The binding of different proteins involved in processes such as transcription, replication and chromatin compaction to regions of the genome is regulated by the structure of DNA. Thus, DNA structure acts as the crucial link modulating evolutionary selection of the DNA sequence based on its own function, and the function of the proteins it encodes. The aim of this work is to examine the role of intrinsic, sequence-dependent structural variations vis-a -vis the protein-induced variations, in allowing DNA to assume geometries necessary for binding by proteins. For this purpose, we carried out analyses of datasets of X-ray crystal structures of free and protein-bound DNA, and molecular dynamics simulation studies of few free DNA structures and a protein-DNA complex. Each of the projects described below will appear as a separate chapter in the thesis. Analysis of X-ray crystal structure datasets Dataset of high-resolution X-ray crystal structures of free and protein-bound DNA This project was initiated with the aim of investigating the variation in A-and B-forms of DNA and the role they play in the binding of proteins. However, a survey of the existing literature indicated that the terms ‘A-DNA’ and ‘B-DNA’ were being used rather loosely and several different parameters at the local structural level were being used by various investigators to characterise these structures. Hence a systematic study was taken up to analyse all high-resolution free DNA structures comprising of sufficient number of contiguous Watson-Crick basepairs, irrespective of how they were classified by the existing databases. We also carried out a study of double-helical, Watson-Crick basepaired, free RNA structures for comparison. The structures in the RNA dataset were observed to rigidly assume the A-form and hence the average values of different parameters for that dataset were used to characterise the A-form. The analysis of free DNA and RNA structures was accompanied by an analysis of protein-bound DNA crystal structures. DNA structures bound to the helix-turn-helix motif in proteins were also analysed separately. The analysis of free DNA and RNA structures allowed us to pinpoint the parameters suitable for discriminating A-and B-forms of DNA at the local structural level. The results illustrated that the free DNA molecule, even in the crystalline state, samples a large amount of conformational space, encompassing both the A-and the B-forms. Most protein-bound DNA structures, including those with large, smooth curvature, were observed to assume the B-form. The A-form was observed to be limited to a small number of dinucleotide steps in DNA structures bound to the proteins belonging to a few specific families. Thus our study highlighted the structural versatility of B-form DNA, which allowed it to take up a range of global geometries to accommodate most DNA-binding protein motifs. Dataset of X-ray crystal structures of the nucleosome The study of high-resolution structures of free and protein-bound DNA was followed by an analysis of a dataset of X-ray crystal structures of the nucleosome, which is the fundamental repeating unit of the eukaryotic chromosome, and has been shown to play an important role in transcription regulation. Our results indicated that there is an ensemble of dinucleotide and trinucleotide level parameters that can give rise to similar global nucleosome structures. We therefore raise doubts about the use of the best resolved nucleosome structure as the template to calculate the energy required by putative nucleosome-forming sequences for adopting the nucleosome structure. Based on our results, we have proposed that the local and global level structural variability of DNA may act as a significant factor influencing the formation of nucleosomes in the vicinity of high-plasticity genes, and in determining the probability of binding by regulatory proteins. Molecular dynamics simulation studies of free and protein-bound DNA structures The analysis of crystal structure databases was complemented by molecular dynamics (MD) studies to investigate the dynamic evolution of the DNA structure in its free and protein-bound states. The following three simulation studies were carried out: Study to examine the biological relevance of the presence of 5-methyl group in thymine nucleotides An investigation of the biological relevance of the 5-methyl group in thymine nucleotides was carried out. For this purpose, comparison of molecular dynamics studies on structures with sequences d(CGCAAAUUUGCG)2and d(CGCAAATTTGCG)2was carried out. Our results showed that the presence of the thymine 5-methyl group was necessary for the A-tract to assume characteristic properties such as a narrow minor groove. It was also shown to modulate local level structural parameters and consequently, the curvature of the longer DNA fragment in which the A-tract was embedded. The analysis also provided possible explanation for the experimentally observed interaction of A-tracts with drugs and DNase-I in the presence and the absence of the thymine 5-methyl group. This project was the first of a series of MD studies, and hence several protocols were tested before finalising the correct protocol. Simulations were carried out using the Berendsen temperature equilibration scheme as well as the Langevin temperature equilibration scheme on both the structures. The Langevin temperature equilibration scheme was found to be unsuitable for nucleic acid simulations, as it caused long-term and possibly permanent disruption of the double-helical structure at the terminal and the neighbouring two positions in the sequence. The Berendsen temperature equilibration scheme was not observed to cause such disruptions. Simulations were also carried out on both structures, with or without initialising the initial ion positions. The position of minimum electrostatic potential, where AMBER8 placed the first counterion, was observed to act as a minimum energy trap from which the counterion could not escape even during the course of several nanoseconds of simulation. Hence, the actual simulations were carried out using the Berendsen temperature equilibration scheme, and after randomisation of initial ion positions. The results of protocol testing have been reported in an appendix. Study of DNA bending and curvature An analysis of DNA bending and curvature was carried out, by MD simulation on structures of three, ∼thirty basepair long sequences, namely, d(G-3(CA4T4G)-C)2, d(G-3(CT4A4G)-C)2and d(T-GACTA5T-GACTA6T-GACTA5T-G). For each molecule, snapshots belonging to a particular global geometry (linear, curved, bent in a particular direction etc.) were grouped together, and the average values of the dinucleotide step parameters for different groups were compared. It was observed that for all the three molecules, the average values for groups corresponding to different global geometries were within 1of each other, indicating that ensemble average values of dinucleotide level parameters are incapable of predicting the global geometry of a DNA molecule. Study of the TraR-Trabox complex The study on DNA bending and curvature was followed by simulations of a protein-DNA complex comprising of the bacterial quorum sensing transcription factor TraR with its promoter region known as Trabox. Simulations of a protein-free wild-type Trabox and a Trabox with two mutations in the spacer region were also carried out. Grouping of DNA snapshots in all the three simulations based on average values of dinucleotide parameters in the spacer region shows how selection of the ‘right’ DNA geometry by proteins works at several levels. The number of snapshots of free mutated Trabox assuming a geometry favourable for protein-binding in terms of average twist alone are less than one-fourth of the corresponding number for free wild-type Trabox. When one applies further selection criteria in terms of other parameters such as roll and slide, the number of mutated Trabox snapshots with a geometry favourable for protein-binding drops to less than 0.5%ofthe total number of MD snapshots. Thus our results highlight how sequence-dependent changes in the structrure of DNA regions, adjacent to those that directly hydrogen-bond to proteins, can also critically influence processes such as transcription. General Conclusion Overall, our results indicate that intrinsic, sequence-dependent structural variations in free B-DNA allow it to sample a large volume of the double-helical conformational space, and assume global geometries that can accomodate most DNA-binding proteins.

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

Understanding the molecular structure of DNA was considered as greatest achievement in modern biology. It helped in understanding fundamental cellular processes such as replication of DNA, nature of the genetic code and transcription. It also led to technological advancements such as DNA sequencing, genetic engineering and gene cloning. The DNA molecule is highly polymorphic in nature and its structure is dependent on environment, base composition and sequence context. B-DNA, A-DNA, Z-DNA and curved or kinked DNA are some of the well characterized double helical polymorphs. B-DNA is the most prevalent structure in vivo and it can undergo small local variations and global variations. In this thesis we refer to distinct structural property of any particular DNA sequence as deviation from fibre model B-DNA structural parameters or random sequence DNA. Structural properties of DNA are an outcome of the linear arrangement of the 4 chemically different nucleotide bases and the characteristic features of the two grooves (minor and major) arising due to the asymmetric position of glycosidic bonds of base pairs. DNA structure and properties are expected to vary along its length. Several structural features have been defined for DNA duplex, while DNA stability, bendability and intrinsic curvature are well studied and found to be biologically relevant. These three sequence dependent properties differ in their nature and information content and can be studied both at local and global levels, depending on the length of DNA fragment being examined. Majority of the work in this thesis focuses on the analysis of these three DNA structural features in promoter regions of different eukaryotic systems and their relationship with gene expression. The thesis work is divided in to five sections briefly described below. The sections discuss prevalence of the three structural features, DNA stability, bendability and intrinsic curvature in the promoter regions of six eukaryotic systems namely S. cerevisiae, D. melanogaster, C. elegans, zebrafish, mouse and human. The relationship between DNA structural features of promoter regions of S. cerevisiae with gene expression variability is discussed, followed by application of the structure-based promoter prediction algorithm ‘PromPredict’ in annotating promoter regions of six different eukaryotes. Finally, an analysis of structural features of the flanking sequences of transcription factor binding sites (TFBSs) of six transcription factors and their relationship with the DNA binding affinity is discussed. Each of the projects described below will appear as a separate chapters in the thesis. An overview of the eukaryotic transcription machinery, promoter elements and different DNA structural properties are discussed in the introduction of the thesis (chapter 1). The structural properties of DNA in the promoter regions of eukaryotic genes (chapter 2)Earlier studies in the lab reported that, apart from sequence motifs, promoter re- gions have distinct structural properties, such as lower stability, lesser bendability and more curvature compared to other genomic regions. But those studies were on small datasets and few model systems. Advancement in high-throughput tech- niques has made availability of transcription start site information for many model systems. This work was initiated with the aim of investigating the structural fea- tures in different eukaryotic systems belonging to different domains of life. The quantitative analysis of three different structural features of promoter regions of six different model systems S. cerevisiae, C. elegans, D. melanogaster, zebrafish, mouse and human has been carried out. Further, the composition of different k-mers (k=3, 4 and 6) A-tracts and G-quadruplexes has been studied. The analysis allowed us to understand the similarities and differences in struc- tural features of promoter sequences in different model systems. The core promoter sequences of S. cerevisiae, C. elegans, D. melanogaster, zebra fish, mouse and hu- man have been observed to be less stable and have lower preference for nucleosome formation. S. cerevisiae, C. elegans and D. melanogaster promoter sequences have been shown to be less bendable whereas zebrafish, mouse and human promoter se- quences are flexible in terms of bendability towards major groove as predicted fDNase 1 sensitivity model. S. cerevisiae, C. elegans, D. melanogaster core promoter regions have AT rich oligomers, whereas mouse and human core promoter regions have GC rich oligomers and G-quadruplex motifs. DNA structural features of TATA-containing andTATA-less promoters (chapter 3)Eukaryotic genes can be broadly classified as TATA-containing and TATA-less based on the presence of TATA-box in their promoter sequences. Experiments on both classes of genes have reported that, they have differences in regulation of gene ex- pression and cellular functions. In this chapter, the differences in compositional and structural features of TATA-containing and TATA-less promoters in the above mentioned model systems are discussed. The results suggested that DNA structural features of TATA-containing and TATA-less promoters are distinctly different in all eukaryotes. The TATA-containing promoters are less stable, more flexible and more curved compared to TATA-less promoters in lower eukaryotes. In mouse and hu- man genes, DNA duplex stability and G-quadruplex motifs are very distinguishing features in the two classes of promoters. DNA structural properties of eukaryotic promoter regions and gene expression variability (chapter 4) Gene expression is regulated by various external (environment and evolution) and internal (genetic) factors. Presence of sequence motifs, such as TFBSs and TATA- box, as well as DNA methylation has been implicated in the regulation of expression of some genes in vertebrates, but a large number of genes lack these sequences. Ear- lier analyses (described in previous sections) in S. cerevisiae, have shown that their promoter sequences have special structural properties, such as low stability, less bendability and more curvature compared to other genomic regions. These strutural features may play a role in transcription initiation and regulation of gene expression. This project was carried out to understand 1. What is the relationship between DNA structural features and gene expres- sion? 2. What is the relationship between gene expression and bidirectionality of a pro- moter region? For this purpose, the information of seven different gene expression variability measures, stochastic noise, responsiveness, stress response, trans variability, mu- tational variance, interstrain variation and expression divergence have been com- pared with structural features in the promoter regions. It is observed that a few of the variability measures of gene expression are linked to DNA structural prop- erties, along with nucleosome occupancy, TATA-box presence and bidirectionality of promoter regions. Interestingly, gene responsiveness is shown to be most, inti- mately correlated with DNA structural features and promoter architecture. The study highlights the importance of sequence dependent structural features in gene regulation. Promoter prediction in eukaryotes using DNA duplex stability (chapter 5) Structural property-based algorithms can discriminate promoter sequences from non-promoter sequences and are far better than sequence motif-based predictors. Compared to other structural features, low stability is found to be the most preva- lent feature in promoter regions. “PromPredict” (in-house algorithm) uses the din- ucleotide free energy values obtained from differential melting stability of DNA du- plexes as a predictor of promoters and has been successfully used earlier to annotate promoter sequences in prokaryotes and rice. Comprehensive analysis of the perfor- mance of PromPredict in S. cerevisiae, D. melanogaster, C. elegans, zebrafish, mouse and human as well as TATA-containing and TATA-less promoter regions of S. cere visiae with TSS data and 48 eukaryotic systems with translation start site (TLS) data revealed that differential stability is a good criterion for promoter prediction. DNA structure in flanking sequences of consensus motifs modulate transcription factor binding (chapter 6) Sequence specific DNA-protein interactions are essential for specific expression pat- terns during the development. There are several factors contribute to DNA-binding specificities of transcription factors (TFs). They include structure and flexibility of TFs, cofactors, chromatin environment and DNA sequence. Along with actual tran- scription factor binding sites (TFBSs), their sequence context (flanking sequences) is also shown to play a major role in gene regulation. Most of the studies have ad- dressed the sequence context at global level but very little is understood about the role of sequences flanking TFBSs in binding of transcription factors. This project was initiated with the aim of understanding the effect of flanking sequences of TFBSs in transcription factor binding affinity. In vitro DNA binding information of six different transcription factors (with three types of DNA bind- ing domains, Zinc finger (GATA4), home domain (AbdA, AbdB and Ubx) and bZIP (fos-jun and Nfil3)) was provided by Aseem Ansari’s lab. The compositional and structural features (minor groove width, propeller twist, wedge and free energy) are compared with the DNA binding profiles of 12mers (or 8mers) of six different transcription factors. It has been observed that some of the DNA structural proper- ties of flanking sequences are strongly correlated with binding affinity. For GATA4 sequences, binding affinity is negatively correlated to GC content or minor groove width at their 5′ -flanking region, showing the significance of narrow minor groove at 5′ -region. On the other hand, the binding affinity of bZIP proteins is negatively correlated to wedge angles, whereas in case of homeodomain proteins, it is posi- tively correlated to propeller twist and GC content. Thus, this study highlights the differential preference for flanking sequences outside the core binding motifs of six different TFs, which interact with DNA through α-helix. ‘The relationship between transcription pre-initiation complexes and gene ex- pression variability in S. cerevisiae’ is briefly described in the appendix section of the thesis. General conclusion Overall, the results presented in this thesis indicate that DNA sequence based structural features are unique to promoter regions and play an important role in gene regulation. Local structural features of flanking sequences of transcription factor binding sites are also instrumental in determining the DNA binding affinity of transcription factors.

Formés à partir de brins d’ADN ou d’ARN riches en guanines, les quadruplexes résultent de l’empilement de tétrades de guanines constituées chacune par l’auto-assemblage dans un même plan de quatre guanines, stabilisées entre elles par un réseau de liaisons hydrogènes. En s’inspirant de cet édifice naturel, il est présenté au long de ce manuscrit de thèse la synthèse et l’étude de molécules de type TASQ (pour template-assembled synthetic G-quartet) hydrosolubles capables de former de manière intramoléculaire une tétrade de guanines synthétique : les DOTASQ, le PorphySQ et le PNADOTASQ. La première application développée pour ces composés est le ciblage des quadruplexes d’ADN et d’ARN, présents dans des régions clefs du génome (télomères, promoteurs d’oncogènes) et du transcriptome (5’-UTR et TERRA), et dont la stabilisation par un ligand pourrait ouvrir de nouvelles perspectives en terme de thérapie antitumorale ciblée. Les résultats in vitro sont présentés et permettent de démontrer que les TASQ hydrosolubles développés sont des composés offrant une bonne sélectivité pour les quadruplexes mais surtout une excellente sélectivité grâce à un mode d’action bioinspiré basé sur une reconnaissance biomimétique. La seconde application mise au point est l’utilisation des TASQ comme catalyseurs pour des réactions de peroxydation : leur architecture même leur permet de mimer l’activité catalytique de l’ADN (ou DNAzyme) ainsi que celle de protéines (enzyme) comme la horseradish peroxidase. Ce processus est dépendant de la formation intramoléculaire de la tétrade de guanines synthétique et ouvre de nombreuses perspectives en terme d’utilisation en biologie ainsi qu’en nanotechnologie. / Natural G-quartets, a cyclic and coplanar array of four guanine residues held together via Hoogsteen H-bond network, have recently received much attention due to their involvement in G-quadruplex-DNA, an alternative higher-order DNA structure strongly suspected to play important roles in key cellular events (chromosomal stability, regulation of gene expression). Besides this, synthetic G-quartets, which artificially mimic native G-quartets, have also been widely studied for their involvement in nanotechnological applications (i.e. nanowires, artificial ion channels, etc.). In contrast, intramolecular synthetic G-quartets, also named template-assembled synthetic G-quartet (TASQ), have been more sparingly investigated, despite a technological potential just as interesting.In this way, we designed and synthesized three series of innovative hydrosoluble TASQ: DOTASQ (for DOTA-Templated Synthetic G-Quartet), PorphySQ (containing a porphyrin template) and the most effective PNADOTASQ where PNA-guanine arms replace native DOTASQ alkyl-guanine arms. We report herein the results of both DNA and RNA interactions (notably their selective recognition of quadruplex-DNA according to a bioinspired process) and peroxidase-like hemin-mediated catalytic activities (either in an autonomous fashion as precatalysts for TASQzyme reactions, or in conjunction with quadruplex-DNA as enhancing agents for DNAzyme processes). These results provide a solid scientific basis for TASQ to be used as multitasking tools for bionanotechnological applications.