本文關(guān)鍵詞：耐輻射奇球菌DNA末端切割過(guò)程的結(jié)構(gòu)與功能學(xué)研究　出處：《浙江大學(xué)》2016年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 耐輻射奇球菌 DNA末端切割 RecJ NurA HerA 同源重組 晶體結(jié)構(gòu)

【摘要】：耐輻射奇球菌作為迄今為止發(fā)現(xiàn)的最具輻射抗性的生物之一,其強(qiáng)大的DNA損傷修復(fù)能力一直被作為研究的焦點(diǎn)。同源重組是細(xì)菌中修復(fù)DNA雙鏈斷裂的重要途徑。細(xì)菌的同源重組主要分為RecBCD和RecFOR兩個(gè)系統(tǒng)。耐輻射奇球菌天生不具備RecBCD途徑,只含RecFOR系統(tǒng),因而耐輻射奇球菌成為研究RecFOR系統(tǒng)最理想的模式菌種。同源重組開(kāi)始階段有一個(gè)重要的過(guò)程,即通過(guò)一些核酸酶/解旋酶處理?yè)p傷的DNA末端,形成3’單鏈DNA末端,繼而入侵同源鏈,造成鏈交換發(fā)生同源重組。此過(guò)程在RecBCD系統(tǒng)中已經(jīng)研究得比較透徹,而在RecFOR系統(tǒng)中卻不甚明確。目前普遍認(rèn)為,核酸酶RecJ、解旋酶RecQ和單鏈結(jié)合蛋白SSB參與了RecFOR系統(tǒng)中的這個(gè)過(guò)程。為了研究該過(guò)程的分子機(jī)制,本文解析了drRecJ與底物DNA復(fù)合體結(jié)構(gòu),drRecJ與產(chǎn)物(dTMP)復(fù)合體結(jié)構(gòu),以及SSBC末端肽段與drRecJ的復(fù)合體結(jié)構(gòu),并進(jìn)行了相應(yīng)的生化實(shí)驗(yàn)驗(yàn)證。drRecJ利用雙金屬離子催化機(jī)制,其底物ssDNA末端堿基與倒數(shù)第二個(gè)堿基形成180度U形拐角,有利于活性位點(diǎn)對(duì)磷酸骨架的固定及打斷�；钚晕稽c(diǎn)的5’磷酸結(jié)合口袋保證了drRecJ對(duì)5’端底物ssDNA的識(shí)別,以及5’-3’外切酶方向的特異性。位于DNA進(jìn)口處的三個(gè)α螺旋構(gòu)成的“門(mén)”,保證了drRecJ只能容納單鏈DNA進(jìn)入活性位點(diǎn)。活性中心區(qū)域DNA結(jié)合通道以及OB結(jié)構(gòu)域多個(gè)殘基對(duì)DNA的穩(wěn)定結(jié)合,保證了DNA的高效運(yùn)輸以及切割的連續(xù)性。SSB C末端肽段結(jié)合在drRecJC端結(jié)構(gòu)域的一個(gè)疏水口袋里,解釋了SSB對(duì)RecJ的招募以及活性加強(qiáng)機(jī)制。RecQ的加入,促進(jìn)了drRecJ對(duì)帶有3'overhang雙鏈DNA的切割,完美地演繹了RecJ, SSB和RecQ三者參與的RecFOR系統(tǒng)對(duì)任何形式DNA末端切割的過(guò)程。另一方面,本文發(fā)現(xiàn)drRecJ的C端結(jié)構(gòu)域還能與HerA互作。HerA,作為一個(gè)ATP酶/DNA運(yùn)輸酶,與另一個(gè)核酸酶,NurA,一起被認(rèn)為參與了古菌同源重組系統(tǒng)的DNA切割過(guò)程。本文研究了耐輻射奇球菌HerA和NurA蛋白的生化及遺傳學(xué)特征。我們發(fā)現(xiàn),和古菌同源蛋白類(lèi)似,drHerA具備ATP酶活性,而drNurA則表現(xiàn)出Mn依賴(lài)性的5’-3’單鏈/雙鏈DNA外切/內(nèi)切酶活性。兩蛋白各自的活性均能被對(duì)方激活。并且,drHerA的N端HAS結(jié)構(gòu)域被鑒定出來(lái)能與drNurA直接互作。herA, nurA敲除菌株均表現(xiàn)出生長(zhǎng)變快(尤其在37℃高溫下),對(duì)絲裂霉素C抗性增強(qiáng)的現(xiàn)象,與recJ突變株的生長(zhǎng)變慢以及絲裂霉素C極其敏感正好相反。體外生化實(shí)驗(yàn)顯示,HerA可以增強(qiáng)RecJ活性,然而NurA的加入可以抑制這種增強(qiáng)。結(jié)合表型結(jié)果,我們認(rèn)為,HerA, NurA這類(lèi)古菌普遍存在的DNA末端處理蛋白出現(xiàn)在耐輻射球菌中,可能對(duì)耐輻射奇球菌的RecFOR系統(tǒng)有一種調(diào)節(jié)作用,具體的調(diào)節(jié)機(jī)制還有待未來(lái)進(jìn)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 偽 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，

本文編號(hào)：1429344