Studies on novel structural motifs using synthetic oligodeoxyribonucleotides

Abstract

The double helical structure of DNA proposed by Watson and Crick was once thought to be rigid and monotonous in structural terms. Although the inherent conformational flexibility in DNA was envisioned through theoretical and fiber diffraction studies about two decades ago, it was only in the last decade that our concepts and knowledge about DNA structure have witnessed dramatic changes. This shift was largely possible due to advancements made in several diverse areas of research, such as the development of synthetic oligonucleotide chemistry, oligonucleotide crystallography, recombinant DNA technology, and the emergence of non-conventional methods such as gel electrophoresis, and chemical and enzymatic probing, which became essential tools for studying DNA structure. Consequently, it became increasingly evident that the structure of DNA could deviate significantly from the ideal right-handed B-conformation to form a variety of unusual structures depending on the primary base sequence, environmental conditions, and protein interactions. As a result, sequence-dependent structural variation in DNA has become a rule rather than an exception. Among the unusual DNA structures identified so far, left-handed Z-DNA, bent DNA, cruciforms, intra- and intermolecular triple helices, and quadruplexes constitute the most prominent classes of structures formed by specific sequence motifs. The unusual structures characterized in earlier studies, which were thought to be interesting purely from a conformational standpoint, have gained further attention following the recent realization that such structures could be formed in vivo by repetitive sequences found in genomic DNA. Therefore, investigating the structural properties, sequence requirements, and stability of unusual DNA structures became of significant interest. In this thesis, studies on two novel DNA structural motifs—B-Z junctions and guanine quadruplexes—are presented. At the time of the author's joining the laboratory, there were research programs aimed at understanding the structure-function relationship of repetitive DNA sequences and sequence-specific structural alterations in DNA. It was well documented that alternating purine-pyrimidine sequences such as (CG)n and (TG)n undergo a transition from the right-handed B-conformation to a left-handed Z-conformation. Since long stretches of CG repeats are not ubiquitous in genomic DNA [Tripathi & Brahmachari (1991), J. Biomol. Struct. Dyn. 9, 387], the formation of left-handed Z-conformation in GC-rich sequences in vivo was thought to depend on the possibility of stabilizing such a structure in short stretches and accommodating them within an overall B-DNA conformation. This would lead to the formation of B-Z junctions at either end of the Z-forming segment. Alternating right-handed B and left-handed Z conformations at every half-turn of the helix with sharp B-Z junctions were shown to be stable in the linear polynucleotide, as well as in a circular plasmid under negative supercoiling [Brahmachari et al. (1989), Nucl. Acids Res. 17, 7273]. Studies on highly supercoiled, zero-link pBR322 form V-DNA carried out in our laboratory using restriction endonucleases and methylases as structural probes revealed that the Z-DNA conformation is not propagated over long stretches of sequence, and such non-B conformations are interrupted by B-DNA conformations [Brahmachari et al. (1987), J. Mol. Biol. 193, 201; Shouche et al. (1990), Nucl. Acids Res. 18, 267]. These studies also indicated that the same Z-potential sequence adopts a non-B conformation depending on the neighboring or junction sequences. The widespread occurrence of short stretches of the most Z-potential sequence (CG)n, where n = 3-5, in both prokaryotic and eukaryotic genomes, prompted us to investigate the effect of junction/neighbouring sequences on the ability of the CGCGCG sequence to adopt the left-handed Z-conformation. In this context, we addressed the following questions: (a) What is the sequence criteria for B-Z junction formation? (b) Is it possible to predict the ability of a CGCGCG sequence to adopt the Z-conformation depending on the flanking/junction sequences? (c) Is it possible to design duplexes that can form such paranemic B-Z structures in the promoter region to study their effect on RNP recognition? In order to find plausible answers to these questions, a series of synthetic oligonucleotides were synthesized, and structural studies using circular dichroism (CD) and enzymatic probing were undertaken. The results of these investigations are presented in Part I of the thesis. Chapter 1 provides a brief account of the major advances made in our understanding of DNA structure. It also reviews existing knowledge regarding unusual DNA structures, with a special focus on left-handed Z-DNA and G-quadruplexes, as well as their probable biological implications. Chapter 2 describes the design, synthesis, and structural studies carried out on a series of oligonucleotides: d(CGCGCGGAATTC), d(CGCGCGGGGCCC), d(CGCGCGATCGAT), d(CGCGCGAGGCCT), d(CGCGCGAAGCCT), d(CGCGCGTACGTA), and d(CGCGCGCATATG). These sequences were designed to understand the effect of flanking sequences on the ability of CGCGCG to adopt the Z-conformation. The sequences were intended to (1) form long concatameric duplexes in solution with alternating Z- and B-helicogenic segments, and (2) include B-helicogenic domains with hexa-cutter restriction enzyme sites for enzymatic probing of the polymer structure. Ultraviolet (UV) melting studies and the ability of these oligonucleotides to form polymers when ligated established that these sequences form stable concatamers in solution. Circular dichroism studies indicated that the oligonucleotides d(CGCGCGGGGCCC), d(CGCGCGAGGCCT), d(CGCGCGGAATTC), and d(CGCGCGATCGAT) adopt a B-Z structure in 5 M NaCl. Circular dichroism salt titrations suggested that the ability of the CGCGCG segment to adopt the Z-conformation varies depending on the flanking sequences. It was found that the propensity of CGCGCG to adopt the Z-conformation is higher when flanked by RRRYYY sequences. The Z-helicogenic CGCGCG domain in the oligonucleotides d(CGCGCGTACGTA) and d(CGCGCGCATATG), despite being flanked by perfectly alternating purine-pyrimidine sequences, did not undergo the B?Z transition with NaCl alone. However, these two oligonucleotides exhibited a cooperative B?Z transition under the combined influence of 5 M NaCl and millimolar concentrations of NiCl2. Ni2+ is known to stabilize the syn conformation of guanines by coordinating to N7 of purines. On the other hand, the CGCGCG segment in the oligonucleotide d(CGCGCGGAATTC), containing an identical number of AT base pairs as the above two sequences, assumes the Z-conformation in the presence of NaCl alone. Most strikingly, the oligonucleotide d(CGCGCGAAGCTT) did not adopt the Z-conformation even in the presence of 5 M NaCl and millimolar concentrations of NiCl2. These results strongly suggest that the ability of the shortest Z-potential sequence (CGCGCG) to adopt the Z-conformation depends primarily on the junction sequences and not solely on the AT content. Chapter 3 discusses probing the structure of these polymers with restriction enzymes. Structure sensitivity of restriction enzymes was exploited to probe the structure of polymers obtained by block ligation of the oligonucleotides used in the previous chapter. Hexamminecobalt(III) chloride (HCC), which is known to stabilize the Z-conformation in (CG)n sequences, was used to induce B-Z junction formation in the polymers, as restriction enzymes remain active in the presence of HCC. Digestion patterns obtained with Hha I (GCGC), which has a site in the Z-helicogenic CGCGCG region, and with the restriction enzymes Apa I, Eco RI, Hae III, Nde I, Sna BI, and Hind III, specific to the B-helicogenic domain of the polymers in the presence and absence of HCC, suggested the formation of multiple B-Z junctions in the polymers d(CGCGCGGGGCCC)n, d(CGCGCGGAATTC)n, d(CGCGCGAGGCCT)n, and d(CGCGCGCATATG)n, induced by HCC. These results, in conjunction with the previous findings, allowed us to arrive at the following hierarchy of DNA sequences that favor the formation of B-Z junctions: GGG > GAG > GGA > GAT > GCA, GTA > GAA. This information was utilized to design a synthetic duplex with short stretches of Z-potential sequences in the promoter region to study the effect of such B-Z structures on RNP recognition. Part II of the thesis deals with another class of unusual DNA structures, namely guanine quadruplexes formed by telomeric sequences. Most strikingly, unlike Z-DNA and other unusual DNA structures, guanine quadruplex structures are stabilized solely by non-Watson-Crick Hoogsteen-type hydrogen bonding interactions. Moreover, among unusual DNA structures, the quadruplex is the second structure that has been visualized at atomic resolution. The crystal structure of an antiparallel hairpin dimer formed by 1.5 repeats of the Oxytricha telomeric sequence d(G4T4G4) was solved by Rich and coworkers [Kang et al., Nature (1992) 356, 126] during the course of the investigations described in Part II of the thesis. Blackburn and coworkers [Blackburn & Szostak (1984), Ann. Rev. Biochem. 53, 163] discovered that the physical ends of the chromosomes of Tetrahymena contain specialized sequences repeated tandemly several times, with one strand being rich in guanines and the complementary strand in cytosines. Subsequently, telomeric sequences of several other eukaryotes, including humans, were determined and found to fall under a subset consensus, d(Ti_3T/AG3_4). The occurrence of tandem repeats with stretches containing contiguous guanines was found to be a conserved feature in most eukaryotic telomeric DNA, indicating a common structural element involved in telomere function. At the onset of the work described in Part II, the telomeric G-rich strand was known to exhibit unusual cohesive properties [Henderson et al. (1987), Cell 51, 899]. Sen and Gilbert [Nature (1988) 334, 364] proposed a parallel four-stranded structure for the G-cluster sequences in the immunoglobulin switch region, and an antiparallel hairpin dimer was suggested for the Tetrahymena telomeric repeat d(T2G4)2 by Sundquist and Klug [Nature (1989) 342, 825]. On the other hand, an intramolecular G-quartet structure was deduced for d(T4G4)4 by Cech and coworkers [Williamson et al. (1989), Cell 59, 871]. However, the sequence requirement for the formation of parallel and antiparallel G-quartet structures was not clear. Hence, experiments were initiated on synthetic oligonucleotide models to understand the various types of G-quartet structures formed by the telomeric sequences. The results on the structure and stability of telomeric sequences are described in Part II. The major aspects addressed in Part II include: (a) What is the effect of the number of thymine residues in the formation and stability of hairpin G-quartet structures? (b) What are the factors that govern the formation of antiparallel or parallel quadruplex structures for telomeric and telomere-like sequences? (c) Intra- and inter-loop interactions and the monovalent cation-dependent structural differences in the Oxytricha telomeric repeat. (d) Structure and stability of human telomeric sequences with special emphasis on the structure and interaction in the loop region. (e) Effect of the sequence, conformation, and orientation of loops on the overall structure and stability of telomeric sequences. A combination of spectroscopic, chemical probing, and gel electrophoretic methods has been extensively used throughout the course of this work to gain insight into the above-mentioned aspects and to seek answers to the questions raised. Chapter 4 deals with the studies aimed at understanding the role of thymine residues in the formation of G-quartet structures for telomeric sequences using model oligonucleotides of the type d(G4TnG4), with n = 1-4. Sequences d(G4T4G4) and d(G4T3G4) adopt a G-quartet structure formed by hairpin dimerization in 70 mM NaCl, as judged by a characteristic circular dichroism signature with a 295 nm positive and 265 nm negative band. Meanwhile, d(G4TG4) adopts a parallel G-quartet structure, like d(G)i2, which exhibits a strong positive band at 260 nm and a negative band at 240 nm. The sequence d(G4T2G4) exhibits a mixture of both conformations. The stability of the hairpin G-quartet structure decreases with a decrease in the number of intervening thymine residues. Potassium permanganate, a single-strand-specific probe, was used to establish the presence of loops composed of T residues in the hairpin G-quartet structures formed by the oligonucleotides d(G4TnG4) with n = 2-4 in 70 mM NaCl. Formation of the hairpin G-quartet structure for the above sequences was further supported by the enhanced electrophoretic mobility observed on non-denaturing polyacrylamide gels. A detailed model for the G-quartet structure, involving a hairpin dimer with alternating syn-anti-syn-anti conformations for the guanine residues both along the chain as well as around the G-tetrad, with interloop T:T base-pairing, was proposed [Balagurumoorthy et al. (1991), J. Biomol. Struct. Dyn. 8, a015; Balagurumoorthy et al., (1992), Nucl. Acids Res. 20, 4061-4067]. This model had many features that were observed in the X-ray crystal structure of d(G4T4G4) [Kang et al. (1992), Nature 356, 126-131]. Such intermolecular associations of short telomeric sequences described in this chapter provide a possible model for chromosomal pairing. Chapter 5 describes the studies aimed at understanding the intra- and inter-loop interactions in the loop region of the quadruplexes. The 3.5-copy Oxytricha telomeric repeat d(G4T4)3G4 was the choice, as the folded structure formed by this sequence would have two thymine loops located at the same end of the G-tetrad stem. A combination of CD and chemical probing was used to arrive at plausible answers. Circular dichroism studies indicated that this sequence adopts an antiparallel intramolecular G-quartet structure, even in the presence of K+ ions, unlike d(G4TnG4) sequences where K+ shifts the equilibrium toward the parallel G-quartet structure. The observed higher thermal stability of d(G4T4)3G4 over d(G4T4G4) was attributed to the additional intra- and inter-loop interactions in the intramolecular antiparallel G-quartet structure of d(G4T4)3G4. Hence, KMnO4 probing experiments were carried out, and the results suggested that the thymine residues in the Na+-induced G-quartet structure of d(G4T4)3G4 are relatively less exposed than those in the K+-induced structure. Outer thymines (i.e., thymine residues adjacent to guanines) in the Na+-induced G-quartet structure of d(G4T4)3G4 were found to be stacked on the last guanine tetrad, suggesting the existence of intra- and interloop interactions. In light of the striking differences observed between the X-ray crystal and NMR structures of d(G4T4G4), it was of interest to study how sequence, conformation, and orientation of loops determine the overall structure and stability of the telomeric sequences. The telomeric DNA of vertebrates, including humans, shares a common sequence motif TTAGGG, repeated several times at each end of the chromosome. It was realized that the human telomeric sequence would be an ideal choice to study the loop interactions and their influence on the overall structure of quadruplexes, as it has the complementary nucleotides A and T in the region intervening guanine tracts. Studies on human telomeric oligonucleotide models are described in Chapter 6. Results of CD experiments showed that human telomeric oligonucleotide models adopt antiparallel G-quartet structures either by intramolecular folding or by dimerization of hairpins, depending upon the number of G-tracts. Studies carried out on truncated human telomeric oligonucleotides d(G3T2A)3G3 (3.5-copy sequence) and d(G3T2AG3) (1.5-copy sequence) showed that adenines occur in the loop. Probing with KMnO4 showed that the TTA loops are oriented at the same end of the G-tetrad stem in the antiparallel hairpin dimer of d(G3T2AG3), in contrast to the loops at opposite ends observed in the hairpin dimer of d(G4T4G4). Concentration-dependent electrophoretic mobility and CD experiments revealed the formation of a dimer of a dimer by end-to-end stacking of G-tetrad planes for d(G3T2AG3). The protection of adenines in d(G3T2AG3) from DEPC indicates that the N7 of adenines is Hoogsteen hydrogen bonded in the hairpin dimer. The partial reactivity of loop adenines with DEPC in d(T2T4G3)4 suggested that the intramolecular G-quartet structure is highly polymorphic, and structures with different loop orientations and topologies are formed in solution. Intra- and inter-loop hydrogen bonding schemes for the TTA loops were proposed to account for the observed DEPC reactivities of adenines. A scheme for the folding pathway has been proposed to rationalize the formation of various quadruplex structures with different loop orientations and topologies depending on the sequence, number of guanine tracts, environmental factors, and the concentration of the oligonucleotide used in the experiment. These studies have provided insight into the structure and stability of telomeric repeats. Chapter 7 is devoted to concluding remarks, highlighting the significance of unusual DNA structures and their possible biological implications.