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，其经历构象变化，如弯曲、扭曲和拉伸。DNA的弯曲导致最明显的构象变化，与细胞内的DNA包装、DNA成环和DNA错配修复有关。这些过程中的任何一个错误都可能导致人类疾病，因此，了解DNA的内在弯曲特性与人类健康有关。为了了解DNA急剧弯曲的频率，我们需要知道它们的能量消耗。人们通过测量线性DNA通过热波动形成环的可能性来估计这种能量。基于这些结果，我们都可以同意需要多少能量才能将500个碱基对长的DNA弯曲成一个圆形，这被认为是正常的弯曲。但实验数据不同意100个碱基对长的DNA，这需要更尖锐的弯曲形成一个圆圈。这些相互矛盾的结果导致了关于DNA如何实现急剧弯曲的不同模型。一个模型表明，双链DNA可以局部融合成未配对的单链，并获得更大的灵活性。然而，这种“泡沫”形成的实验证据是缺乏的。在这个提议中，我们研究了储存的能量， 在小至50个碱基对的DNA环中，并在急剧弯曲状态下测试DNA弯曲的各种模型。关键的想法是形成一个像泪滴一样的小DNA环，并测量它作为一个环停留的时间。将使用称为荧光共振能量转移（FRET）的技术在显微镜上观察环形成或断裂。循环的寿命将取决于它的能量;具有较高能量的循环将不太稳定。因此，我们可以测试一个经典的聚合物模型的DNA被称为蠕虫状链模型的准确性急剧弯曲制度。因为泪滴也有一个尖尖的尖端，不像一个圆形，我们可以把各种DNA内容物放在尖端附近，以比较它们在不同弯曲状态下的弯曲刚度。我们将测量各种序列的弯曲刚度和在正常和急剧弯曲状态下的失配作为温度的函数。我们还将比较我们的结果与DNA熔化理论，以阐明DNA弯曲的DNA熔化的作用。该提案将解决DNA生物物理学中一些最具争议性的话题。