G4 DNA折疊及RecQ5β對(duì)不同DNA解旋的動(dòng)理學(xué)研究
發(fā)布時(shí)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(jí)結(jié)構(gòu),其折疊動(dòng)理學(xué)對(duì)它們的生物學(xué)功能,如在端粒穩(wěn)定性的保持和染色體末端的保護(hù)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(jià)陽(yáng)離子環(huán)境中的折疊動(dòng)理學(xué),并測(cè)定了Na+和K+誘導(dǎo)的G4折疊的具體的動(dòng)理學(xué)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(shí)的動(dòng)理學(xué)過程。更有趣的是,雖然在以前的關(guān)于G4折疊的實(shí)驗(yàn)研究中,Li+經(jīng)常被用作對(duì)照實(shí)驗(yàn)以提供類似的離子環(huán)境,其對(duì)Na+或K+條件下的G4折疊的協(xié)同作用并沒有被考慮進(jìn)去,但是,我們發(fā)現(xiàn),Li+確實(shí)能夠顯著地、以不同的方式影響到Na+和K+誘導(dǎo)的G4折疊的動(dòng)理學(xué)過程,例如,Li+可以改變Na+誘導(dǎo)的G4折疊的比例,并且很大程度地提高K+誘導(dǎo)的G4折疊的速率。我們現(xiàn)在的工作可以對(duì)單價(jià)陽(yáng)離子對(duì)G4折疊動(dòng)理學(xué)產(chǎn)生的影響進(jìn)行新的闡述,并且應(yīng)該會(huì)對(duì)未來G4潛在的折疊機(jī)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(fù)制、DNA修復(fù)、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(duì)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(jìn)展。在本研究工作中,我們使用快速停流方法測(cè)量了RecQ5β解旋酶對(duì)不同結(jié)構(gòu)的DNA底物的解旋動(dòng)理學(xué)和解離動(dòng)理學(xué)。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(diǎn)有關(guān),RecQ5β解旋酶與交叉位點(diǎn)的結(jié)合應(yīng)該非常緊密,并且能夠很好地促進(jìn)自身解旋活性的提高。而且,從全長(zhǎng)型RecQ5βfl與截?cái)嘈蚏ecQ5β1-467的解旋和解離動(dòng)理學(xué)的比較中,我們可以看出,C-末端結(jié)構(gòu)域可能會(huì)強(qiáng)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(huì)對(duì)進(jìn)一步闡明RecQ5β解旋酶的生理功能和工作機(jī)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(xué)位授予單位】：中國(guó)科學(xué)院大學(xué)(中國(guó)科學(xué)院物理研究所)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q523
本文編號(hào)：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(xué)位授予單位】：中國(guó)科學(xué)院大學(xué)(中國(guó)科學(xué)院物理研究所)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q523
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