1400 GQ forming sequences. Additionally, these potential GQ forming motifs were found in close proximity to promoters, rDNA and mitosis- and meiosis-specific double-strand break sites (DSBs). Meiotic recombination in S. cerevisiae as well as humans occurs at meiosis-specific double-strand break (DSBs) sites that are embedded within the G/C-rich sequences. However, much less is known about the structural features and functional significance of DNA quadruplex motifs in sister chromatid alignment N during meiosis. Therefore, one of the aims of the studies described in this thesis was to investigate the relationship between the G/C-rich motif at a meiosis-specific DSB site in S. cerevisiae and its ability to form GQ and i-motif structures. To test this hypothesis, we chose a G/C-rich motif at a meiosis-specific DSB site located between co-ordinates 1242526 to 1242550 on chromosome IV of S. cerevisiae. Using multiple techniques such as native gel electrophoresis, circular dichroism spectroscopy, 2D NMR and chemical foot printing, we show that G-rich motif derived from the meiosis-specific DSB folds into an intramolecular GQ and the complementary C-rich sequence folds into an intramolecular i-motif, the latter under acidic conditions. Interestingly, we found that the C-rich strand folds into i-motif at near neutral pH in the presence of cell-mimicking molecular crowding agents. The NMR data, consistent with our biochemical and biophysical analyses, confirmed the formation of a stable i-motif structure. To further elucidate the impact of these quadruplex structures on DNA replication in vitro, we carried out DNA polymerase stop assay with a template DNA containing either the G-rich or the C-rich sequence. Primer extension assays carried out with Taq polymerase and G-rich template blocked the polymerase at a site that corresponded to the formation of an intramolecular GQ. Likewise, primer extension reactions carried out with KOD-Plus DNA polymerase and C-rich template led to the generation of a stop-product at the site of the formation of intramolecular I -motif under acidic conditions (pH 4.5 and pH 5.5). However, polymerase stop assay performed in the presence of single-walled carbon nanotubes (SWNTs) that stabilize I -motif at physiological pH blocked the polymerase at the site of intramolecular I -motif formation, indicating the possible existence of i-motif in the cellular context. Taken together, these results revealed that the G/C-rich motif at the meiosis-specific DSB site folds into GQ and i-motif structures in vitro. Our in vitro analyses were in line with our in vivo analysis that examined the ability of the G/C-rich motif to fold into quadruplex structures in S. cerevisiae cells. Qualitative microscopic analysis and quantitative analysis with plasmid constructs that harbour the GQ or i-motif forming sequence revealed a significant decrease in the GFP expression levels in comparison to the control. More importantly, all the assays performed with the corresponding mutant sequences under identical experimental conditions did not yield any quadruplex structures, suggesting the involvement of contagious guanine and cytosine residues in the structure formation. Prompted by our earlier results that revealed high binding affinity of Hop1 for GQ, we wished to understand the role of the GQ and i-motif structures during meiosis by analysing their interaction with Hop1 and its truncated variants (HORMA and Hop1CTD). In agreement with our previous observations, Hop1 and Hop1CTD associated preferentially with GQ DNA. Interestingly, whereas the full-length Hop1 showed much weaker binding affinity for i-motif DNA, Hop1 C-terminal fragment but not its N-terminal fragment exhibited robust i-motif DNA binding activity. We have previously demonstrated that Hop1 promotes intermolecular synapsis between synthetic duplex DNA molecules containing a G/C-rich sequence. Hence, to understand the functional role of the quadruplex structures formed at the meiosis-specific G/C-rich motif, we examined the ability of Hop1 to promote pairing between linear duplex DNA helices containing the G/C-rich motif. DNA pairing assay indicated that binding of Hop1 to the G/C-rich duplex DNA resulted in the formation of a side-by-side synapsis product. Under similar conditions, Hop1 was unable to pair mutant duplex DNA molecules suggesting the involvement of the G/C-rich motif in the formation of the synapsis product. Our results were substantiated by the observation that yeast Rad17 failed to promote pairing between duplex DNA molecules with a centrally embedded G/C-rich motif. Altogether, these results provide important structural and functional insights into the role of quadruplex structures in meiotic pairing of homologous chromosomes. The second part of the thesis focuses on the biochemical and functional properties of Red1 protein, a component of S. cerevisiae lateral element. RED1 was identified in a screen for meiotic lethal, sporulation proficient mutants. Genetic, biochemical and microscopic analyses have demonstrated the physical interaction between Hop1 and Red1. Given this, hop1 and red1 mutants display similar phenotypes such as chromosome missegregation and spore inviability and thus are placed under the same epistasis group. However, unlike hop1 mutants, red1 mutants show complete absence of SC. RED1 overexpression suppressed certain non-null hop1 phenotypes, indicating that these proteins may have partially overlapping functions. Further, although the functional significance is unknown, chromatin immunoprecipitation studies have revealed the localization of Red1 to the GC-rich regions (R-bands) in the genome, considered to be meiotic recombination hotspots. Although the aforementioned genetic studies suggest an important role for Red1 in meiosis, the exact molecular function of Red1 in meiotic recombination remains to be elucidated. To explore the biochemical properties of Red1, we isolated the S. cerevisiae RED1 gene, cloned, overexpressed, and purified the protein to near homogeneity. Immunoprecipitation assays using meiotic cells extracts suggested that Red1 exists as a Homodimer linked by disulphide-bonds under physiological conditions. We characterized the DNA binding properties of Red1 by analysing its interaction with recombination intermediates that are likely to form during meiotic recombination. Protein-DNA interaction assays revealed that Red1 exhibits binding preference for the Holliday junction over replication fork and other recombination intermediates. Notably, Red1 displayed ~40-fold higher binding affinity for GQ in comparison with HJ. The observation that Red1 binds robustly to GQs prompted us to examine if Red1 could promote pairing between duplex DNA helices with the G/C-rich sequences similar to Hop1. Interestingly, we found that Red1 failed to promote pairing between dsDNA molecules but potentiated Hop1 mediated pairing between duplex DNA molecules. Our AFM studies with linear and circular DNA molecules along with Red1 suggested a possible role of Red1 in DNA condensation, bridging and pairing of double-stranded DNA helices. Bioinformatics analysis of Red1 indicated the lack of sequence or structural similarity to any of the known proteins. To elucidate structure-function relationship of Red1, we generated several N- and C-terminal Red1 truncations and studied their DNA binding properties. Our results indicated that the N-terminal region comprising of 678 amino acid residues constitutes the DNA-binding region of Red1. The N-terminal region, called RNTF-II, displayed similar substrate specificity comparable to that of full-length Red1. Interestingly, site-directed mutagenesis studies with the Red1 C-terminal region revealed the involvement of two cysteine residues at position 704 and 707 in the disulfide bond mediated intermolecular dimer formation. Finally, to understand the functional significance of Red1 truncations we analyzed the subcellular localization of Red1 and its truncations. We made translation fusions of RED1 and its truncations by placing their corresponding nucleotide sequences downstream of GFP coding sequence in yeast expression vector. Confocal microscopy studies with S. cerevisiae cells transformed with the individual plasmid constructs indicated that the N-terminal variants localized to the nucleus, whereas the C-terminal variants did not localize to the nucleus. These results suggest that NLS-like motifs are embedded in the N-terminal region of the protein. Furthermore, other results indicated that the N-terminal region contains functions such as DNA-binding and intermolecular bridging of non-contiguous DNA segments. Altogether, these findings, on the one hand, provide insights into the molecular mechanism underlying the functions of Hop1 and Red1 proteins and, on the other, support a role for DNA quadruplex structures in meiotic chromosome synapsis and recombination.

Meiosis is a specialized type of cell division where two rounds of chromosome segregation follow a single round of DNA duplication resulting in the formation of four haploid daughter cells. Once the DNA replication is complete, the homologous chromosomes pair and recombine during the meiotic prophase I, giving rise to genetic diversity in the gametes. The process of homology search during meiosis is broadly divided into recombination-dependent (involves the formation of double-strand breaks) and recombination-independent mechanisms. In most eukaryotic organisms, pairing of homologs, recombination and chromosome segregation occurs in the context of a meiosis-specific proteinaceous structure, known as the synaptonemal complex (SC). The electron microscopic visualization of SC has revealed that the structure is tripartite with an electron-dense central element and two lateral elements that run longitudinally along the entire length of paired chromosomes. Transverse filaments are protein structures that connect the central region to the lateral elements. Genetic analyses in budding yeast indicate that mutations in SC components or defects in SC formation are associated with chromosome missegregation, aneuploidy and spore inviability. In humans, defects in SC assembly are linked to miscarriages, birth defects such as Down syndrome and development of certain types of cancer. In Saccharomyces cerevisiae, genetic screens have identified several mutants that exhibit defects in SC formation culminate in a decrease in the frequency of meiotic recombination, spore viability and improper chromosome segregation. Ten meiosis-specific proteins, viz. Hop1, Red1, Mek1, Hop2, Pch2, Zip1, Zip2, Zip3, Zip4 and Rec8, have been shown to be the bona fide components of SC and/or associated with SC function. S. cerevisiae HOP1 (HOmolog Pairing) gene was isolated in a genetic screen for mutants that showed defects in homolog pairing and, consequently, reduced levels of interhomolog recombination (10% of wild-type). Amino acid sequence alignment together with genetic and biochemical analyses revealed that Hop1 is a 70 kDa protein with a centrally embedded essential zinc-finger motif (Cys2/Cys2) and functions in polymeric form. Previous biochemical studies have also shown that Hop1 is a structure-specific DNA binding protein, which exhibits high affinity for the Holliday junction (HJ) suggesting a role of this protein in branch migration of the HJ. Furthermore, Hop1 displays high affinity for G-quadruplex structures (herein after referred to as GQ) and also promotes the formation of GQ from unfolded G-rich oligonucleotides. Strikingly, Hop1 promotes pairing between two double-stranded DNA molecules via G/C-rich sequence as well as intra- and inter-molecular pairing of duplex DNA molecules. Structure-function analysis suggested that Hop1 has a modular organization consisting of a protease-sensitive N-terminal, HORMA domain (characterized in Hop1, Rev7, Mad2 proteins) and protease-resistant C-terminal domain, called Hop1CTD. Advances in the field of DNA quadruplex structures suggest a significant role for these structures in a variety of biological functions such as signal transduction, DNA replication, recombination, gene expression, sister chromatid alignment etc. GQs and i-motif structures that arise within the G/C-rich regions of the genome of different organisms have been extensively characterized using biophysical, biochemical and cell biological approaches. Emerging studies with guanine- and cytosine-rich sequences of several promoters, telomeres and centromeres have revealed the formation of GQs and i-motif, respectively. Although the presence of GQs within cells has been demonstrated using G4-specific antibodies, in general, the in vivo existence of DNA quadruplex structures is the subject of an ongoing debate. However, the identification and isolation of proteins that bind and process these structures support the idea of their in vivo existence. In S. cerevisiae, genome-wide survey to identify conserved GQs has revealed the presence of ~1400 GQ forming sequences. Additionally, these potential GQ forming motifs were found in close proximity to promoters, rDNA and mitosis- and meiosis-specific double-strand break sites (DSBs). Meiotic recombination in S. cerevisiae as well as humans occurs at meiosis-specific double-strand break (DSBs) sites that are embedded within the G/C-rich sequences. However, much less is known about the structural features and functional significance of DNA quadruplex motifs in sister chromatid alignment N during meiosis. Therefore, one of the aims of the studies described in this thesis was to investigate the relationship between the G/C-rich motif at a meiosis-specific DSB site in S. cerevisiae and its ability to form GQ and i-motif structures. To test this hypothesis, we chose a G/C-rich motif at a meiosis-specific DSB site located between co-ordinates 1242526 to 1242550 on chromosome IV of S. cerevisiae. Using multiple techniques such as native gel electrophoresis, circular dichroism spectroscopy, 2D NMR and chemical foot printing, we show that G-rich motif derived from the meiosis-specific DSB folds into an intramolecular GQ and the complementary C-rich sequence folds into an intramolecular i-motif, the latter under acidic conditions. Interestingly, we found that the C-rich strand folds into i-motif at near neutral pH in the presence of cell-mimicking molecular crowding agents. The NMR data, consistent with our biochemical and biophysical analyses, confirmed the formation of a stable i-motif structure. To further elucidate the impact of these quadruplex structures on DNA replication in vitro, we carried out DNA polymerase stop assay with a template DNA containing either the G-rich or the C-rich sequence. Primer extension assays carried out with Taq polymerase and G-rich template blocked the polymerase at a site that corresponded to the formation of an intramolecular GQ. Likewise, primer extension reactions carried out with KOD-Plus DNA polymerase and C-rich template led to the generation of a stop-product at the site of the formation of intramolecular I -motif under acidic conditions (pH 4.5 and pH 5.5). However, polymerase stop assay performed in the presence of single-walled carbon nanotubes (SWNTs) that stabilize I -motif at physiological pH blocked the polymerase at the site of intramolecular I -motif formation, indicating the possible existence of i-motif in the cellular context. Taken together, these results revealed that the G/C-rich motif at the meiosis-specific DSB site folds into GQ and i-motif structures in vitro. Our in vitro analyses were in line with our in vivo analysis that examined the ability of the G/C-rich motif to fold into quadruplex structures in S. cerevisiae cells. Qualitative microscopic analysis and quantitative analysis with plasmid constructs that harbour the GQ or i-motif forming sequence revealed a significant decrease in the GFP expression levels in comparison to the control. More importantly, all the assays performed with the corresponding mutant sequences under identical experimental conditions did not yield any quadruplex structures, suggesting the involvement of contagious guanine and cytosine residues in the structure formation. Prompted by our earlier results that revealed high binding affinity of Hop1 for GQ, we wished to understand the role of the GQ and i-motif structures during meiosis by analysing their interaction with Hop1 and its truncated variants (HORMA and Hop1CTD). In agreement with our previous observations, Hop1 and Hop1CTD associated preferentially with GQ DNA. Interestingly, whereas the full-length Hop1 showed much weaker binding affinity for i-motif DNA, Hop1 C-terminal fragment but not its N-terminal fragment exhibited robust i-motif DNA binding activity. We have previously demonstrated that Hop1 promotes intermolecular synapsis between synthetic duplex DNA molecules containing a G/C-rich sequence. Hence, to understand the functional role of the quadruplex structures formed at the meiosis-specific G/C-rich motif, we examined the ability of Hop1 to promote pairing between linear duplex DNA helices containing the G/C-rich motif. DNA pairing assay indicated that binding of Hop1 to the G/C-rich duplex DNA resulted in the formation of a side-by-side synapsis product. Under similar conditions, Hop1 was unable to pair mutant duplex DNA molecules suggesting the involvement of the G/C-rich motif in the formation of the synapsis product. Our results were substantiated by the observation that yeast Rad17 failed to promote pairing between duplex DNA molecules with a centrally embedded G/C-rich motif. Altogether, these results provide important structural and functional insights into the role of quadruplex structures in meiotic pairing of homologous chromosomes. The second part of the thesis focuses on the biochemical and functional properties of Red1 protein, a component of S. cerevisiae lateral element. RED1 was identified in a screen for meiotic lethal, sporulation proficient mutants. Genetic, biochemical and microscopic analyses have demonstrated the physical interaction between Hop1 and Red1. Given this, hop1 and red1 mutants display similar phenotypes such as chromosome missegregation and spore inviability and thus are placed under the same epistasis group. However, unlike hop1 mutants, red1 mutants show complete absence of SC. RED1 overexpression suppressed certain non-null hop1 phenotypes, indicating that these proteins may have partially overlapping functions. Further, although the functional significance is unknown, chromatin immunoprecipitation studies have revealed the localization of Red1 to the GC-rich regions (R-bands) in the genome, considered to be meiotic recombination hotspots. Although the aforementioned genetic studies suggest an important role for Red1 in meiosis, the exact molecular function of Red1 in meiotic recombination remains to be elucidated. To explore the biochemical properties of Red1, we isolated the S. cerevisiae RED1 gene, cloned, overexpressed, and purified the protein to near homogeneity. Immunoprecipitation assays using meiotic cells extracts suggested that Red1 exists as a Homodimer linked by disulphide-bonds under physiological conditions. We characterized the DNA binding properties of Red1 by analysing its interaction with recombination intermediates that are likely to form during meiotic recombination. Protein-DNA interaction assays revealed that Red1 exhibits binding preference for the Holliday junction over replication fork and other recombination intermediates. Notably, Red1 displayed ~40-fold higher binding affinity for GQ in comparison with HJ. The observation that Red1 binds robustly to GQs prompted us to examine if Red1 could promote pairing between duplex DNA helices with the G/C-rich sequences similar to Hop1. Interestingly, we found that Red1 failed to promote pairing between dsDNA molecules but potentiated Hop1 mediated pairing between duplex DNA molecules. Our AFM studies with linear and circular DNA molecules along with Red1 suggested a possible role of Red1 in DNA condensation, bridging and pairing of double-stranded DNA helices. Bioinformatics analysis of Red1 indicated the lack of sequence or structural similarity to any of the known proteins. To elucidate structure-function relationship of Red1, we generated several N- and C-terminal Red1 truncations and studied their DNA binding properties. Our results indicated that the N-terminal region comprising of 678 amino acid residues constitutes the DNA-binding region of Red1. The N-terminal region, called RNTF-II, displayed similar substrate specificity comparable to that of full-length Red1. Interestingly, site-directed mutagenesis studies with the Red1 C-terminal region revealed the involvement of two cysteine residues at position 704 and 707 in the disulfide bond mediated intermolecular dimer formation. Finally, to understand the functional significance of Red1 truncations we analyzed the subcellular localization of Red1 and its truncations. We made translation fusions of RED1 and its truncations by placing their corresponding nucleotide sequences downstream of GFP coding sequence in yeast expression vector. Confocal microscopy studies with S. cerevisiae cells transformed with the individual plasmid constructs indicated that the N-terminal variants localized to the nucleus, whereas the C-terminal variants did not localize to the nucleus. These results suggest that NLS-like motifs are embedded in the N-terminal region of the protein. Furthermore, other results indicated that the N-terminal region contains functions such as DNA-binding and intermolecular bridging of non-contiguous DNA segments. Altogether, these findings, on the one hand, provide insights into the molecular mechanism underlying the functions of Hop1 and Red1 proteins and, on the other, support a role for DNA quadruplex structures in meiotic chromosome synapsis and recombination.

Meiosis is a specialised form of cell division that occurs specifically in the gonads of sexually reproducing species. It comprises a round of DNA replication followed by two successive rounds of cell division to produce haploid gametes. Each stage is divided into four substages of prophase, metaphase, anaphase and telophase. Prophase I is the longest and most complex stage of meiosis during which homologous chromosomes pair and recombine. The evolution of heteromorphic sex chromosomes has led to a number of changes in meiotic organisation. This includes the non-pairing of sex specific parts of the heteromorphic sex chromosomes and their inactivation in many species. The platypus has a unique set of 10 sex chromosomes with homology to bird sex chromosomes that exist as a chain during meiotic metaphase I. Questions of mode and extent of pairing and the existence of meiotic silencing remained unknown but can inform our understanding of the evolution and mechanisms of meiotic prophase I. Work presented in this thesis provides novel insights into evolution and meiotic organisation of the monotreme sex chromosome complex. The platypus sex chromosome chain forms during zygotene in stepwise manner, with remarkable consistency beginning at the Y5 end of the chain and ending with the X1 (Chapter 1). Synapsis generally relies on 3 main proteins; SYCP1, SYCP2 and SYCP3. Surprisingly platypuses express three different copies of SYCP3 (including a multicopy version on a Y chromosome), genes that generally exist as single isoforms in most other species. Particularly given the SYCP3Y isoform is male specific, this raises the possibility that SYCP3 paralogs may have evolved in relation to the sex chromosome chain during prophase I (Chapter 2). During pachytene, the asynaptic regions of the sex chromosomes adopt a state of folding, similar to that of the avian Z and W chromosomes during synaptic adjustment, albeit without the formation of a central element. During this time the cohesin complex is heavily loaded onto the axial elements of the asynaptic regions of the X and Y regions of the chain. Furthermore at mid-pachytene the asynaptic regions of the chain are pulled to a giant nucleolus at which time the cohesin appears to spread onto the chromosome loops of the asynaptic regions of the chain that are also coincident with DNA condensation (Chapter 3). During platypus pachytene there is global transcriptional downregulation. We observe no localised phosphorylation of the histone H2AX, a hallmark of MSCI but we do observe localised patterns of H2AFY, H3K27me3 and H3K9me3 at a paranucleolar location, however the H2AFY and H3K27me3 showed some colocalisation with sex chromosomes, there was not consistent pattern and H3K9me3 was always associated with a section of chromosome 6 (Chapter 4). Together these results provide novel insights into the meiotic organisation of the monotreme sex chromosome complex and the evolution of MSCI in mammals.
Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2015

Human oocytes can stay dormant for several decades in meiotic arrest, before reactivation and maturation. Similar, Drosophila oocyte is mostly transcriptionally quiescent during prophase I-arrest, transiently reactivating gene expression just before it progresses into metaphase I. Our aim is to better understand such reactivation and its role for oocyte maturation and female fertility. This work is divided in 3 chapters. In chapter I we use an isolated loss of function allele of dkdm5 to characterize the function of this enzyme during oogenesis. The results obtained with the loss of function allele of dkdm5 are consistent, albeit stronger, with the phenotypes previously observed after RNAi depletion and hypomorphic allele of dkdm5. Yet, a qualitatively distinct phenotype has also been identified, suggesting a new function of dkdm5 during oocyte maturation. In chapter II we try to access chromatin quality and synaptonemal complex assembly throughout oocyte development. Proteins from chromatin remodelling complexes, as dkdm5, have been shown to disrupt the synaptonemal complex further leading to problems in meiotic progression. Here we observed that two core components of the pho repressive complex are required for female fertility. Further, we show that specific germline dSfmbt depletion leads to an abnormal increase of Corolla and defective synaptonemal complex morphology in the oocyte chromatin throughout development. Chapter III is an independent chapter where we reveal Nine Teen Complex Protein Salsa as being particularly rate limiting for efficient splicing of short proximal introns and dorsoventral patterning of the Drosophila egg. We observed that, upon specific germline depletion of Salsa, Gurken transcript is poorly spliced leading to an abnormal localization, subsequentially leading to defects in the eggshell dorsoventral patterning and female fertility. Further we show that ectopic Gurken expression can suppress the dorsal ventral patterning defects after Salsa depletion. Our work aims to mechanistically understand our observations.
Os oócitos humanos têm a habilidade de para o seu ciclo celular na meiose durante diversas décadas, num estado de dormência transcricional, antes de reativarem a sua transcrição e maturarem. Semelhante ao que ocorre em humanos, os oócitos de Drosófila também estão, na grande parte do seu desenvolvimento, com o seu ciclo celular parado na prófase I da meiose num estado de quiescência transcricional. Antes da progressão meiótica para a metáfase I, existe uma reativação transiente da transcrição no nucleou do oócito. Este trabalho está dividido em três capítulos. No primeiro capítulo, focamo-nos em utilizar um alelo isolado com a perda de função total de dkdm5 para tentar caracterizar a função desta enzina durante a oogénese. Os resultados obtidos são semelhantes aos observados anteriormente em experiências feitas através de depleção por RNAi ou pelo uso de um alelo hipomórfico. Contudo os fenótipos observados na perda da função total de dkdm5 são mais acentuados. Também, com este trabalho conseguimos desvendar alguns dos processos que são dependentes ou independentes da atividade de demetilase de dkdm5. Contudo, observamos um fenótipo qualitativamente distinto na cromatina do núcleo do oócito o que sugere uma nova função de dkdm5 durante a maturação do oócito. No segundo capítulo, avaliamos a qualidade da cromatina e a formação synaptonemal complex ao longo do desenvolvimento do oócito. Proteínas que integram complexos responsáveis pela remodelação da cromatina, como dkdm5, tem vindo a ser descritos como necessário para a correta formação do synaptonemal complex e subsequentemente uma correta progressão meiótica. Neste capítulo, mostramos que dois dos principais componentes de pho repressive complex, são necessários para a fertilidade feminina. Alem disso, mostramos também que após uma deleção especifica de dSfmbt na linha germinal, existe um aumento da expressão de Corolla e o aparecimento de morfologias defeituosas do SC durante o desenvolvimento do oócito. O terceiro capítulo é um capítulo independente. Neste capítulo nós descobrimos que a proteína Salsa do Nine Teen Complex protein como sendo particularmente limitante para a eficiência de splicing em intrões proximais curtos e padronização dorsoventral do ovo de Drosófila. Observamos que, após depleção especifica da linha germinal de Salsa que, o splincing do transcrito de Gurken não é feito corretamente levando a uma localização anormal desta proteína, subsequentemente levando a defeitos de padronização dorsoventral dos ovos e defeitos na fertilidade feminina. Contudo, mostramos que a expressão ectópica de Gurken pode suprimir os defeitos de padronização dorsoventral após a depleção de Salsa. O nosso objetivo reside em entender os mecanismos que estão por detrás das nossas observações.

Hybrid sterility is one of the reproductive isolation mechanisms restricting gene flow between the related species and leading to speciation. PR domain containing 9 (Prdm9), the only known vertebrate hybrid sterility gene, determines the sites of programmed DNA double-strand breaks (DSBs) and thus specifies hotspots of meiotic recombination but in hybrids between two mouse subspecies causes failure of meiotic chromosome synapsis and hybrid male sterility. In the present study on sterile hybrids, the five smallest autosomes were more prone to asynapsis. To manipulate with the synapsis rate, random stretches of consubspecific homology were inserted into several autosomal pairs. Twenty seven or more megabases of consubspecific sequence fully restore synapsis in a given autosome. Further, at least two symetric DN double-strand breaks per chromosome were necessary for successful synapsis. Moreover, F1 hybrids had sperm when synapsis was rescued in at least three of four segregating chromosomes. To verify the assumption of a lack of symmetric DSBs in meiotic chromosomes of sterile males the chemotherapeutic drug cisplatin was used to induce exogenous DNA DSBs. Cells treated with 5 mg/kg and 10 mg/kg of cisplatin showed increased number of DSBs monitored by immunostaining of RPA and DMC1 sites and...

Meiosis is a specialized type of cell division essential for the production of four normal haploid gametes. In early prophase I of meiosis, the intimate synapsis between homologous chromosomes, and the formation of chiasmata, is facilitated by a proteinaceous structure known as the synaptonemal complex (SC). Ultrastructural analysis of germ cells of a number of organisms has disclosed that SC is a specialized tripartite structure composed of two lateral elements, one on each homolog, and a central element, which, in turn, are linked by transverse elements. Genetic studies have revealed that defects in meiotic chromosome alignment and/or segregation result in aneuploidy, which is the leading cause of spontaneous miscarriages in humans, hereditary birth defects such as Down syndrome, and are also, associated with the development and progression of certain forms of cancer. The mechanism(s) underlying the alignment/pairing of chromosomes at meiosis I differ among organisms. These can be divided into at least two broad pathways: one is independent of DNA double-strand breaks (DSB) and other is mediated by DSBs. In the DSB-dependent pathway, SC plays crucial roles in promoting homolog pairing and disjunction. On the other hand, the DSB-independent pathway involves the participation of telomeres, centromeres and non-coding RNAs in the pre-synaptic alignment, pre-meiotic pairing as well as pairing of homologous chromosomes. Although a large body of literature highlights the central role of SC in meiotic recombination, the possible role of SC components in homolog recognition and alignment is poorly understood. Genetic screens for Saccharomyces cerevisiae mutants defective in meiosis and sporulation lead to the isolation of genes required for interhomolog recombination, including those that encode SC components. In S. cerevisiae, ten meiosis-specific proteins viz., Hop1, Red1, Mek1, Hop2, Pch2, Zip1, Zip2, Zip3, Zip4 and Rec8 have been recognized as bona fide constituents of SC or associated with SC function. Mutations in any of these genes result in defective SC formation, thus leading to reduction in the rate of recombination. HOP1 (Homolog Pairing) encodes a ̴ 70 kDa structural protein, which localizes to the lateral elements of SC. It was found to be essential for the progression of meiotic recombination. In hop1Δ mutants, meiosis specific DSBs are reduced to 10% of that of wild type level and it fails to produce viable spores. It also displays relatively high frequency of inter-sister recombination over inter-homolog recombination. Bioinformatics analysis suggests that Hop1 comprises of an N-terminal HORMA domain (Hop1, Rev7 and Mad2), which is conserved among Hop1 homologs from diverse organisms. This domain is also known to be present in proteins involved in processes like chromosome synapsis, repair and sex chromosome inactivation. Additionally, Hop1 harbors a 36-amino acid long zinc finger 348374 motif (CX2CX19CX2C) which is critical for DNA binding and meiotic progression, and a putative nuclear localization signal corresponding to amino acid residues from 588-594. Previous studies suggested that purified Hop1 protein exists in multiple oligomeric states in solution and displays structure specific DNA binding activity. Importantly, Hop1 exhibited higher binding affinity for the Holliday junction (HJ), over other early recombination intermediates. Binding of Hop1 to the HJ at the core resulted in branch migration of the junction, albeit weakly. Intriguingly, Hop1 showed a high binding affinity for G4 DNA, a non-B DNA structure, implicated in homolog synapsis and promotes robust synapsis between double-stranded DNA molecules. Hop1 protein used in the foregoing biochemical studies was purified from mitotically dividing S. cerevisiae cells containing the recombinant plasmid over-expressing the protein where the yields were often found to be in the low-microgram quantities. Therefore, one of the major limitations to the application of high resolution biophysical techniques, such as X-crystallography and spectroscopic analyses for structure-function studies of S. cerevisiae Hop1 has been the non-availability of sufficient quantities of functionally active pure protein. In this study, we have performed expression screening in Escherichia coli host strains, capable of high level expression of soluble S. cerevisiae Hop1 protein. A new protocol has been developed +2 for expression and purification of S. cerevisiae Hop1 protein, using Ni-NTA and double-stranded DNA-cellulose chromatography. Recombinant S. cerevisiae Hop1 protein thus obtained was >98% pure and exhibited DNA binding activity with high-affinity for Holliday junction. The availability of the bacterial HOP1 expression vector and functionally active Hop1 protein has enabled us to glean and understand several vital biological insights into the structure-function relationships of Hop1 as well as the generation of appropriate truncated mutant proteins. Mutational analyses in S. cerevisiae has shown that sister chromatid cohesion is required for proper chromosome condensation, including the formation of axial elements, SC assembly and recombination. Consistent with these findings, homolog alignment is impaired in red1hop1 strains and associations between homologs are less stable. red1 mutants fail to make any discernible axial elements or SC structures but exhibit normal chromosome condensation, while hop1 mutants form long fragments of axial elements but without any SCs, are defective in chromosome condensation, and produce in-viable spores. Using single molecule and ensemble assays, we found that S. cerevisiae Hop1 organizes DNA into at least four major distinct DNA conformations: (i) a rigid protein filament along DNA that blocks access to nucleases; (ii) bridging of non-contiguous segments of DNA to form stem-loop structures; (iii) intra-and intermolecular long range synapsis between double-stranded DNA molecules; and (iv) folding of DNA into higher order nucleoprotein structures. Consistent with B. McClintock’s proposal that “there is a tendency for chromosomes to associate 2-by-2 in the prophase of meiosis involving long distance recognition of homologs”, these results to our knowledge provide the first evidence that Hop1 mediates the formation of tight DNA-protein-DNA nucleofilaments independent of homology which might help in the synapsis of homologous chromosomes during meiosis. Although the DNA binding properties of Hop1 are relatively well established, comparable knowledge about the protein is lacking. The purification of Hop1 from E. coli, which was functionally indistinguishable from the protein obtained from mitotically dividing S. cerevisiae cells has enabled us to investigate the structure-function relationships of Hop1, which has provided important insights into its role in meiotic recombination. We present several lines of evidence suggesting that Hop1 is a modular protein, consisting of an intrinsically unstructured N-terminal domain and a core C-terminal domain (Hop1CTD), the latter being functionally equivalent to the full-length Hop1 in terms of its in vitro activities. Importantly, however, Hop1CTD was unable to rescue the meiotic recombination defects of hop1null strain, indicating that synergy between the N-terminal and C-terminal domains of Hop1 protein is essential for meiosis and spore formation. Taken together, our findings provide novel insights into the molecular functions of Hop1, which has profound implications for the assembly of mature SC, homolog synapsis and recombination. Several lines of investigations suggest that HORMA domain containing proteins are involved in chromatin binding and, consequently, have been shown to play key roles in processes such as meiotic cell cycle checkpoint, DNA replication, double-strand break repair and chromosome synapsis. S. cerevisiae encodes three HORMA domain containing proteins: Hop1, Rev7 and Mad2 (HORMA) which interact with chromatin during diverse chromosomal processes. The data presented above suggest that Hop1 is a modular protein containing a distinct N-terminal and C-terminal (Hop1CTD) domains. The N-terminal domain of Hop1, which corresponds to the evolutionarily conserved HORMA domain, although, discovered first in Hop1, its precise biochemical functions remain unknown. In this section, we show that Hop1-HORMA domain expressed in and purified from E. coli exhibits preferential binding to the HJ and G4 DNA, over other early recombination intermediates. Detailed functional analyses of Hop1-HORMA domain, using mobility shift assays, DNase I footprinting and FRET, have revealed that HORMA binds at the core of Holliday junction and induces marked changes in its global conformation. Further experimental evidence also suggested that it causes DNA stiffening and condensation. However, like Hop1CTD, HORMA domain alone failed to rescue the meiotic recombination defects of hop1 null strain, indicating that synergy between the N-and C-terminal domains of Hop1 is essential for meiosis as well as for the formation of haploid gametes. Moreover, these results strongly implicate that Hop1 protein harbours a second DNA binding motif, which resides in the HORMA domain at its N-terminal region. To our knowledge, these findings also provide the first insights into the biochemical mechanism underlying HORMA domain activity. In summary, it appears that the C-terminal (CTD) and N-terminal (HORMA) domains of Hop1 may perform biochemical functions similar (albeit less efficiently) to that of the full-length Hop1. However, further research is required to uncover the functional differences between these domains, their respective interacting partners and modulation of the activity of these domains.

Meiosis is a specialized type of cell division essential for the production of four normal haploid gametes. In early prophase I of meiosis, the intimate synapsis between homologous chromosomes, and the formation of chiasmata, is facilitated by a proteinaceous structure known as the synaptonemal complex (SC). Ultrastructural analysis of germ cells of a number of organisms has disclosed that SC is a specialized tripartite structure composed of two lateral elements, one on each homolog, and a central element, which, in turn, are linked by transverse elements. Genetic studies have revealed that defects in meiotic chromosome alignment and/or segregation result in aneuploidy, which is the leading cause of spontaneous miscarriages in humans, hereditary birth defects such as Down syndrome, and are also, associated with the development and progression of certain forms of cancer. The mechanism(s) underlying the alignment/pairing of chromosomes at meiosis I differ among organisms. These can be divided into at least two broad pathways: one is independent of DNA double-strand breaks (DSB) and other is mediated by DSBs. In the DSB-dependent pathway, SC plays crucial roles in promoting homolog pairing and disjunction. On the other hand, the DSB-independent pathway involves the participation of telomeres, centromeres and non-coding RNAs in the pre-synaptic alignment, pre-meiotic pairing as well as pairing of homologous chromosomes. Although a large body of literature highlights the central role of SC in meiotic recombination, the possible role of SC components in homolog recognition and alignment is poorly understood. Genetic screens for Saccharomyces cerevisiae mutants defective in meiosis and sporulation lead to the isolation of genes required for interhomolog recombination, including those that encode SC components. In S. cerevisiae, ten meiosis-specific proteins viz., Hop1, Red1, Mek1, Hop2, Pch2, Zip1, Zip2, Zip3, Zip4 and Rec8 have been recognized as bona fide constituents of SC or associated with SC function. Mutations in any of these genes result in defective SC formation, thus leading to reduction in the rate of recombination. HOP1 (Homolog Pairing) encodes a ̴ 70 kDa structural protein, which localizes to the lateral elements of SC. It was found to be essential for the progression of meiotic recombination. In hop1Δ mutants, meiosis specific DSBs are reduced to 10% of that of wild type level and it fails to produce viable spores. It also displays relatively high frequency of inter-sister recombination over inter-homolog recombination. Bioinformatics analysis suggests that Hop1 comprises of an N-terminal HORMA domain (Hop1, Rev7 and Mad2), which is conserved among Hop1 homologs from diverse organisms. This domain is also known to be present in proteins involved in processes like chromosome synapsis, repair and sex chromosome inactivation. Additionally, Hop1 harbors a 36-amino acid long zinc finger 348374 motif (CX2CX19CX2C) which is critical for DNA binding and meiotic progression, and a putative nuclear localization signal corresponding to amino acid residues from 588-594. Previous studies suggested that purified Hop1 protein exists in multiple oligomeric states in solution and displays structure specific DNA binding activity. Importantly, Hop1 exhibited higher binding affinity for the Holliday junction (HJ), over other early recombination intermediates. Binding of Hop1 to the HJ at the core resulted in branch migration of the junction, albeit weakly. Intriguingly, Hop1 showed a high binding affinity for G4 DNA, a non-B DNA structure, implicated in homolog synapsis and promotes robust synapsis between double-stranded DNA molecules. Hop1 protein used in the foregoing biochemical studies was purified from mitotically dividing S. cerevisiae cells containing the recombinant plasmid over-expressing the protein where the yields were often found to be in the low-microgram quantities. Therefore, one of the major limitations to the application of high resolution biophysical techniques, such as X-crystallography and spectroscopic analyses for structure-function studies of S. cerevisiae Hop1 has been the non-availability of sufficient quantities of functionally active pure protein. In this study, we have performed expression screening in Escherichia coli host strains, capable of high level expression of soluble S. cerevisiae Hop1 protein. A new protocol has been developed +2 for expression and purification of S. cerevisiae Hop1 protein, using Ni-NTA and double-stranded DNA-cellulose chromatography. Recombinant S. cerevisiae Hop1 protein thus obtained was >98% pure and exhibited DNA binding activity with high-affinity for Holliday junction. The availability of the bacterial HOP1 expression vector and functionally active Hop1 protein has enabled us to glean and understand several vital biological insights into the structure-function relationships of Hop1 as well as the generation of appropriate truncated mutant proteins. Mutational analyses in S. cerevisiae has shown that sister chromatid cohesion is required for proper chromosome condensation, including the formation of axial elements, SC assembly and recombination. Consistent with these findings, homolog alignment is impaired in red1hop1 strains and associations between homologs are less stable. red1 mutants fail to make any discernible axial elements or SC structures but exhibit normal chromosome condensation, while hop1 mutants form long fragments of axial elements but without any SCs, are defective in chromosome condensation, and produce in-viable spores. Using single molecule and ensemble assays, we found that S. cerevisiae Hop1 organizes DNA into at least four major distinct DNA conformations: (i) a rigid protein filament along DNA that blocks access to nucleases; (ii) bridging of non-contiguous segments of DNA to form stem-loop structures; (iii) intra-and intermolecular long range synapsis between double-stranded DNA molecules; and (iv) folding of DNA into higher order nucleoprotein structures. Consistent with B. McClintock’s proposal that “there is a tendency for chromosomes to associate 2-by-2 in the prophase of meiosis involving long distance recognition of homologs”, these results to our knowledge provide the first evidence that Hop1 mediates the formation of tight DNA-protein-DNA nucleofilaments independent of homology which might help in the synapsis of homologous chromosomes during meiosis. Although the DNA binding properties of Hop1 are relatively well established, comparable knowledge about the protein is lacking. The purification of Hop1 from E. coli, which was functionally indistinguishable from the protein obtained from mitotically dividing S. cerevisiae cells has enabled us to investigate the structure-function relationships of Hop1, which has provided important insights into its role in meiotic recombination. We present several lines of evidence suggesting that Hop1 is a modular protein, consisting of an intrinsically unstructured N-terminal domain and a core C-terminal domain (Hop1CTD), the latter being functionally equivalent to the full-length Hop1 in terms of its in vitro activities. Importantly, however, Hop1CTD was unable to rescue the meiotic recombination defects of hop1null strain, indicating that synergy between the N-terminal and C-terminal domains of Hop1 protein is essential for meiosis and spore formation. Taken together, our findings provide novel insights into the molecular functions of Hop1, which has profound implications for the assembly of mature SC, homolog synapsis and recombination. Several lines of investigations suggest that HORMA domain containing proteins are involved in chromatin binding and, consequently, have been shown to play key roles in processes such as meiotic cell cycle checkpoint, DNA replication, double-strand break repair and chromosome synapsis. S. cerevisiae encodes three HORMA domain containing proteins: Hop1, Rev7 and Mad2 (HORMA) which interact with chromatin during diverse chromosomal processes. The data presented above suggest that Hop1 is a modular protein containing a distinct N-terminal and C-terminal (Hop1CTD) domains. The N-terminal domain of Hop1, which corresponds to the evolutionarily conserved HORMA domain, although, discovered first in Hop1, its precise biochemical functions remain unknown. In this section, we show that Hop1-HORMA domain expressed in and purified from E. coli exhibits preferential binding to the HJ and G4 DNA, over other early recombination intermediates. Detailed functional analyses of Hop1-HORMA domain, using mobility shift assays, DNase I footprinting and FRET, have revealed that HORMA binds at the core of Holliday junction and induces marked changes in its global conformation. Further experimental evidence also suggested that it causes DNA stiffening and condensation. However, like Hop1CTD, HORMA domain alone failed to rescue the meiotic recombination defects of hop1 null strain, indicating that synergy between the N-and C-terminal domains of Hop1 is essential for meiosis as well as for the formation of haploid gametes. Moreover, these results strongly implicate that Hop1 protein harbours a second DNA binding motif, which resides in the HORMA domain at its N-terminal region. To our knowledge, these findings also provide the first insights into the biochemical mechanism underlying HORMA domain activity. In summary, it appears that the C-terminal (CTD) and N-terminal (HORMA) domains of Hop1 may perform biochemical functions similar (albeit less efficiently) to that of the full-length Hop1. However, further research is required to uncover the functional differences between these domains, their respective interacting partners and modulation of the activity of these domains.