本文關鍵詞： 綠膿桿菌噬菌體PaP1 DNA聚合酶 穩(wěn)態(tài)動力學 穩(wěn)態(tài)前動力學 核苷酸插入　出處：《重慶理工大學》2017年碩士論文　論文類型：學位論文

【摘要】：綠膿桿菌(Pseudomonas aeruginosa)是一種條件致病的耐藥菌,可引起人類多種感染性疾病。近年來由于抗生素的不合理使用甚至濫用,臨床治療比較困難,缺乏有效的抗菌制劑。噬菌體具有宿主特異性的特點,可以應用噬菌體治療多重耐藥菌的感染。最近本實驗室分離出一株綠膿桿菌的裂解性噬菌體PaP1,它能有效侵染并裂解綠膿桿菌,噬菌體基因90編碼的蛋白(gp90)是DNA聚合酶,其可以快速復制產(chǎn)生大量子代噬菌體,并殺死其宿主。但噬菌體DNA復制時會遇到DNA損傷,可能影響DNA復制,進一步會影響噬菌體侵染和裂解綠膿桿菌的能力。為了更好的理解噬菌體的侵染機制和復制效率,我們在體外表達純化噬菌體PaP1的DNA聚合酶gp90,研究DNA聚合酶進行DNA復制以及通過8-oxoG和O~6-MeG兩種DNA損傷的動力學機制。研究目的:本課題研究目的在于構建、表達和純化沒有外切酶活性的綠膿桿菌噬菌體PaP1的DNA聚合酶,在分子層面上研究其進行DNA復制以及跨DNA損傷復制的動力學機制,有助于噬菌體藥物的研發(fā)以及指導合理用藥。研究方法:(1)構建、表達和純化沒有外切酶活性的gp90。本課題組前期已經(jīng)發(fā)現(xiàn)噬菌體PaP1 DNA聚合酶gp90具有單鏈DNA和雙鏈DNA外切酶活性。進行體外動力學實驗,為了排除對錯配切除的影響,僅僅研究聚合酶本征的性質,需要去除外切酶活性。首先構建質粒,其可以表達消除外切酶的DNA聚合酶突變體,然后用IPTG誘導表達,利用鎳柱純化帶有組氨酸標簽的gp90及其突變體。(2)DNA聚合酶參與DNA復制以及通過8-oxoG損傷的動力學機制研究。通過穩(wěn)態(tài)動力學方法,研究全長延伸、單個核苷酸插入和下一位堿基延伸中8-oxoG對DNA復制效率和保真度的影響;采用穩(wěn)態(tài)前動力學方法研究DNA復制通過8-oxoG損傷時單點插入效率;采用生物物理相互作用研究方法,研究8-oxoG對DNA聚合酶與DNA相互作用的影響。(3)DNA聚合酶通過O~6-MeG的動力學機制研究。通過穩(wěn)態(tài)動力學方法,研究全長延伸、單個核苷酸插入以及下一位堿基延伸中O~6-MeG對DNA復制效率和保真度的影響;利用穩(wěn)態(tài)前動力學方法,研究單個核苷酸插入的效率;采用生物物理相互作用研究方法,研究O~6-MeG對聚合酶和DNA相互作用的影響;采用stopped-flow Auto SF-120快速熒光動力學方法研究O~6-MeG對聚合酶構象改變的影響機制。研究結果:(1)根據(jù)生物信息學分析預測外切酶活性位點,通過點突變試劑盒將gp90第60位谷氨酸、第137位天冬氨酸和第234位天冬氨酸分別突變成丙氨酸。然后低溫誘導蛋白表達并用鎳柱純化。用32P同位素標記技術檢測外切酶和聚合酶活性。結果發(fā)現(xiàn)只有gp90 D234A既消除了外切酶活性又保留相當?shù)木酆厦富钚?稱為gp90 exo-。(2)DNA聚合酶gp90 exo-進行DNA復制時,與正常模板G相比,DNA聚合酶通過8-oxoG和O~6-MeG時,延伸產(chǎn)物明顯減少,并伴隨著中間產(chǎn)物生成,這說明DNA氧化損傷和甲基化損傷都會部分抑制gp90 exo-聚合反應。(3)DNA聚合酶在正常模板G上插入dNTP時,采用穩(wěn)態(tài)動力學方法,相對于正配dCTP,發(fā)現(xiàn)單個核苷酸錯配插入的催化效率kcat基本沒變,而Km值增加了103倍,導致錯配率在10-4-10-5之間。對于8-oxoG,DNA聚合酶插入四種核苷酸的效率都降低,但錯配率依然保持在10-4-10-5之間,錯配時Km增加且kcat值減小。通過O~6-MeG損傷時,DNA聚合酶插入四種核苷酸的效率降低,錯配率也增加,尤其是更傾向于錯誤插入dTTP,其插入效率是正配dCTP的67倍,原因是Km減小且kcat值增加。(4)對于錯配與正配對下一位堿基延伸反應的影響,相對于G:C配對,發(fā)現(xiàn)G:A配對導致下一位堿基延伸效率降低8倍,G:T配對效率降低50倍,其中Km基本沒有改變,kcat值降低;對于8-oxoG,不管錯配與否,對下一位堿基插入基本沒有任何影響;O~6-MeG錯誤插入dTTP,反而促進下一位堿基的延伸。(5)DNA聚合酶進行單個dNTP插入的穩(wěn)態(tài)前動力學研究,相對于其它錯配dNTP,正配dCTP優(yōu)先插入到G的對位,并顯示出快速相,說明dCTP插入速率快于DNA聚合酶從DNA上解離的速率。在G的對位插入dATP、dTTP和dGTP,其延伸產(chǎn)物隨著時間呈線性關系。dCTP和dATP優(yōu)先插入8-oxoG的對位并產(chǎn)生快速相,而插入dTTP和dGTP時,其延伸產(chǎn)物隨著時間呈線性關系;dCTP和dTTP優(yōu)先插入O~6-MeG的對位并產(chǎn)生快速相,而插入dATP和dGTP時,其延伸產(chǎn)物隨著時間呈線性關系。(6)DNA聚合酶與DNA的解離常數(shù)(Kd,DNA)用SPR方法測定,研究發(fā)現(xiàn)8-oxoG和O~6-MeG損傷不影響gp90 exo-與DNA的相互作用;但是存在dNTP和Mg2+時有助于DNA聚合酶與DNA的結合,而與正常G相比,8-oxoG和O~6-MeG都會削弱DNA聚合酶和DNA的結合;而錯配會進一步削弱這種結合能力。(7)對于在O~6-MeG對位優(yōu)先插入dTTP,采用SF-120檢測DNA聚合酶的構象改變,發(fā)現(xiàn)插入dTTP時,gp90 exo--DNA-dTTP三元復合物中DNA聚合酶的構象改變很快,從而加快了DNA的復制速度。
[Abstract]:Pseudomonas aeruginosa (Pseudomonas aeruginosa) is a kind of opportunistic drug-resistant bacteria, can cause a variety of human infectious diseases. In recent years, due to the unreasonable use of antibiotics and even abuse, clinical treatment is difficult, the lack of effective antimicrobial agents. With the host specificity of phage, phage can be used treatment of multi drug resistant bacteria infection. Recently, our laboratory isolated a strain of Pseudomonas aeruginosa lytic phage PaP1, which can effectively infect and lyse Pseudomonas aeruginosa phage gene encoding protein 90 (gp90) DNA polymerase, which can produce a large number of progeny phage replication rapidly, and kill its host. But the replication of bacteriophage DNA will encounter DNA injury. May affect the replication of DNA, further will affect the ability of phage infection and lysis of Pseudomonas aeruginosa. In order to better understand the mechanism of infection and phage replication efficiency, in our body appearance As purified phage PaP1 DNA polymerase gp90 of DNA polymerase for DNA replication and dynamic mechanism by 8-oxoG and O~6-MeG two DNA injury. Objective: the purpose of this research is to construct the expression and purification of DNA polymerase, no exonuclease activity of Pseudomonas aeruginosa phage PaP1, study the dynamic mechanism of DNA replication and cross DNA copy the injury at the molecular level, help phage drug development and guide the rational use of drugs. Methods: (1) construction, expression and purification of gp90. exonuclease activity without the prior period have been found PaP1 phage DNA polymerase gp90 with single stranded DNA and double stranded DNA exonuclease activity. In vitro kinetic experiments, in order to to eliminate the effect of mismatch removal, only on the intrinsic nature of the polymerase and exonuclease activity. The need to remove the first plasmid, the expression can be eliminated DNA exonuclease polymerase mutant, and then induced by IPTG. The use of nickel column purification of gp90 and its mutants with a histidine tag. (2) DNA polymerase in DNA replication and the study of mechanism of 8-oxoG injury. By the steady-state kinetic method of full length extension, single nucleotide insertion and a base extension in the effect of 8-oxoG on DNA replication efficiency and fidelity; using pre steady state kinetic studies of DNA replication by 8-oxoG method of injury of single point insertion efficiency; using the research methods of biological physical interactions, 8-oxoG poly synthase and DNA interaction on DNA. (3) through the research on the dynamic mechanism of DNA polymerase O~6-MeG. By steady-state kinetics method, research full length extension, single nucleotide insertion and a base extension in the O~6-MeG effect on DNA replication efficiency and fidelity; using the pre steady state kinetic Study on the efficiency of single nucleotide insertion method; using the research methods of biological physical interactions and effects of O~6-MeG on the polymerase and the interaction of DNA with stopped-flow Auto SF-120; fast fluorescence kinetics method study on the effects of O~6-MeG on polymerase conformational change mechanism. The results of the study: (1) according to the bioinformatics analysis and prediction of exonuclease activity sites, will gp90 sixtieth glutamic acid by point mutation kit, 137th aspartic acid and 234Th aspartic acid were mutated into alanine. Then low temperature induced protein was expressed and purified by nickel column. Using 32P isotope labeling technique for the detection of exonuclease and polymerase activity. The results showed that only gp90 D234A can eliminate the exonuclease activity retains considerable polymerase activity gp90, known as exo-. (2) DNA gp90 exo- polymerase for DNA replication, compared with the normal template G, DNA polymerase by 8-oxoG and O ~6-MeG, the extension product was significantly reduced, and accompanied by the intermediate product, which indicates that the DNA oxidative damage and methylation damage can partially inhibit the polymerization reaction. Gp90 exo- (3) DNA polymerase dNTP is inserted in normal template G, using steady-state kinetics method, compared to positive with dCTP, kcat found that the catalytic efficiency of single nucleotide. With the insertion of the basic did not change, but the Km value increased by 103 times, resulting in mismatch rate between 10-4-10-5. For 8-oxoG, DNA polymerase four nucleotide insertion efficiency are decreased, but the mismatch rate remained at between 10-4-10-5, Km and kcat increased when the mismatch value decreases. By O~6-MeG damage, reduce the efficiency DNA polymerase insertion of four nucleotides, the mismatch rate also increased, especially the more prone to error into dTTP, the insertion efficiency is 67 times is dCTP, the reason is that Km decreased and kcat increased. (4) the mismatch and a base pairing Effect of base extension reaction, relative to the G:C pairing, G:A found a paired lead base extension efficiency is reduced by 8 times, 50 times lower efficiency of G:T pairing, which Km has not changed, the decrease of kcat value; for 8-oxoG, regardless of the mismatch or not, on a base insertion basically no effect; O~6-MeG insertion error dTTP, but to promote the extension under a base. (5) DNA polymerase with single dNTP inserted before the steady state kinetic studies, compared to other mismatch dNTP, is inserted into the G dCTP priority position, and show a rapid rate, dCTP insertion rate is faster than the DNA polymerase from DNA insertion dissociation. DATP in G dTTP and dGTP, the position, extension product with time showed linear relationship between.DCTP and dATP on the first insert 8-oxoG and generate fast phase, and inserted into dTTP and dGTP, the extension product showed a linear relationship with time; dCTP and dTTP. On the insert O~6-MeG and generate fast phase, and the insertion of dATP and dGTP, the extension product showed a linear relationship with time. (6) the dissociation constant of DNA polymerase and DNA (Kd, DNA) was determined by SPR method, the study found that the interaction between 8-oxoG and O~6-MeG damage does not affect the gp90 and DNA exo-; but there are combined dNTP and Mg2+ help DNA polymerase and DNA, and compared with the normal G, the combination of 8-oxoG and O~6-MeG will weaken the DNA polymerase and DNA; and the mismatch will further weaken the binding ability. (7) for the O~6-MeG para preferred to insert dTTP, a conformational change was detected by SF-120 DNA polymerase, found insertion dTTP, the conformation of three yuan compound gp90 exo--DNA-dTTP DNA polymerase change quickly, thereby speeding up the replication rate of DNA.

【學位授予單位】：重慶理工大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R915

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