Interaction and organization of DNA in condensed phases

INTERACTION AND ORGANIZATION OF DNA IN CONDENSED PHASES DAI LIANG NATIONAL UNIVERSITY OF SINGAPORE 2008 INTERACTION AND ORGANIZATION OF DNA IN CONDENSED PHASES DAI LIANG (Ph.D.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTER OF PHILOSOPHY DEPARTMENT OF PHYSICS NATIONAL UNIVERSITY OF SINGAPORE 2008 Acknowledgement I am indebted to the following persons for the completion of this thesis. First and foremost, I would like to thank my supervisor, A/P Johan R. C. van der Maarel for his guidance of conducting this research. I greatly appreciated the relaxed atmosphere created by him, which made the life comfortable and enjoyable. My English skills were also significantly improved during the daily communication with him. In addition, I am grateful to Asst/P Mu Yuguang and Prof. Lars Nordenskiöld in Nanyang Technological University for their instruction on computer simulation, as well as providing computational facilities. I am also grateful to my colleagues, Zhu Xiaoying, Andrej Grimmm, Zhang Ce, Ng Siow Yee and Binu Kundukad for their support. Special thanks should go to Zhu Xiaoying, who made my daily work more productive. Asst/P Yan Jie is also greatly acknowledged for the fruitful discussions. Last but not least, acknowledgement must go to my family members and my girlfriend for their continuous supporting. i List of Publications 1. Charge Structure and Counterion Distribution in Hexagonal DNA Liquid Crystal Liang Dai, Yuguang Mu, Lars Nordenskiöld, Alain Lapp, and Johan R.C. van der Maarel Biophysical Journal, 92, 947-958 (2007) 2. Molecular Dynamics Simulation of Multivalent-ion Mediated Attraction between DNA molecules Liang Dai, Yuguang Mu, Lars Nordenskiold, and Johan R.C. van der Maarel Physical Review Letters, 100, 118301 (2008) ii Table of Contents Acknowledgement i List of Publications ii Table of Contents iii Summary vii List of Tables viii List of Figures ix Chapter Introduction 1.1 General introduction 1.2 DNA interaction 1.3 DNA organization in condensed phases 1.4 Thesis outline 10 References 12 Chapter Methodology 2.1 Large scale preparation of mononucleosomal DNA from calf thymus 22 23 2.1.1 Materials and methods 23 2.1.2 Characterization of isolated DNA 29 2.2 Small angle neutron and X-ray scattering experiments 30 2.2.1 Basic knowledge 30 2.2.2.Quantitative interpretation of scattering intensities 32 2.3 Molecular Dynamics computer simulations 36 iii 2.3.1 Basic principles and the function of molecular dynamics simulations 36 2.3.2 History of molecular dynamics simulation and popular programs 38 2.3.3 Force field parameters 40 2.3.4 Limitations of MD simulations 41 References Chapter Molecular dynamics simulation of DNA attraction mediated by multivalent ions 46 48 3.1 Introduction 48 3.2 Method 52 3.2.1 Molecular dynamics simulation 52 3.2.2 Umbrella sampling 53 3.2.3 Azimuthal orientation correlation 57 3.3 Results and discussions 58 3.3.1 DNA-DNA attraction in presence of multivalent ions 58 3.3.2 Spermine-induced azimuthal orientation correlation 68 3.3.3 Ions dynamics and ion-bridge formation 70 3.4 Conclusions 72 References 75 Chapter Molecular dynamics simulation of DNA fragments under sharp bending conditions 4.1 Introduction 81 4.2 Materials and methods 83 iv 4.3 Results and discussions 85 4.3.1 Structure changes induced by sharp bending 85 4.3.2 Kink formation under moderate curvature 88 4.3.3 Bubble formation 89 4.3.4 Critical bending curvatures for kink and bubble formation 95 4.3.5 Recent force field parmbsc0 96 4.3.6 Twist effect 97 4.4 Conclusions 99 References 99 Chapter Charge structure and counterion distribution in hexagonal DNA liquid crystal 103 5.1 Introduction 103 5.2 Scattering analysis 107 5.2.1 From intensities to structure factors 107 5.2.2 Number and charge structure factor 108 5.2.3 Cell model 110 5.2.4 Radial profiles 112 5.3 Materials and methods 114 5.3.1 Isolation of DNA fragments 114 5.3.2 Small angle neutron scattering 117 5.3.3 Molecular dynamics and Monte Carlo simulations 118 5.4 Results and discussion 120 5.4.1 Molecular dynamics and Monte Carlo simulations 120 5.4.2 SANS data analysis 127 5.4.3 Number and charge structure 130 5.4.4 DNA-counterion and counterion partial structure 134 5.5 Conclusions 138 v References Chapter Conclusions and future work 142 148 6.1 Main research findings 148 6.2 Recommendation of future research 152 References 155 vi Summary In this research, we studied the interaction and organization of DNA through experimental and computational approaches, with a special focus on the interpretation of new and existing experimental results by full-atom molecular dynamics (MD) simulation. This research can be divided into three projects. First, in MD simulations we observed that multivalent ion mediated DNA-DNA attraction is related to the formation of ion bridges, i.e. multivalent ions which are simultaneously bound to the two opposing DNA molecules. The inter-DNA potential was obtained by the umbrella sampling technique. Second, the structure of a base-pair B-DNA duplex under sharp bending conditions was systematically investigated by MD simulations. The DNA duplex exhibited the formation of a kink or bubble under certain bending curvatures. The formation of a kink or bubble was suggested to be the possible mechanism for the unexpected high flexibility of DNA observed in various experiments. Third, the counterion distribution in DNA liquid crystal was measured by small angle neutron scattering and interpreted with the help of molecular dynamics (MD) simulation. The overall research provided understanding of DNA interaction and organization in condensed phases. The research findings demonstrated that the molecular details of DNA molecules are sometimes essential for the understanding of a variety of experimental observations. vii LIST OF TABLES 1. Table 2.1 X-ray and neutron scattering lengths of some elements 32 2. Table 3.1 spring center position, spring constant, simulation time, distance between the two external springs, average inter-duplex distance and standard deviation in inter-duplex distance. The entries in the first row refer to a simulation without the presence of springs 62 3. Table 4.1 Roll and rise give the parameters of the initial DNA structures. Remarks indicate the type of DNA structures after simulations 85 4. Table 5.1 Geometric Parameters DNA in nm 114 5. Table 5.2 Partial molar volumes and scattering lengths 116 6. Table 5.3 Scattering length contrast in 10-12 cm 116 viii Chapter Charge structure and counterion distribution in hexagonal DNA liquid crystal molecule in register with the phosphate moieties, which is not captured by the PB and MC approaches. The DNA-counterion and counterion partial structure factors are fairly sensitive to the distance of closest approach, whereas the effects of the actual shape of the profile and the counterion concentration variation at the cell boundary are minimal. The optimized distance of closest approach agrees with the physical extent of the DNA molecule, hydration shell, and counterion size as shown by the MD profile for those ions which not penetrate the grooves of the DNA molecule. It is also in the range of the values reported for polyamines in isotropic samples of the same DNA fragments (12). 141 Chapter Charge structure and counterion distribution in hexagonal DNA liquid crystal REFERENCES 1. Gelbart, W. M., R. F. Bruinsma, P. A. Pincus, and V. A. 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Extremely precise free energy calculations of amino acid side chain analogos: comparison of common molecular mechanics force fields for proteins. J. Chem. Phys. 119:5740–5761. 147 Chapter Conclusions and future work Chapter Conclusions and future work 6.1 Main research findings The objective of this research was to investigate DNA interaction and organization through the approaches of full-atom molecular dynamics computer simulation and small angle scattering of neutrons. The main focus was to understand and quantitatively interpret various properties of DNA measured by previous and our own experiments with the aid of computer simulations at the atomic scale. Our research went beyond the primitive description, e.g. modeling the DNA structure as a rod-like polyelectrolyte and characterizing the elastic property of DNA by a uniform bending rigidity with a persistence length of about 50 nm. The research findings demonstrated that molecular structures are sometimes essential to understand the behavior of DNA and to explain some experimental observations. The various research findings are listed as followings: i) Molecular dynamics simulations of DNA-DNA attraction mediated by multivalent ions In order to clarify the underlying mechanism in DNA condensation, which 148 Chapter Conclusions and future work commonly occurs when a certain amount of multivalent ions are added to a diluted DNA solution, regardless the length of the DNA molecule, we have done molecular dynamics simulations to study the structure and dynamics of two DNA duplexes in the presence of various multivalent polyamines or cobalt hexamine (3+) ions. It was discovered that DNA-DNA attraction is related to the ion-bridge formation, i.e. one ion binding two DNA molecules simultaneously and temporarily. To the best of our knowledge, it was the first time to observe this phenomenon. The range and strength of the attractive potential between the two DNA molecules were obtained by the technique of umbrella sampling. This study also provided quantitative information about the range and strength of the attraction, as well as new insights to understand the DNA condensation phenomenon. ii) Molecular dynamics simulations of DNA fragments under sharp bending conditions DNA bending flexibility is usually and successfully described by the worm-like chain model with a persistence length of around 50 nm. The bending rigidity depends on the ionic strength and the base pair composition. In recent experiments on DNA under sharp bending conditions, it was observed that DNA was more flexible than expected based on a uniform bending rigidity with a persistence length of the order of 50 nm. Local disruption of base pairs, i.e. a “bubble” with enhanced flexibility has been proposed to explain the unexpected DNA flexibility. Full-atom MD simulation 149 Chapter Conclusions and future work were done to reveal the structures of short DNA fragments under sharp bending conditions and to examine the validity of the bubble model. For bending towards the major groove, the base pair structure was disrupted with a curvature exceeding a certain value, depending on the sequence. For bending towards the minor groove, the DNA duplex generally exhibits a transition from a smoothly bent conformation, through a kink and eventually a bubble structure with increasing curvature. The dihedral torsion energy was substantially reduced by the formation of the bubble. The release of the backbone strain can hence be considered to be the driving force. In addition, sharp bending of the duplex induces under-twisting and vice versa, under-twisting facilitates the dissociation of a base pair. Our simulation results agree with the bubble model to explain the unexpected high flexibility of DNA under sharp bending conditions. This finding is of biological importance, because it gives a possible explanation of how a sharply bent conformation of DNA can be achieved in vivo, such as in the capsid of bacterio phages and in nucleosome core particles. iii) Charge structure and Counterion Distribution in Hexagonal DNA Liquid Crystal DNA, as a polyelectrolyte, always coexists with small ions in biological environments. The conformation and interaction of DNA are strongly influenced by the ion species and ion distribution. In this research, we measured the charge structure and ion distribution in hexagonal DNA liquid crystal by small angle neutron 150 Chapter Conclusions and future work scattering. In addition, we quantitatively interpreted the scattering data with the aid of full-atom MD simulation. The MD simulations showed that the inter-DNA distance fluctuates with a correlation time around ns and a root-mean-square standard deviation of 8.5 % of the interaxial spacing. The MD simulation also showed a distinct double layer structure with more than 95 % of the counterions distributed within half the interaxial spacing away from the DNA spine axis. By comparison of the radial counterion profiles obtained from a one and a nine DNA molecule simulation, it was seen that the effect of inter-DNA distance fluctuations on the counterion distribution is small. Furthermore, the MD simulation showed considerable penetration of the grooves by TMA counterions. Overall, the research addressed several issues of DNA interaction and organization, especially in condensed phases. The knowledge of DNA interaction and organization from a physical point of view, besides genetic information and enzyme reaction, is essential to understand how DNA achieves its biological function, such as transcription, replication, and repair. On other hand, knowledge of DNA, a unique polyelectrolyte, also enriches our understanding of polyelectrolytes and polymers. One highlight in this research is that the full-atom MD simulation was demonstrated to be a powerful and suitable method. It is also worthwhile to mention the limitations of this research. To some extent, 151 Chapter Conclusions and future work this research relied on full-atom dynamics simulations. The imperfectness of the force fields and approximations in the algorithms may result in a incorrect characterization of the properties of DNA. Qualitative disagreements between simulations and experiments were observed. Full-atom MD simulations take account and provide details of molecular structures. This is an advantage, but also results in serious shortcomings compared to other types of computer simulation, e.g. Monte Carlo, Brownian dynamics, and MD simulation with implicit solvent. The limitations in simulation time and system size restrict the scope of the research. Furthermore, the derivation from microscopic properties in MD simulations to macroscopic properties in experiments strongly relies on statistical physics and sometimes this may be not an easy task. For instance, it is difficult to estimate the energy of attraction involved in DNA condensation from the attractive potential between DNA molecules obtained in a simulation, due to the unknown conformational entropy of DNA in the condensed states. 6.2 Recommendation of future research Due to the limited time, many interesting issues in DNA interaction and organization were not explored. A few possible research projects in this area are listed as follows. 152 Chapter Conclusions and future work i) The bending flexibility of kink and bubble in sharply bent DNA In this thesis, we observed kink and bubble formation in DNA fragments under sharp bending conditions. Partial work has also been done to study the flexibility of the kink and bubble. The bubble was much more flexible than the fragment with intact base pairs. More research needs to be done to study the effects of the sequence and salt concentration on the flexibility of the bubble. The flexibility of the kink structure is also of great interest. Further research would give a complete picture of the DNA structure and flexibility under sharp bending conditions. These research findings might agree with the model proposed by Yan et al (1). In this model, a bubble is formed when the local bending curvature exceeds a critical value, about 20.25o/bp, and the bubble is about 20 times more flexible than the intact B-DNA structure. ii) Full-atom molecular dynamics simulation of azimuthal correlation in hexagonally DNA liquid crystal Azimuthal angle refers to the rotation angle of DNA around its axis and azimuthal correlation refers to the correlation of azimuthal angles between different DNA molecules. Azimuthal correlation is a consequence of the interaction between DNA helices in the B-form, in addition to the cholesteric, liquid crystalline structure. Azimuthal correlation has extensively been studied based on the summation of electrostatic interactions between helical lines of charges using screened, Debye-Huckel electrostatics (2-4). In these calculations, specific counterion 153 Chapter Conclusions and future work absorption patterns are always assumed. It would be much more precise to study the azimuthal interaction in full-atom molecular dynamics simulations, which include the counterion absorption patterns and charge correlation in a more realistic way, beyond the framework of the Debye-Huckel theory. Some research has been done and we observed a strong azimuthal correlation when the interhelical distance becomes less than nm. The salt effect on the azimuthal correlation has also been observed. However, a systematic investigation is so far lacking and further analysis is called for. iii) Examine the validity of the theory in which azimuthal frustration is proposed to be the underlying mechanism of hexagonal-cholesteric interaction. As described above, the interaction between the helices in the B-form results in azimuthal correlation. For very close interhelical separation, the optimal azimuthal angle difference between two DNA molecules is non-zero. As a result, the situation of the azimuthal angle of DNA in a hexagonal liquid crystal is like the direction of spin in a spin glass. This situation is called “azimuthal frustration” and has been studied by mapping to the theory for spin glasses (5, 6). These studies obtained the phase behavior of the azimuthal angle in columnar DNA assemblies. On the other side, based on high resolution X-ray scattering, it was proposed that the azimuthal frustration at small inter-DNA separation smears the helical pattern and the optimal inter-axial angle becomes zero. A transition from the cholesteric to the hexagonal 154 Chapter Conclusions and future work phase is predicted, due to the progressive unwinding of the cholesteric pitch (7). A systematic investigation, which combines existing theories of phase behavior (6) and chiral interactions is however lacking. Besides above possible research projects, the inclusion of proteins, e.g. histones, in the simulations of DNA is clearly of interest from a biological point of view. Such simulations demand more computational resources, because of the larger system size. However, some special features of DNA-protein complex forrmation may be characterized by fast dynamics on a temporal scale of a few nanoseconds. In such situation, these issues may be properly studied by MD simulations. References 1. Yan, J., and J. F. Marko. 2004. Localized single-stranded bubble mechanism for cyclization of short double helix DNA. Phys Rev Lett 93:108108. 2. Kornyshev, A. A., and S. Leikin. 1997. Theory of interaction between helical molecules. Journal of Chemical Physics 107:3656-3674. 3. Harreis, H. M., C. N. Likos, and H. Lowen. 2003. Azimuthal frustration and bundling in columnar DNA aggregates. Biophys J 84:3607-3623. 155 Chapter Conclusions and future work 4. Kornyshev, A. A., D. J. Lee, S. Leikin, and A. Wynveen. 2007. Structure and interactions of biological helices. Reviews of Modern Physics 79:943-996. 5. Harreis, H. M., A. A. Kornyshev, C. N. Likos, H. Lowen, and G. Sutmann. 2002. Phase behavior of columnar DNA assemblies. Phys Rev Lett 89:018303. 6. Wynveen, A., D. J. Lee, and A. A. Kornyshev. 2005. Statistical mechanics of columnar DNA assemblies. Eur. Phys. J. E 16:303-318. 7. Strey, H. H., J. Wang, R. Podgornik, A. Rupprecht, L. Yu, V. A. Parsegian, and E. B. Sirota. 2000. Refusing to twist: demonstration of a line hexatic phase in DNA liquid crystals. Phys Rev Lett 84:3105-3108. 8. Kornyshev, A. A., S. Leikin, and S. V. Malinin. 2002. Chiral electrostatic interaction and cholesteric liquid crystals of DNA. European Physical Journal E 7:83-93. 156 [...]... (64), since the frustration of the azimuthal angle at very small inter -DNA separation smears the helical charge pattern and thus the optimal inter-axial angle becomes zero For long DNA molecules, the organization of DNA in condensed phases or confined volumes involves DNA bending As a result, the organization also depends on the elasticity of DNA, in addition to DNA interactions In the case of bacteriophage... an organization similar to the one in the liquid crystalline phase observed in vitro (6) The investigation of these condensed DNA phases can provide valuable insights of DNA interactions and organization in vivo Numerous experimental methods are available to produce condensed DNA phases The easiest way is to dissolve lyophilized DNA in a small amount of water or buffer solution At sufficiently high DNA. .. common methods used in this research, including DNA sample preparation, small angle scattering, computer simulations, are described in chapter 2 The results of this research provide understanding of DNA interactions and organization in dense phases at the atomic scale Furthermore, it will be shown that molecular details of the DNA molecules are essential for the understanding of a variety of experimental... macromolecular species and the 1 Chapter 1 Introduction entanglement of numerous interactions involved in the organization of DNA An alternative experimental approach is to produce condensed DNA phases in vitro (4, 5), which bear some resemblance to the organization of DNA in biology (6, 7) For instance, cryo-electron microscopy images have demonstrated that DNA inside the capsid of T7 bacteriophage is... The research findings are hence of great benefit to understand the behavior of DNA in vivo, e.g DNA packaging inside cells 11 Chapter 1 Introduction Reference 1 Earnshaw, W C., and S R Casjens 1980 DNA packaging by the double-stranded DNA bacteriophages Cell 21:319-331 2 Sipski, M L., and T E Wagner 1977 Probing DNA quaternary ordering with circular dichroism spectroscopy: studies of equine sperm chromosomal... formation of kinks consisting of intact base pairs (70) Occasionally, base pairs were also observed to be disrupted The types and mechanical properties of the defects under certain bending conditions are still unclear 9 Chapter 1 Introduction 1.4 Thesis outline The aim of this research was to investigate the interactions and organization of DNA in condensed phases Traditional approaches in this area are usually... the condensed phases of DNA are liquid crystalline (5) Besides its biological relevance, the investigation of the interactions and organization of DNA also has some physical aspects The negative charge, the double helical structure and the semi-flexibility of the backbone provide opportunities to study chiral interactions between helical molecules (17-19) and to test theoretical concepts pertaining... liquid-crystalline packaging properties (5) (mesophases and transitions between them) More recent studies have discovered that the nucleic acid sequence also plays a role in the DNA interactions (23) 1.2 DNA interactions Due to the negative charge of the DNA phosphate moieties, electrostatic interaction plays a dominant role The electrostatic interaction between DNA molecules strongly depends on the surrounding... replication, recombination and repair (3) The structural organization and dynamics of DNA inside the compacted structures is largely unknown Therefore, it is of great importance to study these issues for the understanding of the mechanisms underlying the basic processes of life It is not easy to study DNA interaction, dynamics and organization in the cellular environment This is due to the abundance of macromolecular... This model is in excellent agreement with single DNA stretching (65, 66) and looping experiments for DNA longer than 230 bp (67) However, in these experiments the extent of the bending is much smaller than required by many cellular processes Two important examples, which involve the formation of DNA loops shorter than 30 nm, are the packaging of DNA into nucleosomes (68) and the regulation of gene expression . understanding of DNA interaction and organization in condensed phases. The research findings demonstrated that the molecular details of DNA molecules are sometimes essential for the understanding. INTERACTION AND ORGANIZATION OF DNA IN CONDENSED PHASES DAI LIANG NATIONAL UNIVERSITY OF SINGAPORE 2008 INTERACTION AND ORGANIZATION OF. investigation of these condensed DNA phases can provide valuable insights of DNA interactions and organization in vivo. Numerous experimental methods are available to produce condensed DNA phases. The