RecA蛋白所介導(dǎo)的同源重組機(jī)制的研究
發(fā)布時間：2019-03-30 13:40
【摘要】：同源重組是發(fā)生于兩條相似或相同DNA鏈之間的遺傳信息的重組,同時,也是DNA損傷修復(fù)的重要途徑之一。對保證基因組完整性和產(chǎn)生遺傳多樣性有著重要意義。而同源重組過程需要重組酶的催化完成,其中大腸桿菌中的重組酶RecA作為典型成員得到了廣泛研究。近年來,單分子技術(shù)不斷快速發(fā)展,單分子熒光共振能量轉(zhuǎn)移技術(shù),磁鑷技術(shù),光鑷技術(shù)等具有實(shí)時、直觀、高精度等優(yōu)點(diǎn),被越來越廣泛地運(yùn)用到生物研究領(lǐng)域,更好地研究一些傳統(tǒng)生化方法難以捕捉和觀測的過程。然而,在同源重組的鏈交換過程中,每反應(yīng)一個核苷酸的長度變化平均約為0.17nm,而同時參與反應(yīng)的核苷酸數(shù)目可達(dá)上百個堿基,目前尚無合適的技術(shù)可以同時兼顧如此高的分辨率要求及測量范圍。利用上述研究技術(shù)手段對同源重組過程的研究結(jié)果認(rèn)為,同源重組可以分為初始識別和后續(xù)鏈交換擴(kuò)展兩個過程,至于鏈交換過程中如何搜尋同源序列,以及如何完成后續(xù)擴(kuò)展至今尚無清晰的結(jié)論,甚至在如擴(kuò)展的速率及特征長度等方面還存在一些爭議性研究。本文的第一部分介紹了一種用于研究同源重組過程的改進(jìn)方案,利用傳統(tǒng)磁鑷結(jié)合DNA發(fā)夾結(jié)構(gòu)的方法就可以觀測同源重組所涉及的多個動態(tài)過程過程。在滿足大觀測范圍的同時,以較好的空間分辨率(5 nm)來觀察各反應(yīng)細(xì)節(jié)。利用該方法,我們不但可以實(shí)時觀測RecA所介導(dǎo)的鏈交換過程,還可以直接觀察RecA第二結(jié)合位點(diǎn)與被置換鏈的動態(tài)相互作用,并且通過鏈交換反應(yīng)曲線的不同來判斷鏈交換的方向性。為深入研究RecA和其他重組酶所介導(dǎo)的DNA重組機(jī)制提供了一個潛在的優(yōu)選方案。第二部分中,利用磁鑷和sm FRET技術(shù)來捕捉同源重組過程中的瞬態(tài)過程,通過設(shè)計不同同源比例DNA鏈,對RecA介導(dǎo)的同源重組所涉及的核心問題進(jìn)行研究。我們利用上述技術(shù)手段不但觀測到同源重組過程中初始同源序列識別的試探過程,而且記錄了初始識別后的異源雙鏈擴(kuò)展的過程,實(shí)現(xiàn)了實(shí)時且完整地觀測同源重組鏈交換的各個反應(yīng)細(xì)節(jié)。結(jié)果表明:1.鏈交換過程是以Rec A為單位,而不是堿基為單位來進(jìn)行的;2.鏈交換過程中鏈交換的尺度與核蛋白絲的結(jié)構(gòu)有很強(qiáng)的相關(guān)性。單次鏈交換長度出現(xiàn)從3nt到24nt不等且都為3nt的整數(shù)倍,其分布期望為9nt,18nt,此結(jié)果統(tǒng)一了此前在相關(guān)領(lǐng)域研究結(jié)果的一些矛盾。此外,根據(jù)實(shí)驗(yàn)結(jié)果,我們推測,同源重組的擴(kuò)展過程存在一個短尺度(~9bp)的快速匹配測試過程和一個長尺度(~18bp)的嚴(yán)格匹配測試過程。
[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.
【學(xué)位授予單位】：中國科學(xué)院大學(xué)(中國科學(xué)院物理研究所)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：Q75
本文編號：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.
【學(xué)位授予單位】：中國科學(xué)院大學(xué)(中國科學(xué)院物理研究所)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：Q75
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 徐悅;陳虎;璩玉杰;Artem K.Efremov;黎明;歐陽鐘燦;劉冬生;嚴(yán)潔;;Mechano-chemical selections of two competitive unfolding pathways of a single DNA i-motif[J];Chinese Physics B;2014年06期
，本文編號：2450130
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