Effect of G-rich Higher Order Structures on Insulin Linked Polymorphism Region

描述(由申请者提供)：该项目的长期目标是通过研究可能与这些疾病有关的DNA复制中短DNA重复的调节机制，改善具有遗传成分的美国人的健康和福祉，如胰岛素依赖型糖尿病(IDDM)、脆性X综合征和亨廷顿氏病。特别是，这个项目探索了新的研究方法来研究G四链，一个二级DNA结构，及其潜在的高阶结构对与IDDM有关的胰岛素连接多态区域(ILPR)长度多态的影响。短DNA重复区域中的二级和高阶结构可能导致滑移，例如，通过停滞复制叉子。滑移是导致长度多态的主要原因之一。模拟结果预测了ILPR区的高阶G四链结构。然而，它们还没有在实验上被观察到，尽管在其他富含G的区域也发现了类似的结构。已知解旋酶可以解开简单的G-四链结构，但当这些结构被某些配体稳定时，解旋酶不能这样做。基于这些事实，该项目提出在ILPR结构域中存在对解旋酶解离具有抵抗性的高阶G四链结构。由于单个生物分子的机械展开具有揭示短寿命中间结构的独特能力，因此我们利用双光束双陷阱激光镊仪在单分子水平上研究了ILPR结构域中的G四链及其潜在的高阶结构。为此，包含ILPR片段的DNA构建将使用作者实验室成功开发的普遍适用的策略来合成。为了确定高阶G四链结构的存在，将采用常规的生化方法。为了帮助未来针对ILPR重复序列的治疗学的发展，将使用单分子和生物化学方法来表征G四链与各种配体之间的相互作用，如卟啉和胰岛素。为了测试RecQ DNA解旋酶是否不能解开单个高阶G四链，将使用激光镊子进行单分子分析。虽然在拟议的实验中使用了RecQ DNA解旋酶和ILPR DNA片段，但该项目的发现将足够普遍，以揭示长度多态的原因，这种多态与包括IDDM、脆性X综合征和亨廷顿病在内的各种疾病有关。公共卫生相关性该项目的长期目标旨在通过研究可能与这些疾病有关的DNA复制中短DNA重复的调节机制，改善具有遗传成分的美国人的健康和福祉，这些疾病包括胰岛素依赖型糖尿病(IDDM)、脆性X综合征和亨廷顿氏病。特别是，这个项目探索了新的研究方法来研究G四链，一个二级DNA结构，及其潜在的高阶结构对与IDDM有关的胰岛素连接多态区域(ILPR)长度多态的影响。

DESCRIPTION (provided by applicant): The long term goal of the project aims to improve the health and well-being of Americans with disorders that have hereditary components, such as Insulin Dependent Diabetes Mellitus (IDDM, type I diabetes), fragile X syndrome and Huntington's disease, by studying regulatory mechanism of short DNA repeats in DNA replication that may be involved in these diseases. In particular, this project explores novel research approaches to investigate the effects of G quadruplex, a secondary DNA structure, and its potential higher order structures on the length polymorphism in insulin linked polymorphic region (ILPR) that is implicated in the IDDM. The secondary and higher order structures in short DNA repeats regions can lead to slippage, for example, by stalling the replication fork. The slippage is one of the primary reasons that lead to length polymorphism. Simulation results have predicted higher order G quadruplex structures in ILPR region. However, they have not been observed experimentally, although similar structures have been found in other G rich regions. It is known that helicase can unwind simple G-quadruplex structures, but fails to do so when these structures are stabilized by certain ligands. Based on these facts, the project proposes that there exist higher order G quadruplex structures that are recalcitrant to the helicase unwinding in the ILPR domain. Since mechanical unfolding of single biomolecules has a unique capability to reveal short-lived intermediate structures, a dual-beam dual-trap laser tweezers instrument is employed to investigate the G quadruplex and its potential higher order structures in the ILPR domain at the single molecular level. For this purpose, DNA constructs containing ILPR fragments will be synthesized using a universally applicable strategy that has been successfully developed in authors' laboratories. To identify the existence of higher order G quadruplex structures, conventional biochemical approaches will be employed. To help future development of therapeutics that targets the ILPR repeats, the interaction between G quadruplexes and various ligands, such as porphyrin and insulin, will be characterized using both single molecule and biochemical approaches. To test whether the RecQ DNA helicase is incapable of unwinding individual higher order G quadruplexes, single molecular assays will be performed using laser tweezers. Although RecQ DNA helicase and ILPR DNA fragments are used in the proposed experiments, the finding from this project will be generic enough to shed light on the cause of length polymorphism that is implicated in various diseases including IDDM, fragile X syndrome and Huntington's disease. PUBLIC HEALTH RELEVANCE The long term goal of the project aims to improve the health and well-being of Americans with disorders that have hereditary components, such as Insulin Dependent Diabetes Mellitus (IDDM, type I diabetes), fragile X syndrome and Huntington's disease, by studying regulatory mechanism of short DNA repeats in DNA replication that may be involved in these diseases. In particular, this project explores novel research approaches to investigate the effects of G quadruplex, a secondary DNA structure, and its potential higher order structures on the length polymorphism in insulin linked polymorphic region (ILPR) that is implicated in the IDDM.