Rozprawy doktorskie na temat „I-motif DNA”

The i-motif is an alternative DNA secondary structure motif formed in sequences rich in cytosine, consisting of four strands, stabilised by hemi-protonated cytosine-cytosine+ base pairs. The motif forms in sequences complimentary to the G-quadruplex however, far less is known about the i-motif and most research to date has focused on its application in nanotechnology. Despite this, recent progress in the field has indicated that the i-motif may be a possible therapeutic target in certain cases. In order to study this structure in more detail a chemical tool box of ligands and conditions is needed, which can be used to probe its potential biological function. Herein the effect of small molecule ligands and cations has been investigated. A previously identified i-motif binding compound BisA has been characterised in detail with a range of biophysical experiments, showing that it does bind to the i-motif but causes the DNA to condense. A high throughput screen has been carried out finding a number of potential new i-motif binding ligands and, through a range of experiments, two lead compounds mitoxantrone and tilorone have been identified with micromolar affinities from which novel i-motif binding analogues and structure activity relationships can be developed. Finally, the effect of cations on the i-motif has been studied, including a wider selection from across the periodic table than has previously been investigated. This has shown that at neutral pH, silver (I) ions have the ability to induce i-motif formation and that this is reversible in the presence of cysteine. While at acidic pH, copper (II) ions have the ability to induce hairpin formation reversibly in the presence of EDTA. This could enable the formation of multiple structures from the same oligonucleotide sequence in response to different conditions. The combination of these results should provide useful tools to further study the i-motif structure.

Heme proteins carry out a diverse array of functions in vivo while maintaining a well-conserved 3-over-3 α-helical structure. Human hemoglobin (Hb) is well-known for its oxygen transport function. Type 1 non-symbiotic hemoglobins (nsHb1) in plants and bacterial flavohemoglobins (fHb) from a variety of bacterial species have been predicted to carry out a nitric oxide dioxygenase function. In nsHb1 and fHb this function has been linked to protection from nitrosative stress. Herein, I combine photoacoustic calorimetry (PAC), transient absorption spectroscopy (TA), and classical molecular dynamics (cMD) simulations to characterize molecular mechanism of diatomic ligand interactions with a hexa-coordinate globin from plant (rice hemoglobin), bacterial flavohemoglobins and human hemoglobin. In rice type 1 non-symbiotic hemoglobin (rHb1), the dynamics and energetics of structural changes associated with ligand photodissociation is strongly impacted by solvent and temperature, namely CO escape from the protein matrix is slower at pH = 6.0 compare to neutral pH (ns) due to the CD loop reorganization which forms a pathway for ligand escape. In human hemoglobin, exogenous allosteric effectors modulate energetics of conformational changes associated with the CO and O2 escape although the effectors impact on rate constants for ligand association is small. The conformational dynamics associated with ligand photorelease from fHbs from Cupriavidus necator (FHP) and Staphylococcus aureus (HMPSa) are strongly modulated by the presence of azole drugs indicating that drug association modulates structural properties of the heme binding pocket. In addition, we carried out a study of the formation of the DNA intercalated motif (i-motif). The formation of the structure is strongly favored at acidic pH; therefore, PAC was combined with a 2-nitrobenzaldehyde pH-jump to probe formation of the i-motif on fast timescales. i-Motif folding is two-step process with the initial protonation of cytosine residues being endothermic with ΔHfast=8.5 ± 7.0 kcal mol-1 and ΔVfast=10.4 ± 1.6 mL mol-1 and subsequent nucleation/i-motif folding (τ = 140 ns) with ΔHslow=-51.5 ± 4.8 kcal mol-1 and ΔVslow=-6.6 ± 0.9 mL mol-1. The above results indicate that PAC can be employed to study diverse biochemical reactions such as DNA folding, drug binding and ligand photorelease from proteins.

The i-motif is a DNA structure formed by cytosine-rich sequences that consists of parallel- stranded duplexes held together by intercalated base pairs. The in vitro formation of this structure in DNA sequences corresponding to the promoter regions of several oncogenes, such as c-kit, c-myc or bcl-2, has been demonstrated. Recently, the first direct evidence for its in vivo presence in human cells and control regulatory functions has been proven. This structure is not only interesting from a biophysical and biomedical point of view, but also for their potential application in Analytical Chemistry or Nanotechnology. The present Doctoral Thesis deals with the application of analytical and chemometric methodologies to study complex bioanalytical processes involving DNA i-motif structures. The sequences studied correspond to those found at cytosine-rich regions found near the promoter regions of the nmyc and SMARCA4 genes. On the one hand, the stability of the i- motif structures formed by these sequences according to variations of pH, temperature, ionic strength, or presence of ligands in steady-state conditions has been studied. On the other hand, the potential of ultrafast spectroscopies for the study of fast kinetic processes triggered by light has been evaluated. Through the Thesis, curve resolution methods, either based on soft-, hard- or hybrid-modelling have been used extensively to model the biochemical processes of interest. The steady-state studies have demonstrated that the stability against pH or temperature variations of the three different kinds of cytosine-rich sequences mentioned above is strongly dependent on the number of the C·C+ base pairs, but also on the contribution of other factors, such as the base composition and length of the loops and the presence of additional stabilising structures (hairpins) in the DNA sequence. The studies performed at ultrafast time scales have revealed that the photochemical process induced by UV-lamp irradiation and monitored by rapid-scan FTIR involves the formation of dimeric photoproducts in folded and unfolded sequences. The study of processes monitored by time-resolved fluorescence in the scale of picoseconds has shown that i-motif relaxation is detected by the presence of fast lifetimes in the pH range between 4 and 6, associated with intrinsic conformational changes at the fluorescent site. In this last study, one or two different i-motif structures have been detected in the nmyc and in the shortest DNA sequences studied, respectively. Finally, the application of multivariate resolution methods, based either on hard- or soft- modelling, has allowed the recovery of valuable chemical information from evolutionary processes of DNA. Besides, the adaptation and application of hybrid hard- and soft- modelling has been shown to be a useful approach to detect intermediate temperature- dependent conformational transitions and to avoid the effect of baseline drifts in the estimation of the melting temperature, as well to retrieve rate constants from the kinetic information present in rapid-scan FTIR difference spectra.
La estructura de l’ADN coneguda com “i-motif” es forma en seqüències riques en bases citosina (C). L’esquelet de l’i-motif està format per parells de bases C·C+ intercalats i estabilitzats per ponts d’hidrogen. S'ha demostrat la formació in vitro d'aquesta estructura en seqüències d'ADN corresponents a les regions promotores de diversos oncògens, com el c-kit, el c-myc o el bcl-2. Recentment, s'ha demostrat la primera evidència de la seva presència in vivo. La present Tesi Doctoral tracta de l'aplicació de metodologies analítiques i quimiomètriques per estudiar processos bioanalítics complexos que en els que intervenen aquestes estructures. Les seqüències estudiades corresponen a les regions promotores dels gens nmyc i SMARCA4. D'una banda, s'ha estudiat l'estabilitat de les estructures formades per aquestes seqüències segons variacions de pH, temperatura, força iònica o presència de lligands en condicions d'estat estacionari. D'altra banda, s'ha avaluat el potencial d'espectroscòpia ultraràpida per a l'estudi de processos cinètics ràpids provocats per la llum. Al llarg de la Tesi, els mètodes de resolució multivariant, ja sigui basats en models flexibles, rígids o híbrids, s'han utilitzat àmpliament per modelitzar els processos d'interès. Els estudis d'estat estacionari han demostrat que l'estabilitat davant el pH o les variacions de temperatura de les tres diferents seqüències riques en citosina esmentades anteriorment depèn molt del nombre de parells de bases de C·C+, però també de la contribució d'altres factors, com ara la composició de la base i la longitud dels bucles. A partir d'estudis ultraràpids, el procés fotoquímic induït per la irradiació de llum UV i l'IR d'escaneig ràpid s'ha relacionat amb la formació de fotoproductes dimèrics en seqüències plegades i desplegades. L'estudi dels processos seguits mitjançant fluorescència resolta en el temps ha demostrat l’existència de més d’una espècie associada amb l’estructura i-motif en el cas de les seqüencies curtes. Finalment, l'aplicació de mètodes de resolució multivariant, basats tant en models rígids o flexibles, han permès extreure informació química valuosa dels processos evolutius de l'ADN. A més, s'ha demostrat que l'adaptació i l'aplicació del modelatge híbrid ha permet calcular les constants cinètiques i detectar transicions dependents de la temperatura.

The nature of DNA has captivated scientists for more than fifty years. The discovery of the double-helix model of DNA by Watson and Crick in 1953 not only established the primary structure of DNA, but also provided the mechanism behind DNA function. Since then, the demonstration of DNA secondary structure formation has allowed for the proposal that the dynamics of DNA itself can function to modulate transcription. We demonstrate for the first time the i-motif DNA secondary structure formed from an element within the Bcl-2 promoter region has potential to serve as a cellular molecular target for modulation of gene expression. Unlike typical oncogenes, Bcl-2 acts by promoting cellular survival rather than increasing cellular proliferation. The over-expression of Bcl-2, most notably in lymphomas, has been associated with the development of chemoresistance.Transcriptional regulation of Bcl-2 is highly complex and a guanine- and cytosine-rich (GC-rich) region directly upstream of the P1 site has been shown to be integral to Bcl-2 promoter activity. We have demonstrated that the C-rich strand is capable of forming an intramolecular i-motif DNA secondary structure with a transition pH of 6.6 and a predominant 8:5:7 loop using mutational studies coupled with circular dichroic spectra and thermal stability analyses. In addition, a novel assay involving the sequential incorporation of a fluorescent thymine analog at each thymine position provided evidence of a capping structure within the top loop region of the i-motif. Two different classes of steroids either stabilize or destabilize the i-motif structure and this differential interaction results in the activation or repression of Bcl-2 expression. The i-motif stabilizing steroid significantly up-regulated Bcl-2 gene and protein expression in BJAB Burkitt's lymphoma cells while the destabilizing steroid down-regulated Bcl-2 expression in B95.8 Burkitt's and Granta-519 mantle cell lymphoma cells, as well as in a SCID mouse lymphoma model. More importantly, the down-regulation of Bcl-2 led to chemosensitization of etoposide-resistant lymphoma cells demonstrating that Bcl-2 i-motif interactive small molecules can act as chemosensitizing agents. Conversely, compounds that up-regulate Bcl-2 by stabilization of the i-motif have potential for use as neuroprotective agents.

Angiogenesis is known to be induced and maintained in tumors by the constant expression of the hypoxia inducible factor 1 alpha (HIF-1α) and human vascular endothelial growth factor (VEGF). In fact, tumor recurrence, aggressive metastatic legions and patient mortality rates are known to be positively correlated with overexpression of these two proteins. The HIF-1α and VEGF promoters contain a polypurine/polypyrimidine (pPu/pPy) tract, which are known to play critical roles in their transcriptional regulation, and are structurally dynamic where they can undergo a conformational transition between B-DNA, single stranded DNA and atypical secondary DNA structures such as G-quadruplexes and i-motifs. We hypothesize that the i-motif and G-quadruplex structures can form within the pPu/pPy tracts of the HIF-1α and VEGF proximal promoters, which play important roles in the transcriptional regulation of these genes by acting as scaffolds for alternative transcription factor binding sites. The purpose of this dissertation was to elucidate the transcriptional regulation of the HIF-1α and VEGF genes through the atypical DNA structures that form within the pPu/pPy tracts of their proximal promoters. We investigated the interaction of the C-rich and guanine-rich (G-rich) strands of both of these tracts with transcription factors heterogeneous nuclear ribonucleoprotein (hnRNP) K and nucleolin, respectively, both in vitro and in vivo and their potential role in the transcriptional control of HIF-1α and VEGF. In this dissertation, we demonstrate that both nucleolin and hnRNP K bind selectively to the G- and C-rich sequences, respectively, in the pPu/pPy tract of the HIF-1α and VEGF promoters. Specifically, the small interfering RNA-mediated silencing of either nucleolin or hnRNP K resulted in the down-regulation of basal VEGF gene, and the opposite effect was seen when the transcription factors were overexpressed, suggesting that they act as activators of VEGF transcription. Taken together, the identification of transcription factors that can recognize and bind to atypical DNA structures within pPu/pPy tracts will provide new insight into mechanisms of transcriptional regulation of the HIF-1α and VEGF gene.

The recent explosion of DNA sequence information has provided compelling evidence for the following facts. (1) Simple repetitive sequences-microsatellites and minisatellites occur commonly in the human genome and (2) these repetitive DNA sequences could play an important role in the regulation of various genetic processes including modulation of gene expression. These sequences exhibit extensive polymorphism in both length and the composition between species and between organisms of the same species and even cells of the same organism. The repetitive DNA sequences also exhibit structural polymorphism depending on the sequence composition. The functional significance of repetitive DNA is a well-established fact. The work done in many laboratories including ours has conclusively documented the functional role played by repetitive sequences in various cellular processes. Structural studies have established the sequence requirement for various non-B DNA structures and the functional significance of these unusual DNA structures is becoming increasingly clear. The structures that were characterised earlier purely from conformation point of view have aroused interest after the recent realisation that these structures could be formed in vivo when cloned in a supercoiled plasmid. The discovery of novel type of dynamic mutations where intragenic amplifications of trinucleotide repeats is associated with phenotypic changes causing many neurodegenerative disorders has provided the most compelling evidence for the importance of simple repeats in the etiology of these disorders. Secondary structures adopted by these simple repeats is a common causative factor in the mechanism of expansion of these repeats. This realisation prompted many investigations into the relationship between the DNA sequence, structure and molecular basis of dynamic mutation. Many experimental evidences have implicated paranemic DNA structures in various biological processes, especially in the regulation of gene expression. Earlier work done in our laboratory on the structure function relationship of repetitive DNA sequences provided experimental evidence for the role of paranemic DNA structure in the regulation of gene expression. It was demonstrated that intramolecular triplex potential sequences within a gene downregulate its expression in vivo (Sarkar and Brahmachari (1992) Nucleic Acids Res., 20, 5713-5718). Similarly the effect of cruciform structure forming sequences on gene expression was also documented. Sequence specific alterations in DNA structures were studied in our laboratory using a variety of biophysical and biochemical techniques. An intramolecular, antiparallel tetraplex structure was proposed for human telomeric repeat sequences (Balagurumoorthy, et al., (1994) J. Biol. Chem., 269, 21858-21869). The telomeric repeats are not only present at the end of chromosomes but they are also present at many interstitial sites in the human genome. Database search reveals that the human telomeric sequences as well as similar sequences with minor variations are present at many locations in the human genome. Telomeric repeats are GC rich sequences with the G rich strand protruding as a 3' end overhang at the end of chromosomes. When human telomeric repeats are cloned in a supercoiled plasmid, the C rich strand adopts a hairpin like conformation where as the G-rich strand extrudes into a quadruplex structure. However, the biological significance of these structures in vivo still remains to be elucidated completely. The role of a putative tetraplex DNA structure in the insulin gene linked polymorphic region of the human insulin gene in vivo in the regulation of expression of the insulin gene has been suggested. In this context, we have addressed the question whether the telomeric repeats when present within a gene affect its expression in vivol If so, what would be the possible mechanism? An attempt has been made to understand the effect of presence of telomeric repeats within a gene on its expression. The details of these studies have been presented in Chapter 2 of this thesis. Contrary to telomeric repeats which provide stability to the chromosomes, recently expansion of a GC rich dodecamer repeat upstream of cystatin B gene (chromosome 21q) has been shown to be the most common mutation associated with Progressive Myoclonus Epilepsy (EPM1) of Unverricht-Lundberg type. Two to three copies of the repeat (CCCCGCCCCGCG)n are present in normal individuals whereas the affected individuals have 30-75 copies of this repeat. The expression of cystatin B gene is reduced in patients in a cell specific manner. The repeat also shows intergenerational variability. The exact mechanism of expansion of this repeat is not known. In the case of trinucleotide repeat expansion, it is shown that the structure adopted by the repeat plays an important role in the mechanism of expansion and that some of the secondary structures adopted by trinucleotide repeats could be inherently mutagenic conformations. In order to understand the mechanism of expansion EPM1 dodecamer repeat, the work reported in this thesis was carried out with the following objectives. • To understand the structure of G rich and C-rich strands of EPM1 repeat. • To understand the variations in the structure with the increase in the length and its possible implications in the mechanism of expansion of EPM 1 repeat. Studies aimed with these objectives are presented in chapters 3, 4 and 5 of the thesis. Chapter 1 provides a general introduction to repetitive DNA, the various structures adopted by repetitive DNA sequences in the genome, the functional significance of the various simple repetitive DNA sequences in the genome has been presented. An account of trinucleotide repeat expansion and associated disorders, non-trinucleotide repeat expansions and associated disorders has been presented. The various non B-DNA structures adopted these repeats and their implications in the mechanism of expansion have been discussed. Chapter 2 describes in frame cloning of human telomeric repeats d(G3T2A)3G3 in the N-terminal region of β-galactosidase gene. The effect of such repeat Sequences on transcription elongation in vivo has been studied using E.coli as a model system. The 3.5 copies of human telomeric repeat sequences were cloned in the sense strand of plasmid pBluescriptllSK+ so as to create plasmid clone pSBQ8 and in the template strand of plasmid pBluescriptHKS+ so as to create clone pSBRQ8. One dimensional chloroquine gel shift assay indicated presence of an unwound structure in pSBQ8 and pSBRQ8. β-galactosidase activity assay suggested downregulation of the gene in vivo. In the case of plasmid pSBQ8 the difference in β-galactosidase activity was approximately 6 fold as compared to the parent plasmid pBluescriptIISK+ whereas in the case of pSBRQ8 the difference in β-galactosidase activity was approximately 8 fold as compared to the control pBluescriptIIKS+. The analysis of β-galactosidase transcript showed that full length transcript was formed in the case of pSBQ8. Full length transcript was not formed in the case of pSBRQ8. We propose that in the case of pSBQ8 the gene expression is inhibited in steps subsequent to transcription elongation. In the case of pSBRQ8, we propose that quadruplex structure may be formed by the template strand at the DNA level thereby blocking transcription elongation step. Chapter 3 describes studies aimed at understanding the structure of G-rich strand (referred to as G strand) of Progressive Myoclonus Epilepsy (EPM1) repeat. The sequence of the G strand of dodecamer EPM1 repeat is d(GGGGCGGGGCGC)n. Oligoucleotides containing one (12mer), two (24mer) and three(36mer) were synthesised. These oligonucleotides are referred to as dG12, dG24 and dG36 respectively. Structural studies were carried out using CD spectroscopy, UV melting, non-denaturing gel electrophoresis and chemical and enzymatic probing. The G strand oligonucleotides showed enhanced gel elecrophoretic mobility in the presence of monovalent cations KCl and NaCl. Oligonucleotide dG12 also showed retarded species on non-denaturing gel in the presence of 70mM KCl indicating intermolecular associations. Oligonucleotides dG24 and dG36 predominantly formed intramolecular structures which migrated anomalously faster than the expected size. The CD spectrum for dG12 showed an intense positive band at 260nm and a negative band at 240nm in the presence of KCl indicative of an intermolecular, parallel G quartet structure. The CD spectra of dG24 and dG36 showed 260nm positive peak, 240nm negative peak along with a positive band around 290nm. This is indicative of folded back structure. These findings support the results of non-denaturing gel electrophoresis of G strand oligonucleotides. The UV melting profiles suggested increase in the stability with the increase in the length. These structures were further characterised by PI nuclease and chemical probing using DMS and DEPC. The structural studies with G-rich strand of EPM1 dodecamer repeat showed that this repeat motif adopts intramolecularly folded structures with increase in the length of the repeat thereby favouring slippage during replication. Chapter 4 deals with the studies aimed at understanding the structure at acidic pH of C-rich strand (referred to as C strand) of Progressive Myoclonus Epilepsy (EPM1) repeat. The sequence of the C strand of dodecamer EPM1 repeat is d(CCCCGCCCCGCG)n. The C rich oligonucleotides are known to form a four stranded structure called i-motif at acidic pH involving intercalated base pairs. The i-motif consists of two parallel stranded, base paired duplexes are arranged in an antiparallel orientation. Since, the base pairs of one base paired duplex intercalate into those of the other duplex, the structure is called as i-motif. We have investigated structure of C strand of EPM1 repeat by circular dichroism (CD), native polyacrylamide gel electrophoresis and UV melting. Oligonucleotide dC12 showed two bands of which the major band was retarded on the native gel (pH 5.0) at low temperature suggesting that dC12 predominantly formed intermolecular structure, Oligonucleotides dC24 and dC36 migrated anomalously faster than the expected size indicating formation of compact, intramolecularly folded structures. Circular dichroism studies indicate that, all the oligonucleotides displayed an intense positive band near 285nm, a negative band around 260nm with a cross over at 270nm, This is a characteristic CD signature for an i-motif structure and reflects the presence of secondary structure due to formation of hydrogen bonded pairs between protonated cytosines. All the C strand oligonucleotides showed hyperchromism at 265nm, which is an isobestic wavelength for C protonation. Studies described in this chapter suggest an intramolecular i-motif structure for dC24 and dC36 and an intermolecular i-motif for oligonucleotide dC12. In addition, it was interesting to note that inspite of the presence of G residues, the stretch of C residues could adopt i-motif structure. Although these structures are formed at an acidic pH, it is indicative of formation of possible intramolecularly folded structure. Many reports have suggested the possibility of cytosine rich sequences adopting i-motif structure even at neutral pH. In order to test this possibility, structural studies were carried out on the C strand EPM1 oligonucleotides at pH 7.2 in the presence of 70mM NaCl. These studies have been described in Chapter 5. The investigations were done using CD spectroscopy, UV melting, native polyacrylamide gel electrophoresis, and chemical probing using hydroxylamine and PI nuclease. These studies indicate that all the C strand oligonucleotides form intramolecular, hairpin structure at physiological pH. All the three C strand oligonucleotides migrated anomalously faster on the native gel indicating the presence of a compact structure. The CD spectra at pH 7.2 showed a blue shift as compared to those at pH 5.0. This indicated absence of base pairs. The hydroxylamine chemical probing suggested presence of G-C Watson-Crick base pairs. The loop residues of the folded back hairpin structures were probed with PI nuclease. The C strand oligonucleotides showed possibility of formation of multiple hairpin structures with the increase in the length of the repeat. The propensity to form hairpin structures suggests a possibility of formation of slip loop structures during the replication process thereby promoting expansion of this repeat. Formation of folded back hairpin like structures is significant in terms of mechanism of expansion of this repeat. Chapter 6 is devoted to concluding remarks highlighting the significance of the experimental results presented in this thesis and their possible biological implications in the light of contemporary research.

The recent explosion of DNA sequence information has provided compelling evidence for the following facts. (1) Simple repetitive sequences-microsatellites and minisatellites occur commonly in the human genome and (2) these repetitive DNA sequences could play an important role in the regulation of various genetic processes including modulation of gene expression. These sequences exhibit extensive polymorphism in both length and the composition between species and between organisms of the same species and even cells of the same organism. The repetitive DNA sequences also exhibit structural polymorphism depending on the sequence composition. The functional significance of repetitive DNA is a well-established fact. The work done in many laboratories including ours has conclusively documented the functional role played by repetitive sequences in various cellular processes. Structural studies have established the sequence requirement for various non-B DNA structures and the functional significance of these unusual DNA structures is becoming increasingly clear. The structures that were characterised earlier purely from conformation point of view have aroused interest after the recent realisation that these structures could be formed in vivo when cloned in a supercoiled plasmid. The discovery of novel type of dynamic mutations where intragenic amplifications of trinucleotide repeats is associated with phenotypic changes causing many neurodegenerative disorders has provided the most compelling evidence for the importance of simple repeats in the etiology of these disorders. Secondary structures adopted by these simple repeats is a common causative factor in the mechanism of expansion of these repeats. This realisation prompted many investigations into the relationship between the DNA sequence, structure and molecular basis of dynamic mutation. Many experimental evidences have implicated paranemic DNA structures in various biological processes, especially in the regulation of gene expression. Earlier work done in our laboratory on the structure function relationship of repetitive DNA sequences provided experimental evidence for the role of paranemic DNA structure in the regulation of gene expression. It was demonstrated that intramolecular triplex potential sequences within a gene downregulate its expression in vivo (Sarkar and Brahmachari (1992) Nucleic Acids Res., 20, 5713-5718). Similarly the effect of cruciform structure forming sequences on gene expression was also documented. Sequence specific alterations in DNA structures were studied in our laboratory using a variety of biophysical and biochemical techniques. An intramolecular, antiparallel tetraplex structure was proposed for human telomeric repeat sequences (Balagurumoorthy, et al., (1994) J. Biol. Chem., 269, 21858-21869). The telomeric repeats are not only present at the end of chromosomes but they are also present at many interstitial sites in the human genome. Database search reveals that the human telomeric sequences as well as similar sequences with minor variations are present at many locations in the human genome. Telomeric repeats are GC rich sequences with the G rich strand protruding as a 3' end overhang at the end of chromosomes. When human telomeric repeats are cloned in a supercoiled plasmid, the C rich strand adopts a hairpin like conformation where as the G-rich strand extrudes into a quadruplex structure. However, the biological significance of these structures in vivo still remains to be elucidated completely. The role of a putative tetraplex DNA structure in the insulin gene linked polymorphic region of the human insulin gene in vivo in the regulation of expression of the insulin gene has been suggested. In this context, we have addressed the question whether the telomeric repeats when present within a gene affect its expression in vivol If so, what would be the possible mechanism? An attempt has been made to understand the effect of presence of telomeric repeats within a gene on its expression. The details of these studies have been presented in Chapter 2 of this thesis. Contrary to telomeric repeats which provide stability to the chromosomes, recently expansion of a GC rich dodecamer repeat upstream of cystatin B gene (chromosome 21q) has been shown to be the most common mutation associated with Progressive Myoclonus Epilepsy (EPM1) of Unverricht-Lundberg type. Two to three copies of the repeat (CCCCGCCCCGCG)n are present in normal individuals whereas the affected individuals have 30-75 copies of this repeat. The expression of cystatin B gene is reduced in patients in a cell specific manner. The repeat also shows intergenerational variability. The exact mechanism of expansion of this repeat is not known. In the case of trinucleotide repeat expansion, it is shown that the structure adopted by the repeat plays an important role in the mechanism of expansion and that some of the secondary structures adopted by trinucleotide repeats could be inherently mutagenic conformations. In order to understand the mechanism of expansion EPM1 dodecamer repeat, the work reported in this thesis was carried out with the following objectives. • To understand the structure of G rich and C-rich strands of EPM1 repeat. • To understand the variations in the structure with the increase in the length and its possible implications in the mechanism of expansion of EPM 1 repeat. Studies aimed with these objectives are presented in chapters 3, 4 and 5 of the thesis. Chapter 1 provides a general introduction to repetitive DNA, the various structures adopted by repetitive DNA sequences in the genome, the functional significance of the various simple repetitive DNA sequences in the genome has been presented. An account of trinucleotide repeat expansion and associated disorders, non-trinucleotide repeat expansions and associated disorders has been presented. The various non B-DNA structures adopted these repeats and their implications in the mechanism of expansion have been discussed. Chapter 2 describes in frame cloning of human telomeric repeats d(G3T2A)3G3 in the N-terminal region of β-galactosidase gene. The effect of such repeat Sequences on transcription elongation in vivo has been studied using E.coli as a model system. The 3.5 copies of human telomeric repeat sequences were cloned in the sense strand of plasmid pBluescriptllSK+ so as to create plasmid clone pSBQ8 and in the template strand of plasmid pBluescriptHKS+ so as to create clone pSBRQ8. One dimensional chloroquine gel shift assay indicated presence of an unwound structure in pSBQ8 and pSBRQ8. β-galactosidase activity assay suggested downregulation of the gene in vivo. In the case of plasmid pSBQ8 the difference in β-galactosidase activity was approximately 6 fold as compared to the parent plasmid pBluescriptIISK+ whereas in the case of pSBRQ8 the difference in β-galactosidase activity was approximately 8 fold as compared to the control pBluescriptIIKS+. The analysis of β-galactosidase transcript showed that full length transcript was formed in the case of pSBQ8. Full length transcript was not formed in the case of pSBRQ8. We propose that in the case of pSBQ8 the gene expression is inhibited in steps subsequent to transcription elongation. In the case of pSBRQ8, we propose that quadruplex structure may be formed by the template strand at the DNA level thereby blocking transcription elongation step. Chapter 3 describes studies aimed at understanding the structure of G-rich strand (referred to as G strand) of Progressive Myoclonus Epilepsy (EPM1) repeat. The sequence of the G strand of dodecamer EPM1 repeat is d(GGGGCGGGGCGC)n. Oligoucleotides containing one (12mer), two (24mer) and three(36mer) were synthesised. These oligonucleotides are referred to as dG12, dG24 and dG36 respectively. Structural studies were carried out using CD spectroscopy, UV melting, non-denaturing gel electrophoresis and chemical and enzymatic probing. The G strand oligonucleotides showed enhanced gel elecrophoretic mobility in the presence of monovalent cations KCl and NaCl. Oligonucleotide dG12 also showed retarded species on non-denaturing gel in the presence of 70mM KCl indicating intermolecular associations. Oligonucleotides dG24 and dG36 predominantly formed intramolecular structures which migrated anomalously faster than the expected size. The CD spectrum for dG12 showed an intense positive band at 260nm and a negative band at 240nm in the presence of KCl indicative of an intermolecular, parallel G quartet structure. The CD spectra of dG24 and dG36 showed 260nm positive peak, 240nm negative peak along with a positive band around 290nm. This is indicative of folded back structure. These findings support the results of non-denaturing gel electrophoresis of G strand oligonucleotides. The UV melting profiles suggested increase in the stability with the increase in the length. These structures were further characterised by PI nuclease and chemical probing using DMS and DEPC. The structural studies with G-rich strand of EPM1 dodecamer repeat showed that this repeat motif adopts intramolecularly folded structures with increase in the length of the repeat thereby favouring slippage during replication. Chapter 4 deals with the studies aimed at understanding the structure at acidic pH of C-rich strand (referred to as C strand) of Progressive Myoclonus Epilepsy (EPM1) repeat. The sequence of the C strand of dodecamer EPM1 repeat is d(CCCCGCCCCGCG)n. The C rich oligonucleotides are known to form a four stranded structure called i-motif at acidic pH involving intercalated base pairs. The i-motif consists of two parallel stranded, base paired duplexes are arranged in an antiparallel orientation. Since, the base pairs of one base paired duplex intercalate into those of the other duplex, the structure is called as i-motif. We have investigated structure of C strand of EPM1 repeat by circular dichroism (CD), native polyacrylamide gel electrophoresis and UV melting. Oligonucleotide dC12 showed two bands of which the major band was retarded on the native gel (pH 5.0) at low temperature suggesting that dC12 predominantly formed intermolecular structure, Oligonucleotides dC24 and dC36 migrated anomalously faster than the expected size indicating formation of compact, intramolecularly folded structures. Circular dichroism studies indicate that, all the oligonucleotides displayed an intense positive band near 285nm, a negative band around 260nm with a cross over at 270nm, This is a characteristic CD signature for an i-motif structure and reflects the presence of secondary structure due to formation of hydrogen bonded pairs between protonated cytosines. All the C strand oligonucleotides showed hyperchromism at 265nm, which is an isobestic wavelength for C protonation. Studies described in this chapter suggest an intramolecular i-motif structure for dC24 and dC36 and an intermolecular i-motif for oligonucleotide dC12. In addition, it was interesting to note that inspite of the presence of G residues, the stretch of C residues could adopt i-motif structure. Although these structures are formed at an acidic pH, it is indicative of formation of possible intramolecularly folded structure. Many reports have suggested the possibility of cytosine rich sequences adopting i-motif structure even at neutral pH. In order to test this possibility, structural studies were carried out on the C strand EPM1 oligonucleotides at pH 7.2 in the presence of 70mM NaCl. These studies have been described in Chapter 5. The investigations were done using CD spectroscopy, UV melting, native polyacrylamide gel electrophoresis, and chemical probing using hydroxylamine and PI nuclease. These studies indicate that all the C strand oligonucleotides form intramolecular, hairpin structure at physiological pH. All the three C strand oligonucleotides migrated anomalously faster on the native gel indicating the presence of a compact structure. The CD spectra at pH 7.2 showed a blue shift as compared to those at pH 5.0. This indicated absence of base pairs. The hydroxylamine chemical probing suggested presence of G-C Watson-Crick base pairs. The loop residues of the folded back hairpin structures were probed with PI nuclease. The C strand oligonucleotides showed possibility of formation of multiple hairpin structures with the increase in the length of the repeat. The propensity to form hairpin structures suggests a possibility of formation of slip loop structures during the replication process thereby promoting expansion of this repeat. Formation of folded back hairpin like structures is significant in terms of mechanism of expansion of this repeat. Chapter 6 is devoted to concluding remarks highlighting the significance of the experimental results presented in this thesis and their possible biological implications in the light of contemporary research.

Cancer diseases are increasing worldwide and more than 20 million new cancer cases per year are expected by 2025. At these days the treatment of neoplastic forms took advantage of classic approaches, based on chemotherapeutics and radiotherapeutics agents. However they are characterized by numerous limitations as remarkable side effects, toxicity and selection of resistant phenotypes to such therapies. This prompted the development of so-called targeted therapies, where selective chemical entities (small molecules, monoclonal antibodies, miRNAs, siRNAs etc.) hit a single molecular target of the tumor phenotype. Despite, these therapies have proven to be efficient alternatives they also present several limitations that make them quite ineffective. In order to overcome these remarkable drawbacks, he modulation of the gene expression, that exploits the ability of nucleic acids to assume different conformations, defined as non-canonical, became extremely interesting. Among these non-canonical conformations, extremely fascinating are the tetrahelical conformations known as G-quadruplex (G4) and i-Motif (iM), that seem to be involved in the blockage of the cancer development. G4 structures occur at DNA and RNA sequences presenting a high abundance of consecutive guanines that interact each other through Hoogstein hydrogen bonds to generate a planar structure called G-tetrads. Stacking interaction between two or more G tetrads create the overall structure. Bioinformatics studies revealed that prevalently these regions are contained along the telomeres and within the untranslated region (UTR) or within the promoter sites of several oncogenes (approximately 40%) directly implicated in the development of tumor phenotypes. The UTR domains, as the promoter regions, are double-stranded DNA sequences. Therefore the complementary strand results enriched in cytosine, that under specific environmental conditions can folds into a tetrahelical conformation, known as i-Motif. Unlike the G4s, the building block of the entire structure is a dimer of cytosine mainly stabilized by the presence of three Hoogstein hydrogen bonds. The in vivo formation of G4 and iM leads to a steric hindrance at the DNA level; this suggests an inhibition/activation effect on the elongation process of the telomere or on the gene expression process Under the supervision of Dr. Laurence J. Hurley, the structural characterization of the cytosine rich sequence contained within the NHE(III)1 region of MYC promoter was completed. In particular, was assessing the effect of the loop composition on the stability and folding process of the already characterized iM. Since it was proved that this conformation in vivo is involved in the transcriptional activation, the possibility to target it by using a selected compound (IMC-30) was considered. Furthermore, we took into consideration the possibility to use this compound (IMC-30) as an anticancer drug by testing its ability to induce the apoptosis process in a cancer cell line in which the selected gene was overexpressed. Besides the several evidence reported for the tetrahelical conformations assumed by the GC-rich promoter regions, more recently the efforts moved forward to the G-rich tract contained in the untranslated (UTR) domains, both the 5’- and the 3’-UTR, of the primary transcript. Since, they can act as modulators of the translation process. Based on this evidence, in this project, the guanine rich sequences contained in the 5'-UTR region, both at the DNA and RNA levels, of the BCL2 gene were considered. In particular, the structural characterization study was initially carried out on the minimal sequences (dBcl2_G and rBcl2_G), then the effect exerts by the presence of additional nucleotides on the folding process towards the G-quadruplex was taken into consideration (dBcl2_G + 3 WC, rBcl2_G + 3 WC and rBcl2_48). Additionally, the cytosine rich tract contained on the DNA complementary strand was considered and characterized. Our data have shown that the dBcl2_G and rBcl2_G are able to assume multiple G4 conformations. While, the presence of additional nucleotides strongly modulates their ability to assume the non-canonical conformation. Indeed, we proved that the presence of 3 WC pairing partially prevents the formation of G4 both in the DNA and in the RNA, while the addition of a greater number of bases (rBcl2_48) leads to the formation of a different conformation that competes with the G4 structure. Regarding the cytosine rich string, its conformational equilibria have been taken into consideration both in a mildly acidic environment and in an environment that mimics the physiological condition. Finally, we implemented our work, by screening a library of compounds on each tested sequences in order to find a ligand that selectively recognizes and stabilizes one conformation. From the acquired data it emerged the feasibility to stabilize/induce the iM using the Bisanthrene compound and its derivative Bis 1-8. For the guanine rich sequences, Sanguinarine and Chelerythrine provide the best results on each tested tracts, therefore they cannot be considered selective compounds. Similarly, also the Bisanthrene derivatives recognize and interact with each tested guanine tracts, although with different selectivity.
Oggigiorno una delle “piaghe” che affligge maggiormente la popolazione mondiale è il cancro. Il trattamento di queste forme neoplastiche sfrutta agenti chemioterapici e radioterapici, caratterizzati da numerose limitazioni legate ai notevoli effetti collaterali, alla tossicità e alla selezione di fenotipi resistenti a tali terapie. Ciò ha portato allo sviluppo delle targeted therapy, che sfruttano entità chimiche (small molecules, anticorpi monoclonali, miRNA, siRNA ecc.) selettive per un bersaglio molecolare caratteristico del fenotipo tumorale. Nonostante più mirati anche questi approcci presentano degli effetti collaterali Pertanto la modulazione dell’espressione genica che sfrutta la capacità degli acidi nucleici di assumere differenti conformazioni, definite non canoniche, ha destato sempre più interesse. Tra le possibili strutture non canoniche di notevole interesse sono le conformazioni tetraelicoidali note come G-quadruplex (G4) e i-Motif (iM). La struttura G4 è propria di sequenze di DNA e RNA contenenti un’elevata abbondanza di guanine consecutive che, mediante legami a idrogeno di tipo Hoogstein, generano delle strutture planari chiamate tetradi. Dall’’impilamento di due o più tetradi si genera la struttura a tetraelica. Poiché il DNA è una doppia elica, il filamento complementare a queste regioni G ricche presenta un’elevata abbondanza di citosine. Anche questi domini in particolari condizioni ambientali, possono generare una conformazione tetraelicoidale, nota come i-Motif. A differenza del G4, il building block dell’intera struttura è un dimero di citosine stabilizzato dalla presenza di tre legami a idrogeno. In vivo l’esistenza di queste conformazioni, genera una sorta d’ingombro sterico a livello del DNA e ciò presuppone un effetto d’inibizione/attivazione del processo di elongazione del telomero o del processo trascrizionale. Sotto la supervisione del Dott. Laurence J. Hurley, è stata implementata la caratterizzazione strutturale della stringa di citosine contenute nel promotore del gene MYC. In seguito un selezionato ligando è stato testato con l’idea di poter modulare il processo di folding/unfolding alla base dell’attivazione trascrizionale. Infine, l’effetto mediato da questo composto sul processo apoptotico è stato preso in considerazione lavorando su una selezionata linea cellulare. Di notevole interesse sono le regioni GC-ricche contenute nella porzione non tradotta del trascritto primario (mRNA). Sulla base di ciò, in questo progetto, sono state prese in considerazioni, le stringhe di guanina e citosina contenute nella regione del 5’-UTR, sia a livello del DNA sia del RNA, del gene BCL2. Inizialmente è stato condotto uno studio di caratterizzazione sulle sequenze minimali dBcl2_G, dBcl2_C e rBcl2_G. In seguito è stato preso in considerazione l’effetto della presenza di nucleotidi adiacenti sul processo di folding verso il G-quadruplex (dBcl2_G + 3WC, rBcl2_G + 3WC e rBcl2_48). I dati ottenuti dimostrano che le sequenze dBcl2_G e rBcl2_G sono in grado di assumere molteplici conformazioni G4. La presenza di nucleotidi addizionali modula la loro capacità di assumere queste conformazioni. In particolare, la presenza di tre appaiamenti WC impedisce parzialmente la formazione del G4 sia nel DNA, che nel RNA mentre, l’aggiunta di un maggior numero di basi (rBcl2_48) sposta l’equilibrio conformazionale verso una conformazione in forte competizione con il G4. Per la sequenza ricca di citosine, l’equilibrio conformazionale è stato valutato sia in ambiente blandamente acido, che in un ambiente che mima la condizione fisiologica. Infine, poiché negli ultimi anni è stata dimostrata la capacità di alcuni ligandi sintetici/naturali, di spostare gli equilibri conformazionali del DNA, dalla classica forma a doppio filamento, verso queste conformazioni tetraelicoidali, una selezionata libreria di composti è stata, scrinata allo scopo di individuare un ligando in grado di riconoscere e stabilizzare selettivamente una conformazione al pari di un'altra.

Intramolecular i-motifs of the form C3L3-8C3L3-8C3L3-8C3, where C3 denotes the cytosine stretch and L3-8 are “loop” regions containing any DNA base (L) including cytosine, were studied to understand the effects of loop length on i-motif stability. It contrast to the previously held notion that long-looped i-motifs are more stable, it was found that i-motif structures with short loops exhibit higher thermal stabilities and transitional pH values. The stability of long-looped i-motifs are then shown to increase with the addition of [Ru(phen)2dppz]2+ (phen = 1,10-phenanthroline, dppz = dipyrido [3,2-a:2',3'-c] phenazine); a polypyridyl complex that has a potential for photodynamic therapy. Addition of the complex enhances the stability of d(C3T838)3C3 but not that of d(C3T383)3C3, implying that loop lengths are important in defining i-motif-ligand interactions. The effects of loop base composition on the stability of i-motifs have also been presented. It is shown that when d(C3XYZ)3C3 sequences are used (where X and Z are adenine, thymine and guanine, and Y is any of the four DNA bases), pyrimidine-rich sequences form more stable i-motif structures. However, when guanine is X, only two out of 12 d(C3XYG)3C3 sequences were able to form i-motifs. Change in sequence direction also resulted in different thermal and pH stabilities; emphasising the role of loop base composition on not only the i-motif’s stability but also in its formation. X-ray crystallography was used to further understand the effects of loop bases on i-motif structures. The study focuses on four tetramolecular i-motifs; two of which were solved in the mid1990’s; (d(C4)4 and d(C3T)4 but have now been re-examined using improved experimental approaches. Two novel i-motif structures of d(C3A)4 and [d(C3A) + d(C3T)] are presented. Following the X-ray diffraction of d(C3T)4 crystals to 0.68 Å resolution (previously reported at 1.4 Å) at beamline I02 (Diamond Light Source Ltd.), a novel neutron diffraction study on the particular i-motif was conducted. Single crystal neutron diffraction was carried out at MaNDi beamline (Spallation Neutron Source) to find the distribution of the proton between the hemiprotonated cytosine+·cytosine base pairs and to understand the role that H-bonded water can play in stabilising the i-motif structure.

Els àcids nucleics poden existir a la natura en una varietat de formes estructurals potencialment rellevants per al funcionament i la regulació dels gens. Aquesta tesi doctoral versa sobre una estructura no canònica del DNA: l'estructura quàdruplex anomenada bi-loop. Es tracta d'un motiu que implica la interacció de dues cadenes que es disposen antiparal·lelament i es mantenen unides per quatre parells de bases intermoleculars. Els parells s'enfronten pel solc menor formant dues tètrades no planes. Aquest motiu havia estat observat en l'estructura cristal·logràfica d'un heptàmer lineal i en les estructures en solució de diversos octàmers cíclics.
Un dels objectius d'aquesta tesi doctoral era aprofundir en l'estudi del bi-loop per tal de determinar-ne els requeriments seqüencials i intentar observar-lo per primera vegada en solució amb oligonucleòtids lineals.
Inicialment s'estudià el motiu bi-loop heterolineal, format per un oligonucleòtid cíclic i un altre lineal, partint de l'heterodímer d+d. Mitjançant RMN, s'observà que la formació del complex heterolineal és més favorable quan l'oligonucleòtid lineal es troba tallat entre els nucleòtids aparellats. Tot i així, el seu extrem 3' és flexible i un dels parells de bases només es troba parcialment format. En tenir un oligonucleòtid lineal tallat pel llaç, sembla que també s'estructura en forma de bi-loop en presència del motlle cíclic, però aquesta estructuració es perd en allargar-ne els extrems.
Per altra banda, s'ha vist que el bi-loop pot estar estabilitzat per parells no canònics, com els GT. Les dades de RMN permeten afirmar que l'octàmer cíclic d s'estructura en forma de bi-loop estabilitzat per una tètrada GTGT directa i una GCGC desplaçada. Un altre octàmer cíclic, d, pot autoassociar-se de dues formes diferents donant lloc a dos dímers: un estabilitzat per dues tètrades mixtes GCGT desplaçades i el segon estabilitzat per una tètrada GTGT desplaçada i una GCGC desplaçada.
L'observació del motiu en fragments lineals en solució seria de gran importància com a pas previ per a establir-ne la rellevància biològica. Amb aquest objectiu es dissenyaren cinc seqüències lineals derivades d'octàmers cíclics que formen bi-loops força estables però que no poden formar estructures monomèriques. D'aquestes, només dues d'elles s'estructuren en solució en forma de bi-loop: d(TGCTTCGT) i d(TCGTTGCT). La seva estructura es va determinar mitjançant dinàmica molecular restringida i s'assembla molt a totes les estructures de la mateixa família. Aquests dímers, però, són molt menys estables que el seu anàleg cíclic degut al cost entròpic de doblegar les cadenes.
El segon objectiu d'aquesta tesi era emprar el motiu bi-loop com a motlle per a la ciclació assistida. Inicialment es va assajar la ciclació d'oligonucleòtids lineals fosforilats en un dels seus extrems en presència d'un motlle cíclic que afavorís la formació d'un motiu heterolineal, sense èxit. S'observà la massa del producte de ciclació, però aquest no es va poder aïllar, de manera que es conclou que el motiu no és prou estable o és massa flexible. Posteriorment es va assajar la ciclació d'oligonucleòtids lineals fosforilats en 3' que s'autoestructuren en forma de bi-loop. Usant EDC o bromur de cianogen es va poder observar la formació del motiu tant per espectrometria de masses com per electroforesi en gel de poliacrilamida.
Per últim, s'ha intentat aplicar la ciclació assistida per motlle a la fase sòlida, per superar les limitacions de mida del cicle i aprofitar els avantatges de treball i purificació de la fase sòlida, especialment el desancoratge selectiu de les cadenes. No s'ha pogut assolir l'objectiu desitjat degut a la dificultat d'activar un fosfat diester en un medi aquós.
Non-canonical forms of DNA may play important roles in gene function and regulation. This thesis is focused on a type of non-canonical quadruplex called bi-loop. The bi-loop results from interaction of two chains and it is stabilized by four intermolecular base pairs. The base pairs face each other through their minor groove sides giving rise to two non-planar tetrads. This motif has been observed in the crystal structure of a lineal heptamer and in the solution structures of several cyclic octamers.
In this thesis we wished to further define the sequence requirements for the bi-loop formation, and to observe it in solution with linear oligonucleotides for the first time. The study started with the so-called heterolineal complexes, formed by a cyclic octamer and a linear one. Using NMR, we observed that the formation of the bi-loop is favoured when the nick is between the base pairs, although the 3'-end is flexible. We also observed that the bi-loop can be stabilized by non-canonical base-pairs, such as GT. The cyclic octamers d and d form bi-loops stabilized by GT and GC pairs, giving rise to three new types of tetrads (direct GTGT, slipped GTGT and slipped GCGT).
Observation of the motif in solution with linear oligonucleotides would be very important to reinforce its potential biological relevance. For this purpose, five linear sequences that cannot form hairpins were designed. Two of these sequences were found to form a bi-loop motif in solution: d(TGCTTCGT) and d(TCGTTGCT). Their structures were solved by RMD and proved to be very similar to those formed by cyclic molecules, though less stable due to the entropic cost.
Secondly, we wanted to use the bi-loop motif as a template for assisted cyclization. We started with the heterolineal complexes and we were not lucky in achieving the ligation. In contrast, we succeeded in the cyclization of linear phosphorylated oligonucleotides that formed bi-loop homodimers.
Finally, the solid-phase template-directed cyclization of oligonucleotides was also assayed. We were not successful because of the difficulty in activating a phosphate diester moiety in an aqueous medium.

Yes
There are hundreds of ligands which can interact with G-quadruplex DNA, yet very few which target i-motif. To appreciate an understanding between the dynamics between these structures and how they can be affected by intervention with small molecule ligands, more i-motif binding compounds are required. Herein we describe how the drug mitoxantrone can bind, induce folding of and stabilise i-motif forming DNA sequences, even at physiological pH. Additionally, mitoxantrone was found to bind i-motif forming sequences preferentially over double helical DNA. We also describe the stabilisation properties of analogues of mitoxantrone. This offers a new family of ligands with potential for use in experiments into the structure and function of i-motif forming DNA sequences.

碩士
國立中山大學
化學系研究所
98
The study for surfaced-immobilized nucleic acid probes in nanometer region in response to hybridization and to discrimination ofdifferent target nuclei acids. The hairpin locked nucleic acid (LNA-HP) isselected to be the probe molecule, and target molecules include perfect complementary (PC) and single mismatch (1MM). The self-assembledLNA-HP molecular nanospot is successfully prepared by liquid phaseAFM (Atomic Force Microscope)-based nanolithography technique, then in situ hybridization is carried out by using different targets (PC/1MM).To obtain the information of structure change, we use AFM to analyze therelative heights in the process of hybridization. The experimental results point out that (1) the structure changes of surface probe molecules maycorrelate with the AFM signal when target sequence hybridizes to the probe, (2) miniaturization of the size of the nucleic acid probe may promote hybridization efficiency and enhance the discrimination between PC and 1MM. Studies on whether the different chemical impetus in solution can affect conformation of the human telomeric DNA of sequence is conducted. A human talomeric DNA composed of ( 5’-TTAGGG-3’:5’-CCCTAA-3’ ) repeats, with a 100-200 nt ( T2AG3 ) repetitive unit overhang at 3’ ends is chosen. This extended single-stranded sequence is called G-rich DNA, which forms the special G-quadruplex structure in solution containing sodium ions or potassium ions. The single-stranded sequence composed of ( C3TA2 ) repetitive units called C-rich DNA displays the i-motif folded structure in the low pH environment. These biomimetic DNA’s are thiol-modified to self-assemble on gold surfaces. Separate measurements with AFM (the molecular thickness and rootmean- square roughness of the self-assembly monolayer of DNA ) and CD( circular dichroism ) ( structure characterization ) confirm the conformational changes of G-rich and C-rich DNA’s on gold surface are indeed dependent of the presence of cations and protons.

A number of studies have established that DNA targeting is a successful strategy in anticancer therapy. In recent years, however, the focus has shifted from double-stranded DNA to alternative DNA motifs such as G-quadruplex and i-motif structures that are often found in the telomeric and transcriptional regulatory regions of genes in almost all eukaryotic organisms. The mutually exclusive formation of G-quadruplex and i-motif structures by G/C rich sequences, which in turn impact a variety of DNA transactions, suggest that stabilization of such structures by small molecules offer alternative options for the treatment of genetic and life-style diseases. Indeed, a large number of G-quadruplex stabilizing ligands have been extensively studied for their anti-cancer activity both in vitro and in vivo. Acetyl-CoA carboxylase catalyses the ATP-dependent carboxylation of cytosolic acetyl-CoA to malonyl-CoA. The synthesis of malonyl-CoA is the first committed step for de novo fatty acid biosynthesis pathway. Numerous lines of evidence suggest that the expression and specific activity of acetyl-CoA carboxylase is highly regulated at transcriptional, translational, and post-translational levels. The expression of human acetyl-CoA carboxylase 1 gene (ACC1) is regulated by three alternative promoters (PI, PII, and PIII), but all three promoters produce the same protein coding sequence. Notably, promoter 1 and 2, but not promoter 3, harbour G/C-rich cis-elements whose secondary structure and function remain unknown. In the present study, using multiple complementary methods such as CD spectroscopy, FRET, electrophoretic mobility shift assay, chemical foot printing and computational methods we show that G-rich cis-elements of PI and PII promoters fold into thermodynamically stable G-quadruplex structures, and then establish unambiguously the topologies of these structures. Furthermore, we found that PI promoter folds into two distinct G-quadruplex structures with 1:1:1 loop arrangement, while PII promoter folds into 1:4:1 loop arrangement. Human nucleolin, a conserved major nuclear protein is known to regulate gene expression through interaction with G/C-rich sequences in the genome. We show that nucleolin binds to G-quadruplex DNA formed by ACC1 promoters with high specificity and in a dose-dependent manner. Most importantly, G-quadruplex formation in ACC1 gene promoter region blocks DNA replication, suppresses transcription, and this effect was further augmented by G-quadruplex stabilizing ligands. Taken together, these results not only demonstrate the existence of G-quadruplex structures in ACC1 promoters, but also attest to their functional significance. The formation of a G-quadruplex structure within a genomic duplex DNA region requires the separation of the G-rich strand from its complementary C-rich strand. As our study revealed that the G-rich sequences in ACC1 promoters, PI and PII, fold into G-quadruplex DNA, then the question arises whether the C-rich strand folds into i-motif structures. Using multiple complementary methods such as CD spectroscopy, FRET and electrophoretic mobility shift assay, we show that the C-rich sequences of PI and PII promoters fold into intramolecular imotif structures. The results of chemical foot printing assays indicated that whereas PI promoter folds into two distinct i-motif structures with 2:2:2 loop arrangements, PII promoter folds into 2:3:2 loop arrangement. Consistent with the significance of I-motif structures in the cellular context, we found that molecular crowding agents abet the formation of I-motif structures. These findings were ascertained by luciferase reporter activity assay. The data revealed that the C-rich sequence complementary to the G-rich sequence in ACC1 promoters markedly attenuated luciferase expression. The attenuated activity of the reporter could be unleashed by mutations in the C-rich sequence of ACC1 promoters. Several classes of small molecules that selectively stabilize G-quadruplex structures over duplex DNA are known. G-quadruplexes adopt a wide range of conformations and thus the ligands that can stabilize one structure may not stabilize others. Previously, we have shown that benzimidazole-carbazole conjugates selectively bind and stabilize the telomeric G quadruplex structures over B-form DNA and inhibit telomerase activity and proliferation of human cancer cells. In addition, our previous studies have shown that these ligands induce topological conversion from non-parallel to parallel forms in the human telomeric G quadruplex structure. In the present study, we have investigated the interaction of bezimidazole-carbazole ligands with c-MYC, c-KIT1, c-KIT2, VEGF and BCL2 promoter G quadruplex structures. CD measurements suggested that ligands induce topological changes from hybrid to stable parallel G-quadruplex DNA. Our CD melting and FID assays revealed that these ligands show higher affinity and confers stability to promoter G-quadruplexes. Further, to investigate the probable modes of binding of the ligands to various G4 DNAs at the promoter and telomeric regions, we have performed the docking studies, and which shows ligands interacts with promoter G-quadruplex. Overall, this study suggests that ligands that are earlier shown to interact with telomeric G-quadruplex DNA also stabilize promoter G-quadruplexes. The development of ideal anti-cancer therapies that are highly efficient and exhibit minimal systemic toxicity is an important research area. The pro-drug approach has generated significant interest for selective targeting of cancerous cells. Photodynamic therapy (PDT) is one of the novel pro-drug approaches that involve activation of the pro-drug by a light stimulus. PDT involves light and a photosensitizer (PS) that in conjunction with molecular oxygen elicits cell death. A large number of ruthenium complexes have been examined for their DNA binding properties and photo-reactivities. In the present study, we have synthesized and characterized four new ruthenium azo-8-hydroxyquinoline complexes, their DNA binding properties and anticancer activities. Our studies revealed that these complexes can be stimulated by visible light to induce ROS mediated DNA photocleavage activity in a cellular environment. Interestingly, these complexes display potent cytotoxic activity in cancer cells, which is further augmented by exposure to visible light. Thus, we propose that the new Ru-complexes have the potential to be used in photodynamic therapy and as anticancer agents. Thus, the current work not only provides new insights into the regulation of ACC1 gene expression, but also identified a promising set of ruthenium metal complexes.

abstract: The communication of genetic material with biomolecules has been a major interest in cancer biology research for decades. Among its different levels of involvement, DNA is known to be a target of several antitumor agents. Additionally, tissue specific interaction between macromolecules such as proteins and structurally important regions of DNA has been reported to define the onset of certain types of cancers. Illustrated in Chapter 1 is the general history of research on the interaction of DNA and anticancer drugs, most importantly different congener of bleomycin (BLM). Additionally, several synthetic analogues of bleomycin, including the structural components and functionalities, are discussed. Chapter 2 describes a new approach to study the double-strand DNA lesion caused by antitumor drug bleomycin. The hairpin DNA library used in this study displays numerous cleavage sites demonstrating the versatility of bleomycin interaction with DNA. Interestingly, some of those cleavage sites suggest a novel mechanism of bleomycin interaction, which has not been reported before. Cytidine methylation has generally been found to decrease site-specific cleavage of DNA by BLM, possibly due to structural change and subsequent reduced bleomycin-mediated recognition of DNA. As illustrated in Chapter 3, three hairpin DNAs known to be strongly bound by bleomycin, and their methylated counterparts, were used to study the dynamics of bleomycin-induced degradation of DNAs in cancer cells. Interestingly, cytidine methylation on one of the DNAs has also shown a major shift in the intensity of bleomycin induced double-strand DNA cleavage pattern, which is known to be a more potent form of bleomycin induced cleavages. DNA secondary structures are known to play important roles in gene regulation. Chapter 4 demonstrates a structural change of the BCL2 promoter element as a result of its dynamic interaction with the individual domains of hnRNP LL, which is essential to facilitate the transcription of BCL2. Furthermore, an in vitro protein synthesis technique has been employed to study the dynamic interaction between protein domains and the i-motif DNA within the promoter element. Several constructs were made involving replacement of a single amino acid with a fluorescent analogue, and these were used to study FRET between domain 1 and the i-motif, the later of which harbored a fluorescent acceptor nucleotide analogue.
Dissertation/Thesis
Doctoral Dissertation Chemistry 2014

Helicobacter pylori, a human pathogen dominating gastric microbial population, displays differential gene expression during various stages of stomach colonization. Topoisomerases play a crucial role in maintaining DNA superhelicity and therefore gene expression. H. pylori has only two topoisomerases: DNA gyrase and Topoisomerase I, as opposed to four in most other prokaryotes. The current study focuses on studying the biochemical and mechanistic details of H. pylori Topoisomerase I (HpTopoI) catalysis. Sequence comparison of HpTopoI with EcTopoI shows the presence of four zinc finger motifs (ZFs) at the carboxyl terminal domain (CTD) unlike three in EcTopoI. To understand the role of ZFs in HpTopoI function, the ZFs were sequentially deleted from the carboxyl terminus. It was observed that the third and fourth ZFs are dispensable for HpTopoI function. DNA relaxation activity was hampered considerably when only one zinc finger (ZF1) was present. Deletion of all ZFs, however, drastically reduced DNA binding and abolished DNA relaxation. These results highlight the importance of ZF1 in catalyzing the relaxation of DNA. Furthermore, the annotated active site tyrosine residue in HpTopoI when mutated to phenylalanine retained its DNA relaxation activity. Intriguingly, the CTD HpTopoI alone (all four ZFs) could relax the supercoiled DNA although with a specific activity 8.28 fold less than the WT. Gel filtration chromatography analysis suggests that the CTD occurs as dimer in solution and has an ability to form multimers as shown by glutaraldehyde crosslinking. The amino terminal ‘Toprim’ domain houses an acidic triad DxDxE which co-ordinates a Mg2+ cation that is indispensable for the re-ligation activity during the DNA relaxation process. The deletion of Toprim domain or the triple mutation of the acidic triad residues DxDxE to AxAxE reduced the DNA relaxation activity by 14 fold and 9.5 fold, respectively. HpTopoI has a total of 31 tyrosines. An HpTopoI mutant with all the tyrosines mutated to phenylalanine was over-expressed and purified. This mutant is completely inactive which reinstates the fact that it is indeed tyrosine(s) which bring out the nucleophilic attack on the DNA which is essential for DNA relaxation. A comprehensive analysis of 77 fully sequenced strains of H. pylori revealed the presence of multiple copies of HpTopoI. Out of 77 strains, H. pylori strain XZ274 has emerged as a unique paradigm. This strain was isolated from a gastric cancer patient from Tibet. It has 3 genes annotated as TopoI. This strain lacks a FL copy of HpTopoI. Intriguingly, one of the variant is 317 bp in length encodes for protein with only two zinc fingers. Topoisomerases have been known to interact with several proteins involved in replication, transcription and recombination. In H. pylori, DprA is a crucial protein involved in natural transformation. This study shows that HpDprA stimulates HpTopoI activity at a lower concentration and interacts physically with HpTopoI as demonstrated by SPR and microscale thermophoresis. This indicates a possible role of HpTopoI during the process of natural transformation. Taken together, this study reports the biochemical characterization of HpTopoI and sheds light on the unusual role of zinc finger motifs in enzyme catalysis. Analysis of redundant TopoI copies across several H. pylori strains corroborates with the in vitro results that HpTopoI CTD alone can function as a DNA relaxase

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.