Recognition and self-assembly of DNA aggregates

DNA聚集体的识别和自组装

• 批准号: 8351094
• 负责人: Sergey Leikin
• 金额: $ 9.8万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8351094
• 关键词: Accounting; Affect; Agreement; Archaea; Bacteria; Base Pairing; Base Sequence; Binding; Biological; Cell Death; Cells; Charge; Circular DNA; DNA; DNA Repair; DNA Structure; DNA lesion; DNA reverse gyrase; Dependence; Development; Electrostatics; Environment; Fiber; Gene Activation; Gene Silencing; Genetic Materials; Genetic Recombination; Genome; Goals; Hand; Lead; Left; Life; Liquid substance; Malignant Neoplasms; Measures; Modeling; Molecular Conformation; Motion; Nucleosomes; Nucleotides; Oligonucleotides; Organism; Osmotic Pressure; Paper; Pattern; Perception; Phase; Physical condensation; Physics; Play; Process; Proteins; Publishing; Reporting; Role; Sequence Homologs; Sequence Homology; Sodium Chloride; Solutions; Specificity; Speed; Structure; Sugar Phosphates; Surface; Temperature; Testing; Universities; Variant; Vertebral column; Virus; base; design; expectation; in vivo; interest; intermolecular interaction; macromolecule; prevent; research study; self assembly; single molecule; theories

For years, interactions between double stranded (duplex) DNA were presumed to be independent of the DNA structure and base pair sequence because the nucleotides are buried inside the double helix and shielded by the highly charged sugar-phosphate backbone. In discussion of such interactions, duplex DNA was explicitly or implicitly modeled as a uniformly charged cylinder. However, this concept was based on intuitive perception rather than experiments or rigorous theory. In reality, the experimental evidence, e.g., transformation of duplex DNA from a non-ideal helix with 10.5 base pairs/turn in solution into a nearly ideal helix with 10.0 bp/turn in aggregates, suggested that this concept may be wrong. Starting from the classical paper of Rhodes and Klug published in 1980, it became clear that interactions between duplex DNA not only depend on but also affect the double helix structure. To account for possible effects of the structure of the sugar phosphate backbone on DNA-DNA interactions, over the last decade we have been developing a theory of electrostatic interactions between macromolecules with helical patterns of surface charges. Even the simplest models, which did not account for dynamic variations in the structure, e.g., due to the thermal motion, already suggested possible explanations for many observations. Such observations included the torsional deformation of the double helix upon aggregation mentioned above, counterion-specificity of DNA condensation, multiple liquid crystalline phases in DNA aggregates, and measured intermolecular forces. We, therefore, continued development of this theory and its applications to various phenomena. Most importantly, this theory predicted that the dependence of the backbone structure on the nucleotide sequence might be sufficiently strong to affect DNA-DNA interactions. The structure-specific DNA-DNA interactions result from preferential juxtaposition of the negatively charged sugar phosphate backbone with counterions bound in grooves on the opposing molecule. Our analysis of x-ray diffraction experiments confirmed such juxtaposition of parallel DNA molecules in fibrous, hydrated aggregates. Furthermore, statistical analysis and comparison of known structures of DNA oligonucleotides in crystals (determined by x-ray diffraction) and in solution (determined by NMR) revealed changes in DNA structure within the crystals consistent with predictions of this theory and allowed us to evaluate essential parameters of the theory. However, it remained unclear how sequence-dependent interactions might be affected by thermal fluctuations, particularly by DNA bending. In the last two years, we developed and published a comprehensive statistical theory, which predicted dramatic effects of thermal bending. Contrary to our expectations, thermal undulations of DNA strongly amplify rather than weaken the sequence-dependent interactions. The undulations enhance the structural adaptation of DNA, leading to better alignment of neighboring molecules and pushing the geometry of the DNA backbone closer to that of an ideal helix. Quantitative comparison revealed good agreement of the theoretical predictions with measured osmotic pressures in DNA aggregates. During the last year, we utilized these results for refining DNA fiber x-ray diffraction theory. Comparison of the latter theory with available experimental diffraction patterns further supported our predictions for the relationship of the double helix sequence and structure with DNA-DNA interactions. The effects of the sequence on interactions between duplex DNAs, e.g., the predicted direct recognition of sequence homology between 100 base pair (bp) or longer sequences, may have significant biological implications. For instance, the speed and accuracy of sequence homology recognition is crucial for DNA repair, preventing DNA lesions that lead to cell death and cancer. In 2008, we published the first experimental evidence for homologous pairing of 300 bp, intact DNA double helices in liquid crystalline aggregates. These experiments and published reports, which indicated that homologous, nucleosomes-free regions of duplex DNA might preferentially interact in vivo, suggested that local, transient pairing of homologous sequences in intact DNAs may precede double strand breaks, further recognition by protein-covered single strands, and strand crossover. In late 2009, the group of M. Prentiss from Harvard University published elegant single-molecule studies, which revealed selective binding of 1-5 kb duplex DNA fragments to homologous regions on much longer molecules, further supporting our theory. Surprisingly, however, they observed this binding in monovalent salt, at conditions at which pairing or aggregation of duplex DNAs was never observed before and was considered to be theoretically impossible. In our theory, a stable parallel juxtaposition of two homologous DNA duplexes was expected to occur in the presence of some divalent and most polyvalent counterions, but not in monovalent salt. To resolve the potential discrepancy between our predictions and the experimental observations of M. Prentiss group, we revisited the theory of electrostatic pairing between duplex DNA. Specifically, we eliminated the simplifying assumption that DNA duplexes remain straight and parallel to each other when they form a stable pair. We found that DNA molecules will tend to supercoil, forming a braid. During the last year, we completed and published a theory for the electrostatic energy of such braids, demonstrating that formation of stable braided pairs of homologous double helices may be energetically favorable even in monovalent salt, depending on counterion environment. Our predictions for the dependence of such pairing on counterions, salt concentration and temperature closely matched the experimental observations of the Prentiss group. Furthermore, this theory suggested possible interpretation for a number of other puzzling phenomena. For instance, electrostatic stabilization of left-handed braids predicted by this theory may be an important factor in explaining why hyperthermophilic bacteria and archea need reverse gyrases to promote left-handed supercoiling of circular DNA, which provides more stable conformation (essential for protecting the genome at temperatures above 100 C). We are presently conducting experiments designed to test some of these ideas.

多年来，双链 DNA 之间的相互作用被认为与 DNA 结构和碱基对序列无关，因为核苷酸埋藏在双螺旋内部并受到高电荷糖磷酸骨架的屏蔽。在讨论此类相互作用时，双链 DNA 被明确或隐含地建模为均匀带电的圆柱体。然而，这个概念是基于直觉感知，而不是实验或严格的理论。事实上，实验证据，例如，双链 DNA 从溶液中 10.5 个碱基对/转的非理想螺旋转化为聚集体中 10.0 bp/转的近乎理想的螺旋，表明这个概念可能是错误的。从1980年Rhodes和Klug发表的经典论文开始，我们就清楚地认识到双链DNA之间的相互作用不仅取决于而且影响双螺旋结构。 为了解释磷酸糖主链结构对 DNA-DNA 相互作用的可能影响，在过去十年中，我们一直在发展具有表面电荷螺旋模式的大分子之间的静电相互作用理论。即使是最简单的模型，没有考虑结构的动态变化，例如由于热运动，也已经为许多观察结果提出了可能的解释。这些观察包括上述聚集时双螺旋的扭转变形、DNA凝聚的反离子特异性、DNA聚集体中的多个液晶相以及测量的分子间力。因此，我们继续发展这一理论及其在各种现象中的应用。 最重要的是，该理论预测主链结构对核苷酸序列的依赖性可能足够强，足以影响 DNA-DNA 相互作用。结构特异性的 DNA-DNA 相互作用是由带负电荷的糖磷酸主链与结合在相反分子凹槽中的反离子优先并置产生的。我们对 X 射线衍射实验的分析证实了纤维状水合聚集体中平行 DNA 分子的这种并置。此外，对晶体中（通过 X 射线衍射测定）和溶液中（通过 NMR 测定）的 DNA 寡核苷酸的已知结构进行统计分析和比较，揭示了晶体内 DNA 结构的变化与该理论的预测一致，并使我们能够评估该理论的基本参数。然而，目前尚不清楚热波动（尤其是 DNA 弯曲）如何影响序列依赖性相互作用。 在过去两年中，我们开发并发布了综合统计理论，预测了热弯曲的巨大影响。与我们的预期相反，DNA 的热波动强烈增强而不是减弱序列依赖性相互作用。这些起伏增强了 DNA 的结构适​​应性，导致相邻分子更好地排列，并使 DNA 主链的几何形状更接近理想螺旋的几何形状。定量比较表明理论预测与 DNA 聚集体中渗透压的测量结果非常吻合。去年，我们利用这些结果完善了 DNA 纤维 X 射线衍射理论。后一种理论与现有实验衍射图的比较进一步支持了我们对双螺旋序列和结构与 DNA-DNA 相互作用之间关系的预测。 序列对双链 DNA 之间相互作用的影响，例如，预测的 100 碱基对 (bp) 或更长序列之间序列同源性的直接识别，可能具有重要的生物学意义。例如，序列同源识别的速度和准确性对于 DNA 修复、防止导致细胞死亡和癌症的 DNA 损伤至关重要。 2008 年，我们发表了第一个关于液晶聚集体中 300 bp、完整 DNA 双螺旋同源配对的实验证据。这些实验和已发表的报告表明，双链 DNA 的同源、无核小体区域可能优先在体内相互作用，表明完整 DNA 中同源序列的局部、瞬时配对可能先于双链断裂、被蛋白质覆盖的单链进一步识别和链交叉。 2009 年末，哈佛大学的 M. Prentiss 小组发表了精彩的单分子研究，揭示了 1-5 kb 双链 DNA 片段与更长分子上的同源区域的选择性结合，进一步支持了我们的理论。然而，令人惊讶的是，他们在单价盐中观察到了这种结合，而在此条件下，双链 DNA 的配对或聚集以前从未观察到，并且被认为理论上是不可能的。在我们的理论中，两个同源 DNA 双链体的稳定平行并置预计会在一些二价和大多数多价抗衡离子存在的情况下发生，但在一价盐中则不会发生。 为了解决我们的预测与 M. Prentiss 小组的实验观察之间的潜在差异，我们重新审视了双链 DNA 之间的静电配对理论。具体来说，我们消除了 DNA 双链体在形成稳定对时保持笔直且彼此平行的简化假设。我们发现DNA分子会倾向于超螺旋，形成辫子。去年，我们完成并发表了此类编织物的静电能理论，证明即使在单价盐中，形成稳定的同源双螺旋编织对也可能在能量上有利，具体取决于反离子环境。我们对这种配对对反离子、盐浓度和温度的依赖性的预测与 Prentiss 小组的实验观察结果非常吻合。此外，这一理论还为许多其他令人费解的现象提出了可能的解释。例如，该理论预测的左手辫子的静电稳定性可能是解释为什么超嗜热细菌和古细菌需要反向回旋酶来促进环状DNA的左手超螺旋的一个重要因素，这提供了更稳定的构象（对于在100℃以上的温度下保护基因组至关重要）。我们目前正在进行旨在测试其中一些想法的实验。