Effect of sequence on DNA structure : Insights from molecular dyanmics studies

Abstract

DNA is a flexible molecule, known to adopt a wide variety of structures ranging from right-handed A, B, C to left-handed Z DNA and higher-order structures like quadruplexes, depending on the sequence and environment. This conformational flexibility of DNA has been utilized in many biological processes including transcription, packaging, replication, and repair. For more than two decades, several experimental and theoretical studies have attempted to understand the sequence-structure relationship. In particular, several studies have focused on sequences containing stretches of AT bases, because of their unique structural properties and their implications in biological processes. For example, A-tracts are reported to play a crucial role in nucleosome structure, in forming complexes with regulatory proteins, and as sites for minor groove-binding drugs. Incidentally, the first reported crystal structure of B-form DNA also contained an AT-rich region in the middle. The crystal structure revealed many sequence-dependent structural features, one of them being the narrow minor groove width.

However, recently it was suggested that sequence-dependent localization of counterions could also be the cause for the observed narrow minor groove width. This indicated that a better understanding of the sequence-dependent structural properties of DNA was necessary. The study presented in this thesis aims to explore the sequence-dependent structural features of DNA, with particular reference to A-tracts and alternating AT and CG sequences.

One of the most useful tools available to study the dynamic properties of biomolecules is Molecular Dynamics (MD) simulation. Chapter 1 of the thesis gives a brief overview of different polymorphic forms of DNA and the use of MD simulation techniques to understand the dynamic structural properties of DNA. Chapter 2 details the methods used during the course of this study.

To understand the dynamics, hydration, and ion-binding features of A-tract and non–A-tract sequences, a 7 ns MD study was performed on three dodecamer sequences: d(CGCAAATTTGCG)? (A3T3) and on heteropolymers containing AT and CG base pairs, d(CGCATATATGCG)? ((AT)?) and d(CGCGCGCGCGCG)? ((CG)?) (Chapters 3 and 4). The results of the MD on the A-tract sequence suggest that the intrusion of Na? ions into the minor groove is a rare event and the structure is not very sensitive to the location of the sodium ions. The MD simulation carried out for a longer time scale unambiguously revealed the formation of sequence-dependent hydration patterns in the minor groove, often called the “spine of hydration” near the A-rich region and “ribbon of hydration” near the GC regions. The characteristic narrowing of the minor groove in the A-tract region is seen to precede the formation of the spine of hydration. The occurrence of cross-strand C2-H2···O2 hydrogen bonds in the minor groove of A-tract sequences is also confirmed, and these are found to occur even before the narrowing of the minor groove, indicating that such interactions are an intrinsic feature of A-tract sequences.

Compared to the A-tract sequence, the minor groove widths of the non–A-tract sequences ((CG)? and (AT)?) are wider. They are even wider than that of the starting fiber model. This variation in the minor groove width is also reflected in the hydration patterns. In contrast to the “spine” of hydration at A-tract sequences, a “ribbon” of hydration is observed in the minor groove of alternating AT as well as CG sequences. Strikingly, the hydration pattern is more well defined in the (AT)? MD structure than in the crystal structure. Variations in the values of the propeller twist of A·T base pairs are also seen among A-tracts and AT-tracts. The A·T base pairs in the (AT)? oligomer adopt smaller propeller twist values than in the A3T3 structure, possibly due to the absence of the stabilizing cross-strand hydrogen bonds. The MD studies conclude that the duplex structure formed by the alternating (AT)? sequence is more flexible than that of oligo A-tract and CG-tract. This has consequences for DNA curvature and bendability, features that are important for its biological function.

All three MD studies showed fluctuations in the backbone torsion angles, which correspond to one of the conformational substates of B-DNA, known as BII conformation. BII conformational substate is the most predominantly observed conformational substate of B-DNA and has been reported to play a key role in protein-DNA recognition. One example from NMR, signifying the importance of BII, is for the NF-?B binding site, where BI/BII equilibrium in the flanking steps is shown to induce dynamic curvature, necessary for DNA-protein recognition. BI and BII conformations are easily identified by the ?–? difference, which is negative in BI and positive in BII. Detailed analysis has been carried out to understand the sequence preference for BII conformation using MD and crystal structures (Chapter 5). Our analysis shows that BII conformation is sequence-specific and the dinucleotides GC, CG, CA, and TG show higher preference to adopt BII conformation than TT, TC, CT, and CC. The local geometrical parameters of a dinucleotide step with BII backbone conformation are considerably different from those of a step with BI conformation and are characterized by high twist, negative roll, and positive slide values.

Interestingly, the magnitude of variation in the dinucleotide parameters depends mainly on two factors: the magnitude of ?–? difference and the presence or absence of BII conformation across the Watson-Crick base-paired dinucleotide step. Based on the possible conformation in the base pair, three possible backbone combinations could be both BI (BI·BI), BI/BII·BII/BI (BI+BII), and both BII (BII·BII). Based on the magnitude of ?–? difference, both BII can be further classified as strong BII and weak BII. Strong BII conformation is observed to have large twist, high positive slide, and negative roll values, which can contribute to groove opening/closing and thus modulate protein-DNA interactions. Steps with weak BII take values similar to that of the hybrid BI+BII combination. The study also reveals the rare occurrence of BII conformation; thus, it can be concluded that both BI and BI+BII backbone combinations are more favorable than BII·BII.

Recently, based on NMR study results, a structural role for BII nucleotides has also been proposed in C-form DNA. Since the proposed NMR model for C-form DNA mainly contained BII backbone, the role of BII conformation in C-form DNA has also been examined. A systematic modeling exercise has been carried out for various sets of dinucleotide parameters (roll, twist, and slide) using different sequences. The final model for C-form DNA has been built for the dodecamer d(CGCGCGCGCGCG)? with all the steps in BII backbone conformation with C-DNA helical parameters. The stability of the model was checked by carrying out a 7 ns MD study using the AMBER program. Even during the early period of the dynamics, some steps showed a conformational transition from BII to BI, and all were observed to fluctuate throughout the dynamics. RMSD statistics also indicated that the structures deviate from the starting C-form DNA model and tend to converge towards B-form DNA. As observed in previous MD studies, BII conformation is not observed simultaneously in any two contiguous steps and is rarely seen on both strands together. Thus, the present study indicates that the proposed model with BII backbone in all the steps may not be stable, and hence C-form DNA should have a mixture of BI and BII backbone conformations. The details of these results are described in Chapter 6.

Summary and future prospects are presented in Chapter 7.

Part of the work presented in this thesis has been reported in the following publications:

A. Madhumalar and M. Bansal. Structural Insights into the Effect of Hydration and Ions on A-Tract DNA: A Molecular Dynamics Study. Biophysical Journal, 2003, 85:1805–1816.

A. Madhumalar and M. Bansal. Sequence Dependent Structural Features of A3T3 vs (AT)? from Molecular Dynamics Study. Recent Trends in Biophysical Research, 2003, (Ed. M. Maiti), 10–16.

A. Madhumalar and M. Bansal. Sequence Preference for BI/BII Conformations in DNA: MD and Crystal Structure Data Analysis (communicated).

Symposia Presentations:

Presented a poster titled “Structural Insights into A-Tract DNA – A Molecular Dynamics Study,” A. Madhumalar and M. Bansal, at the International Symposium on Crystallography and Bioinformatics in Structural Biology, Indian Institute of Science and National Centre for Biological Sciences, Bangalore, India, November 22–25, 2001.

Presented a poster titled “The Role of BII Nucleotides in C-Form DNA,” A. Madhumalar and M. Bansal, at the 48th Biophysical Society Annual Meeting, February 14–18, 2004, Baltimore, Maryland, USA.