RecA蛋白所介导的同源重组机制的研究
发布时间：2019-03-30 13:40
【摘要】：同源重组是发生于两条相似或相同DNA链之间的遗传信息的重组,同时,也是DNA损伤修复的重要途径之一。对保证基因组完整性和产生遗传多样性有着重要意义。而同源重组过程需要重组酶的催化完成,其中大肠杆菌中的重组酶RecA作为典型成员得到了广泛研究。近年来,单分子技术不断快速发展,单分子荧光共振能量转移技术,磁镊技术,光镊技术等具有实时、直观、高精度等优点,被越来越广泛地运用到生物研究领域,更好地研究一些传统生化方法难以捕捉和观测的过程。然而,在同源重组的链交换过程中,每反应一个核苷酸的长度变化平均约为0.17nm,而同时参与反应的核苷酸数目可达上百个碱基,目前尚无合适的技术可以同时兼顾如此高的分辨率要求及测量范围。利用上述研究技术手段对同源重组过程的研究结果认为,同源重组可以分为初始识别和后续链交换扩展两个过程,至于链交换过程中如何搜寻同源序列,以及如何完成后续扩展至今尚无清晰的结论,甚至在如扩展的速率及特征长度等方面还存在一些争议性研究。本文的第一部分介绍了一种用于研究同源重组过程的改进方案,利用传统磁镊结合DNA发夹结构的方法就可以观测同源重组所涉及的多个动态过程过程。在满足大观测范围的同时,以较好的空间分辨率(5 nm)来观察各反应细节。利用该方法,我们不但可以实时观测RecA所介导的链交换过程,还可以直接观察RecA第二结合位点与被置换链的动态相互作用,并且通过链交换反应曲线的不同来判断链交换的方向性。为深入研究RecA和其他重组酶所介导的DNA重组机制提供了一个潜在的优选方案。第二部分中,利用磁镊和sm FRET技术来捕捉同源重组过程中的瞬态过程,通过设计不同同源比例DNA链,对RecA介导的同源重组所涉及的核心问题进行研究。我们利用上述技术手段不但观测到同源重组过程中初始同源序列识别的试探过程,而且记录了初始识别后的异源双链扩展的过程,实现了实时且完整地观测同源重组链交换的各个反应细节。结果表明:1.链交换过程是以Rec A为单位,而不是碱基为单位来进行的;2.链交换过程中链交换的尺度与核蛋白丝的结构有很强的相关性。单次链交换长度出现从3nt到24nt不等且都为3nt的整数倍,其分布期望为9nt,18nt,此结果统一了此前在相关领域研究结果的一些矛盾。此外,根据实验结果,我们推测,同源重组的扩展过程存在一个短尺度(~9bp)的快速匹配测试过程和一个长尺度(~18bp)的严格匹配测试过程。
[Abstract]:Homologous recombination is the recombination of genetic information between two similar or same DNA chains, and it is also one of the important ways to repair DNA damage. It is of great significance to ensure genome integrity and produce genetic diversity. The homologous recombination process needs to be catalyzed by recombinant enzymes, in which the recombinant enzyme RecA in Escherichia coli has been widely studied as a typical member. In recent years, monomolecular technology has been developed rapidly. Monomolecular fluorescence resonance energy transfer technology, magnetic tweezers technology, optical tweezers technology and other advantages, such as real-time, intuitive, high-precision, have been more and more widely used in the field of biological research. Better study some of the traditional biochemical methods difficult to capture and observe the process. However, in the chain exchange process of homologous recombination, the average length change of one nucleotides per reaction is about 0.17 nm, and the number of nucleotides involved in the reaction can reach hundreds of bases at the same time. At present, there is no suitable technology which can take into account both the high resolution requirement and the measurement range. The results show that homologous recombination can be divided into two processes: initial identification and subsequent chain exchange extension. As for how to search for homologous sequences in chain switching process, the results show that the process of homologous recombination can be divided into two processes: initial identification and subsequent chain exchange extension. There are still no clear conclusions about how to complete the subsequent expansion, and there are still some controversial studies in such aspects as the rate of expansion and the length of features. In the first part of this paper, we introduce an improved scheme to study the homologous recombination process. By using the traditional magnetic tweezers combined with the DNA hairpin structure, we can observe the multiple dynamic processes involved in the homologous recombination. At the same time, a good spatial resolution (5 nm) was used to observe the details of the response. By using this method, we can not only observe the chain exchange process mediated by RecA in real time, but also directly observe the dynamic interaction between the second binding site of RecA and the replaced chain. And the direction of chain exchange can be judged by the different curve of chain exchange reaction. It provides a potential optimal scheme for further study of the mechanism of DNA recombination mediated by RecA and other recombinant enzymes. In the second part, magnetic tweezers and sm FRET techniques are used to capture the transient process of homologous recombination. The core issues involved in RecA-mediated homologous recombination are studied by designing DNA chains with different homology ratios. Using the above-mentioned techniques, we not only observed the exploratory process of identifying initial homologous sequences in the process of homologous recombination, but also recorded the process of heterogenous double-stranded expansion after initial recognition. Real-time and complete observation of the reaction details of homologous recombination chain exchange has been achieved. The results show that: 1. The chain exchange process is carried out in Rec A units, not in base units; 2. There is a strong correlation between the scale of chain exchange and the structure of nuclein filament in the process of chain exchange. The single chain switching length varies from 3nt to 24nt and is an integer multiple of 3nt, and its distribution is expected to be 9 NT and 18 NT. This result unifies some contradictions of previous research results in related fields. In addition, according to the experimental results, we speculate that there is a short-scale (~ 9bp) fast matching test process and a long-scale (~ 18bp) strict matching test process in the extended process of homologous recombination.
【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】：Q75
本文编号：2450130
[Abstract]:Homologous recombination is the recombination of genetic information between two similar or same DNA chains, and it is also one of the important ways to repair DNA damage. It is of great significance to ensure genome integrity and produce genetic diversity. The homologous recombination process needs to be catalyzed by recombinant enzymes, in which the recombinant enzyme RecA in Escherichia coli has been widely studied as a typical member. In recent years, monomolecular technology has been developed rapidly. Monomolecular fluorescence resonance energy transfer technology, magnetic tweezers technology, optical tweezers technology and other advantages, such as real-time, intuitive, high-precision, have been more and more widely used in the field of biological research. Better study some of the traditional biochemical methods difficult to capture and observe the process. However, in the chain exchange process of homologous recombination, the average length change of one nucleotides per reaction is about 0.17 nm, and the number of nucleotides involved in the reaction can reach hundreds of bases at the same time. At present, there is no suitable technology which can take into account both the high resolution requirement and the measurement range. The results show that homologous recombination can be divided into two processes: initial identification and subsequent chain exchange extension. As for how to search for homologous sequences in chain switching process, the results show that the process of homologous recombination can be divided into two processes: initial identification and subsequent chain exchange extension. There are still no clear conclusions about how to complete the subsequent expansion, and there are still some controversial studies in such aspects as the rate of expansion and the length of features. In the first part of this paper, we introduce an improved scheme to study the homologous recombination process. By using the traditional magnetic tweezers combined with the DNA hairpin structure, we can observe the multiple dynamic processes involved in the homologous recombination. At the same time, a good spatial resolution (5 nm) was used to observe the details of the response. By using this method, we can not only observe the chain exchange process mediated by RecA in real time, but also directly observe the dynamic interaction between the second binding site of RecA and the replaced chain. And the direction of chain exchange can be judged by the different curve of chain exchange reaction. It provides a potential optimal scheme for further study of the mechanism of DNA recombination mediated by RecA and other recombinant enzymes. In the second part, magnetic tweezers and sm FRET techniques are used to capture the transient process of homologous recombination. The core issues involved in RecA-mediated homologous recombination are studied by designing DNA chains with different homology ratios. Using the above-mentioned techniques, we not only observed the exploratory process of identifying initial homologous sequences in the process of homologous recombination, but also recorded the process of heterogenous double-stranded expansion after initial recognition. Real-time and complete observation of the reaction details of homologous recombination chain exchange has been achieved. The results show that: 1. The chain exchange process is carried out in Rec A units, not in base units; 2. There is a strong correlation between the scale of chain exchange and the structure of nuclein filament in the process of chain exchange. The single chain switching length varies from 3nt to 24nt and is an integer multiple of 3nt, and its distribution is expected to be 9 NT and 18 NT. This result unifies some contradictions of previous research results in related fields. In addition, according to the experimental results, we speculate that there is a short-scale (~ 9bp) fast matching test process and a long-scale (~ 18bp) strict matching test process in the extended process of homologous recombination.
【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】：Q75
【参考文献】
相关期刊论文 前1条
1 徐悦;陈虎;璩玉杰;Artem K.Efremov;黎明;欧阳钟灿;刘冬生;严洁;;Mechano-chemical selections of two competitive unfolding pathways of a single DNA i-motif[J];Chinese Physics B;2014年06期
，本文编号：2450130
本文链接：https://www.wllwen.com/shoufeilunwen/jckxbs/2450130.html
