耐辐射奇球菌DNA末端切割过程的结构与功能学研究

发布时间：2018-01-15 17:27

  本文关键词：耐辐射奇球菌DNA末端切割过程的结构与功能学研究　出处：《浙江大学》2016年博士论文　论文类型：学位论文

  更多相关文章： 耐辐射奇球菌 DNA末端切割 RecJ NurA HerA 同源重组 晶体结构

【摘要】：耐辐射奇球菌作为迄今为止发现的最具辐射抗性的生物之一,其强大的DNA损伤修复能力一直被作为研究的焦点。同源重组是细菌中修复DNA双链断裂的重要途径。细菌的同源重组主要分为RecBCD和RecFOR两个系统。耐辐射奇球菌天生不具备RecBCD途径,只含RecFOR系统,因而耐辐射奇球菌成为研究RecFOR系统最理想的模式菌种。同源重组开始阶段有一个重要的过程,即通过一些核酸酶/解旋酶处理损伤的DNA末端,形成3’单链DNA末端,继而入侵同源链,造成链交换发生同源重组。此过程在RecBCD系统中已经研究得比较透彻,而在RecFOR系统中却不甚明确。目前普遍认为,核酸酶RecJ、解旋酶RecQ和单链结合蛋白SSB参与了RecFOR系统中的这个过程。为了研究该过程的分子机制,本文解析了drRecJ与底物DNA复合体结构,drRecJ与产物(dTMP)复合体结构,以及SSBC末端肽段与drRecJ的复合体结构,并进行了相应的生化实验验证。drRecJ利用双金属离子催化机制,其底物ssDNA末端碱基与倒数第二个碱基形成180度U形拐角,有利于活性位点对磷酸骨架的固定及打断。活性位点的5’磷酸结合口袋保证了drRecJ对5’端底物ssDNA的识别,以及5’-3’外切酶方向的特异性。位于DNA进口处的三个α螺旋构成的“门”,保证了drRecJ只能容纳单链DNA进入活性位点。活性中心区域DNA结合通道以及OB结构域多个残基对DNA的稳定结合,保证了DNA的高效运输以及切割的连续性。SSB C末端肽段结合在drRecJC端结构域的一个疏水口袋里,解释了SSB对RecJ的招募以及活性加强机制。RecQ的加入,促进了drRecJ对带有3'overhang双链DNA的切割,完美地演绎了RecJ, SSB和RecQ三者参与的RecFOR系统对任何形式DNA末端切割的过程。另一方面,本文发现drRecJ的C端结构域还能与HerA互作。HerA,作为一个ATP酶/DNA运输酶,与另一个核酸酶,NurA,一起被认为参与了古菌同源重组系统的DNA切割过程。本文研究了耐辐射奇球菌HerA和NurA蛋白的生化及遗传学特征。我们发现,和古菌同源蛋白类似,drHerA具备ATP酶活性,而drNurA则表现出Mn依赖性的5’-3’单链/双链DNA外切/内切酶活性。两蛋白各自的活性均能被对方激活。并且,drHerA的N端HAS结构域被鉴定出来能与drNurA直接互作。herA, nurA敲除菌株均表现出生长变快(尤其在37℃高温下),对丝裂霉素C抗性增强的现象,与recJ突变株的生长变慢以及丝裂霉素C极其敏感正好相反。体外生化实验显示,HerA可以增强RecJ活性,然而NurA的加入可以抑制这种增强。结合表型结果,我们认为,HerA, NurA这类古菌普遍存在的DNA末端处理蛋白出现在耐辐射球菌中,可能对耐辐射奇球菌的RecFOR系统有一种调节作用,具体的调节机制还有待未来进一步探索。
[Abstract]:As one of the most radiation-resistant organisms found to date, Radiococci is one of the most radiation-resistant organisms in the world. Homologous recombination is an important way to repair DNA double strand breaks in bacteria. Homologous recombination of bacteria is mainly divided into RecBCD and RecFO. S. radiodurans have no RecBCD pathway. Due to the presence of only RecFOR system, S. radiodurans has become the most ideal model strain for the study of RecFOR system. There is an important process in the initial stage of homologous recombination. In other words, the damaged DNA terminal was treated with some nucleases / helicases to form a 3'single-stranded DNA terminal, and then to invade the homologous chain. This process has been thoroughly studied in RecBCD system, but not very clear in RecFOR system. It is widely believed that nuclease RecJ. Helicase RecQ and single-stranded binding protein SSB are involved in this process in the RecFOR system. In this paper, the structure of drRecJ / substrate DNA complex, the complex structure of SSBC terminal peptide and drRecJ, and the structure of SSBC terminal peptide with drRecJ were analyzed. The corresponding biochemical experiments were carried out to verify that the substrate ssDNA terminal base and the reciprocal second base formed 180-degree U-shaped corner using bimetallic ion catalysis mechanism. The 5'phosphoric acid binding pocket of the active site ensures the recognition of 5'substrate ssDNA by drRecJ. And the specificity of 5 '-3'exonuclease direction. The "door" is composed of three 伪 helix at the entrance of DNA. The drRecJ can only accommodate single strand DNA into the active site, the DNA binding channel in the active center region and the stable binding of multiple residues to DNA in the OB domain. It ensures the efficient transport of DNA and the continuity of cleavage. SSB C-terminal peptides are bound in a hydrophobic pocket of the drRecJC terminal domain. It was explained that the recruitment of RecJ by SSB and the addition of reactivity enhancement mechanism. RecQ promoted the drRecJ to cut the double-stranded DNA with 3o overhang. It perfectly deduces the process of RecFOR system in which Recj, SSB and RecQ participate in any form of DNA end cutting. On the other hand. It is found that the C-terminal domain of drRecJ can also interact with HerA as a transporter of ATP and another nuclease. This paper studies the biochemical and genetic characteristics of HerA and NurA proteins of A. radiodurans. Similar to the homologous protein of Archaea, Hera has ATP enzyme activity. On the other hand, drNurA showed Mn-dependent activity of 5 '-3'single / double stranded DNA exonuclease / endonuclease. The activity of each protein was activated by each other. The N-terminal HAS domain of drHerA was identified to interact directly with drNurA. HerA and nurA knockout strains all showed rapid growth (especially at 37 鈩，

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