G4 DNA折叠及RecQ5β对不同DNA解旋的动理学研究
发布时间：2019-04-18 10:20
【摘要】：G4结构是人类端粒末端存在的一种特殊的DNA二级结构,其折叠动理学对它们的生物学功能,如在端粒稳定性的保持和染色体末端的保护等方面,都有非常重要的影响。G4结构种类很多,受到环境因素的影响非常大,如离子种类、溶液的拥挤程度等。在本工作中,我们主要研究了G4 DNA在不同的单价阳离子环境中的折叠动理学,并测定了Na+和K+诱导的G4折叠的具体的动理学参数,和G4从Na+条件下的反平行-篮式构型,到K+条件下的杂交构型的结构变化时的动理学过程。更有趣的是,虽然在以前的关于G4折叠的实验研究中,Li+经常被用作对照实验以提供类似的离子环境,其对Na+或K+条件下的G4折叠的协同作用并没有被考虑进去,但是,我们发现,Li+确实能够显著地、以不同的方式影响到Na+和K+诱导的G4折叠的动理学过程,例如,Li+可以改变Na+诱导的G4折叠的比例,并且很大程度地提高K+诱导的G4折叠的速率。我们现在的工作可以对单价阳离子对G4折叠动理学产生的影响进行新的阐述,并且应该会对未来G4潜在的折叠机制方面的研究有一定帮助。RecQ5β解旋酶是人类体内存在的一种必不可少的RecQ解旋酶,它能够在DNA复制、DNA修复、DNA重组和RNA转录等过程中发挥非常重要的作用。到目前为止,我们对RecQ5β解旋酶的解旋活性和底物特异性方面的研究一直没有取得很大进展。在本研究工作中,我们使用快速停流方法测量了RecQ5β解旋酶对不同结构的DNA底物的解旋动理学和解离动理学。我们发现,RecQ5β解旋酶可以高效地解旋单双链DNA、岔口DNA和霍利迪连结体DNA,但其解旋平末端DNA和G4结构DNA的效率却很低。RecQ5β解旋酶的这种解旋底物特异性很可能与DNA底物具有的交叉位点有关,RecQ5β解旋酶与交叉位点的结合应该非常紧密,并且能够很好地促进自身解旋活性的提高。而且,从全长型RecQ5βfl与截断型RecQ5β1-467的解旋和解离动理学的比较中,我们可以看出,C-末端结构域可能会强烈影响RecQ5β的解旋活性和与DNA底物的结合亲和力。这些结论可能会对进一步阐明RecQ5β解旋酶的生理功能和工作机理有很大帮助。
[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.
【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】：Q523
本文编号：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.
【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】：Q523
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