用单分子技术研究BLM解旋G-四聚体

发布时间：2019-04-25 16:43

【摘要】：近年来,单分子技术在分子生物学领域发挥了巨大的作用。作为单分子操纵术中的磁镊技术因为构建方便,精度较高而受到青睐。但当磁力小于10 p N时,传统磁镊的分辨率会受到磁球布朗运动的限制。为了提高磁镊的分辨率,我们把全内反射荧光技术引入到磁镊系统中,我们同时发展了一种新的单分子连接系统:“磁球-手柄-荧光球-待测底物”。改进的磁镊可以实现小力下纳米精度的空间分辨率,同时在长时间观测中保持较高的时间分辨率。G-四聚体(G-quadruplex;G4)是广泛存在于细胞基因组中的一种DNA结构,在DNA的代谢如复制、转录、同源重组等过程中起重要作用。随着单分子技术的发展,越来越多的的关于G4的研究被发表,但是一些基本的问题还不是很清楚。G4解旋酶近年来受到广泛研究,其中BLM解旋酶的研究已经相当丰富。BLM解旋酶在同源重组修复过程中维持了基因的稳定性,研究表明BLM可以高效的结合和解旋G4。我们应用全内反射瞬逝场照明磁镊对BLM解旋G4的动力学过程进行深入研究,观察到了BLM解旋G4的分步过程。相对于单分子荧光共振能量转移技术而言,磁镊的长时间观测能力使我们在近饱和ATP浓度的实验体系中观察到BLM长时间反复解开G4或者长时间维持G4在打开状态的两种作用方式。我们使用相同的实验条件作了单分子荧光共振能量转移实验,确定了加载2-3 pN的外力不会对BLM解旋G4的过程产生影响。通过对BLM解旋G4后G4去折叠态的驻留时间的研究,我们发现BLM解旋G4的过程对应于一个长驻留时间过程和一个短驻留时间过程。我们发现ATP水解和HRDC结构域同单链DNA的结合是BLM解旋G4的长驻留时间过程的原因。我们的数据让我们提出了HRDC对BLM解旋G4过程影响的模型。当HRDC和G4序列相结合时,BLM可以使G4长时间保持非折叠态;当HRDC处于自由态时,BLM就会快速的重复解旋G4。
[Abstract]:In recent years, monomolecular technology has played an important role in the field of molecular biology. As a monomolecular manipulation technique, magnetic tweezers are favored because of their convenient construction and high precision. However, when the magnetic force is less than 10 PN, the resolution of the traditional tweezers will be limited by the Brownian motion of the magnetic sphere. In order to improve the resolution of magnetic tweezers, we introduced the total internal reflection fluorescence technique into the magnetic tweezers system. At the same time, we developed a new monomolecular joining system: magnetic sphere-handle-fluorescent sphere-substrate to be measured. The improved magnetic tweezers can achieve the spatial resolution of nano-precision under small force while maintaining a high temporal resolution in long-time observation. G-tetramer (G-tetramer); G4) is a kind of DNA structure which widely exists in the cell genome. It plays an important role in the metabolism of DNA such as replication transcription homologous recombination and so on. With the development of monomolecular technology, more and more researches on G4 have been published, but some basic problems are still unclear. G4 racemases have been extensively studied in recent years. Among them, the study of BLM helicase has been quite abundant. BLM helicase has maintained the gene stability in homologous recombination repair process. The results show that BLM can efficiently bind and decompose G4. The results show that BLM can efficiently bind and decompose G4 gene in the process of homologous recombination repair. The kinetic process of BLM de-spin G _ 4 was studied deeply by using the total internal reflection transient field illumination tweezers. The step-by-step process of BLM de-spin G _ 4 was observed. Compared to the single molecule fluorescence resonance energy transfer technique, The ability of long-term observation of magnetic tweezers led us to observe two ways of BLM repeatedly unlocking G4 or maintaining G4 in open state for a long time in the experimental system of near-saturated ATP concentration. We use the same experimental conditions to do a single molecule fluorescence resonance energy transfer experiment, and it is determined that the external force loading of 2 ~ 3 pN will not affect the process of BLM de-spin G _ 4. By studying the residence time of the de-folded state of G4 after BLM de-spin, it is found that the process of BLM de-rotating G4 corresponds to a process of long residence time and a process of short residence time. It is found that ATP hydrolysis and the binding of HRDC domain with single strand DNA are the reasons for the long residence time of BLM despin G4. Our data allow us to propose a model of the effect of HRDC on the BLM de-spin G4 process. When HRDC and G4 sequences are combined, BLM can keep G4 in non-folded state for a long time, and when HRDC is in free state, BLM will rapidly repeat G4.
【学位授予单位】：中国科学院大学(中国科学院物理研究所)
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q6-3

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