Investigation of energetics of sharp DNA bending

DNA急剧弯曲的能量学研究

• 批准号: 9031121
• 负责人: Harold D Kim
• 金额: $ 25.06万
• 依托单位: GEORGIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-06 至 2020-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9031121
• 关键词: Address; Base Pair Mismatch; Base Pairing; Biological Assay; Biological Process; Biophysics; Cells; Chromatin Loop; Complex; Cyclization; DNA; DNA Packaging; DNA Repair; Data; Data Set; Dinucleoside Phosphates; Exhibits; Fluorescence Resonance Energy Transfer; Gene Expression Regulation; Genome; Goals; Health; Human; Investigation; Kinetics; Lead; Learning; Length; Link; Measures; Microscope; Mismatch Repair; Modeling; Nature; Outcome; Ploidies; Polymers; Probability; Process; Property; Research; Role; Shapes; Stretching; Techniques; Temperature; Testing; base; career; cost; disease-causing mutation; ds-DNA; flexibility; human disease; in vivo; melting; novel; physical property; single-molecule FRET; theories

DESCRIPTION (provided by applicant): DNA is a dynamic molecule that goes through conformational changes such as bending, twisting, and stretching. Bending of DNA, which leads to most noticeable conformational changes, is associated with DNA packaging, DNA looping and DNA mismatch repair inside a cell. Errors in any of these processes can cause human diseases, and therefore, understanding intrinsic bending properties of DNA is related to human health. To understand how frequently sharp DNA bends can arise, we need to know their energy cost. People have estimated this energy by measuring how likely it is for a linear DNA to form a loop by thermal fluctuation. Based on the results, we can all agree on how much energy is needed to bend a 500 base-pair long DNA into a circle, which is considered as normal bending. But experimental data disagree regarding a 100 base-pair long DNA, which requires sharper bending to form a circle. These contradictory results prompted different models about how DNA achieves sharp bending. One model suggests that double-stranded DNA can locally melt into unpaired single strands and acquire increased flexibility. However, experimental evidence for this "bubble" formation is lacking. In this proposal, we investigate the energy stored in a DNA loop as small as 50 base pairs and test various models of DNA bending in the sharp bending regime. The key idea is to form a small DNA loop shaped like a teardrop, and measure how long it stays as a loop. Loop formation or breakage will be observed on a microscope using a technique called Fluorescence Resonance Energy Transfer (FRET). The lifetime of the loop will depend on its energy; a loop with a higher energy will be less stable. Therefore, we can test a classical polymer model of DNA known as the worm-like chain model with accuracy in the sharp bending regime. Because a teardrop also has a pointy tip unlike a circle, we can place various DNA content near the tip to compare their bending rigidity in different bending regimes. We will measure bending rigidity of various sequences and mismatches in normal and sharp bending regimes as a function of temperature. We will also compare our results against DNA-melting theories to elucidate the role of DNA melting in DNA bending. This proposal will address some of the most controversial topics on DNA biophysics.

描述（由申请人提供）：DNA是一种动态分子，经过构象变化，例如弯曲，扭曲和拉伸。 DNA的弯曲，导致最明显的构象变化，与DNA包装，DNA循环和DNA不匹配修复有关。这些过程中的任何一个中的错误都会引起人类疾病，因此，了解DNA的内在弯曲特性与人类健康有关。要了解急剧的DNA弯曲的频率，我们需要知道它们的能源成本。人们通过测量线性DNA通过热波动形成循环的可能性来估计这种能量。根据结果​​，我们都可以就需要多少能量弯曲500碱基对长DNA成圆，这被认为是正常弯曲的。但是实验数据在100个碱基对长的DNA上不同意，这需要尖锐的弯曲才能形成一个圆。这些矛盾的结果促使有关DNA如何实现锋利弯曲的不同模型。一种模型表明，双链DNA可以局部融化成未配对的单链并获得提高的柔韧性。但是，缺乏这种“气泡”形成的实验证据。在此提案中，我们研究了存储的能量 在小至50个碱基对的DNA环中，测试了尖锐弯曲方案中各种DNA弯曲模型。关键的想法是形成形状像泪珠形状的小型DNA环，并测量其作为循环的时间。使用称为荧光共振能量转移（FRET）的技术，将在显微镜上观察到循环形成或断裂。循环的寿命将取决于其能量。能量较高的循环将不那么稳定。因此，我们可以测试具有尖锐弯曲状态准确性的DNA的经典聚合物模型。由于泪珠还具有与圆圈不同的尖尖，因此我们可以在尖端附近放置各种DNA含量，以比较它们在不同的弯曲状态下的弯曲刚度。我们将在正常和锋利的弯曲方案中测量各种序列和不匹配的弯曲刚度随温度的函数。我们还将比较我们的结果与DNA熔融理论，以阐明DNA熔化在DNA弯曲中的作用。该提案将解决有关DNA生物物理学最具争议的主题。