G4 DNA折疊及RecQ5β對不同DNA解旋的動理學研究
發(fā)布時間：2019-04-18 10:20
【摘要】：G4結(jié)構(gòu)是人類端粒末端存在的一種特殊的DNA二級結(jié)構(gòu),其折疊動理學對它們的生物學功能,如在端粒穩(wěn)定性的保持和染色體末端的保護等方面,都有非常重要的影響。G4結(jié)構(gòu)種類很多,受到環(huán)境因素的影響非常大,如離子種類、溶液的擁擠程度等。在本工作中,我們主要研究了G4 DNA在不同的單價陽離子環(huán)境中的折疊動理學,并測定了Na+和K+誘導的G4折疊的具體的動理學參數(shù),和G4從Na+條件下的反平行-籃式構(gòu)型,到K+條件下的雜交構(gòu)型的結(jié)構(gòu)變化時的動理學過程。更有趣的是,雖然在以前的關(guān)于G4折疊的實驗研究中,Li+經(jīng)常被用作對照實驗以提供類似的離子環(huán)境,其對Na+或K+條件下的G4折疊的協(xié)同作用并沒有被考慮進去,但是,我們發(fā)現(xiàn),Li+確實能夠顯著地、以不同的方式影響到Na+和K+誘導的G4折疊的動理學過程,例如,Li+可以改變Na+誘導的G4折疊的比例,并且很大程度地提高K+誘導的G4折疊的速率。我們現(xiàn)在的工作可以對單價陽離子對G4折疊動理學產(chǎn)生的影響進行新的闡述,并且應該會對未來G4潛在的折疊機制方面的研究有一定幫助。RecQ5β解旋酶是人類體內(nèi)存在的一種必不可少的RecQ解旋酶,它能夠在DNA復制、DNA修復、DNA重組和RNA轉(zhuǎn)錄等過程中發(fā)揮非常重要的作用。到目前為止,我們對RecQ5β解旋酶的解旋活性和底物特異性方面的研究一直沒有取得很大進展。在本研究工作中,我們使用快速停流方法測量了RecQ5β解旋酶對不同結(jié)構(gòu)的DNA底物的解旋動理學和解離動理學。我們發(fā)現(xiàn),RecQ5β解旋酶可以高效地解旋單雙鏈DNA、岔口DNA和霍利迪連結(jié)體DNA,但其解旋平末端DNA和G4結(jié)構(gòu)DNA的效率卻很低。RecQ5β解旋酶的這種解旋底物特異性很可能與DNA底物具有的交叉位點有關(guān),RecQ5β解旋酶與交叉位點的結(jié)合應該非常緊密,并且能夠很好地促進自身解旋活性的提高。而且,從全長型RecQ5βfl與截斷型RecQ5β1-467的解旋和解離動理學的比較中,我們可以看出,C-末端結(jié)構(gòu)域可能會強烈影響RecQ5β的解旋活性和與DNA底物的結(jié)合親和力。這些結(jié)論可能會對進一步闡明RecQ5β解旋酶的生理功能和工作機理有很大幫助。
[Abstract]:G4 structure is a special DNA secondary structure in human telomere terminal. The folding kinetics of G4 on its biological function, such as the maintenance of telomere stability and the protection of chromosome terminus, and so on. There are many kinds of G4 structure, which are greatly influenced by environmental factors, such as ion species, solution crowding and so on. In this work, we mainly studied the folding kinetics of G4 DNA in different monovalent cation environments, and measured the specific kinetic parameters of G4 folding induced by Na and K, and the antiparallel-basket configuration of G4 from Na condition. The kinetic process of the structure change of the hybrid configuration under K condition. More interestingly, although in previous experimental studies on G4 folding, Li was often used as a control experiment to provide a similar ionic environment, its synergistic effect on G4 folding under Na or K conditions was not taken into account, however, We found that Li did significantly affect the kinetic process of G4 folding induced by Na and K in different ways. For example, Li could change the ratio of Na-induced G4 folding. Moreover, K-induced folding rate of G _ 4 was increased to a great extent. We are now working on a new account of the effect of monovalent cations on the folding kinetics of G4, And it should be helpful to study the potential folding mechanism of G4 in the future. RecQ5 尾-racemase is an essential RecQ helicase in human body, which can replicate in DNA and repair DNA. DNA recombination and RNA transcription play a very important role. Up to now, we have not made great progress in the study of the activity and substrate specificity of RecQ5 尾 helicase. In this work, we measured the desolation kinetics and dissociation kinetics of RecQ5 尾-helicase on different structures of DNA substrates by rapid stop-flow method. We found that RecQ5 尾 helicase can efficiently decompose the single and double stranded DNA, fork DNA and the Holiday linker DNA,. However, the efficiency of cleavage of DNA and G4 structure DNA is very low. The specificity of RecQ5 尾 helicase is probably related to the cross site of DNA substrate, and the binding of RecQ5 尾 helicase to cross site should be very close. And it can promote the improvement of self-despinning activity. Furthermore, from the comparison between full-length RecQ5 尾 fl and truncated RecQ 5 尾 1-467, we can see that the C-terminal domain may strongly affect the activity of RecQ5 尾 and its binding affinity with DNA substrate. The results show that the C-terminal domain may strongly affect the despin activity and binding affinity of RecQ 5 尾 1-467 with the truncated RecQ 5 尾 1-467. These conclusions may be helpful for further elucidating the physiological function and working mechanism of RecQ5 尾 helicase.
【學位授予單位】：中國科學院大學(中國科學院物理研究所)
【學位級別】：博士
【學位授予年份】：2017
【分類號】：Q523
本文編號：2459972
[Abstract]:G4 structure is a special DNA secondary structure in human telomere terminal. The folding kinetics of G4 on its biological function, such as the maintenance of telomere stability and the protection of chromosome terminus, and so on. There are many kinds of G4 structure, which are greatly influenced by environmental factors, such as ion species, solution crowding and so on. In this work, we mainly studied the folding kinetics of G4 DNA in different monovalent cation environments, and measured the specific kinetic parameters of G4 folding induced by Na and K, and the antiparallel-basket configuration of G4 from Na condition. The kinetic process of the structure change of the hybrid configuration under K condition. More interestingly, although in previous experimental studies on G4 folding, Li was often used as a control experiment to provide a similar ionic environment, its synergistic effect on G4 folding under Na or K conditions was not taken into account, however, We found that Li did significantly affect the kinetic process of G4 folding induced by Na and K in different ways. For example, Li could change the ratio of Na-induced G4 folding. Moreover, K-induced folding rate of G _ 4 was increased to a great extent. We are now working on a new account of the effect of monovalent cations on the folding kinetics of G4, And it should be helpful to study the potential folding mechanism of G4 in the future. RecQ5 尾-racemase is an essential RecQ helicase in human body, which can replicate in DNA and repair DNA. DNA recombination and RNA transcription play a very important role. Up to now, we have not made great progress in the study of the activity and substrate specificity of RecQ5 尾 helicase. In this work, we measured the desolation kinetics and dissociation kinetics of RecQ5 尾-helicase on different structures of DNA substrates by rapid stop-flow method. We found that RecQ5 尾 helicase can efficiently decompose the single and double stranded DNA, fork DNA and the Holiday linker DNA,. However, the efficiency of cleavage of DNA and G4 structure DNA is very low. The specificity of RecQ5 尾 helicase is probably related to the cross site of DNA substrate, and the binding of RecQ5 尾 helicase to cross site should be very close. And it can promote the improvement of self-despinning activity. Furthermore, from the comparison between full-length RecQ5 尾 fl and truncated RecQ 5 尾 1-467, we can see that the C-terminal domain may strongly affect the activity of RecQ5 尾 and its binding affinity with DNA substrate. The results show that the C-terminal domain may strongly affect the despin activity and binding affinity of RecQ 5 尾 1-467 with the truncated RecQ 5 尾 1-467. These conclusions may be helpful for further elucidating the physiological function and working mechanism of RecQ5 尾 helicase.
【學位授予單位】：中國科學院大學(中國科學院物理研究所)
【學位級別】：博士
【學位授予年份】：2017
【分類號】：Q523
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