绿脓杆菌噬菌体PaP1 DNA聚合酶通过DNA损伤的动力学机制研究

发布时间：2018-01-20 08:17

本文关键词： 绿脓杆菌噬菌体PaP1 DNA聚合酶稳态动力学稳态前动力学核苷酸插入　出处：《重庆理工大学》2017年硕士论文　论文类型：学位论文

【摘要】：绿脓杆菌(Pseudomonas aeruginosa)是一种条件致病的耐药菌,可引起人类多种感染性疾病。近年来由于抗生素的不合理使用甚至滥用,临床治疗比较困难,缺乏有效的抗菌制剂。噬菌体具有宿主特异性的特点,可以应用噬菌体治疗多重耐药菌的感染。最近本实验室分离出一株绿脓杆菌的裂解性噬菌体PaP1,它能有效侵染并裂解绿脓杆菌,噬菌体基因90编码的蛋白(gp90)是DNA聚合酶,其可以快速复制产生大量子代噬菌体,并杀死其宿主。但噬菌体DNA复制时会遇到DNA损伤,可能影响DNA复制,进一步会影响噬菌体侵染和裂解绿脓杆菌的能力。为了更好的理解噬菌体的侵染机制和复制效率,我们在体外表达纯化噬菌体PaP1的DNA聚合酶gp90,研究DNA聚合酶进行DNA复制以及通过8-oxoG和O~6-MeG两种DNA损伤的动力学机制。研究目的:本课题研究目的在于构建、表达和纯化没有外切酶活性的绿脓杆菌噬菌体PaP1的DNA聚合酶,在分子层面上研究其进行DNA复制以及跨DNA损伤复制的动力学机制,有助于噬菌体药物的研发以及指导合理用药。研究方法:(1)构建、表达和纯化没有外切酶活性的gp90。本课题组前期已经发现噬菌体PaP1 DNA聚合酶gp90具有单链DNA和双链DNA外切酶活性。进行体外动力学实验,为了排除对错配切除的影响,仅仅研究聚合酶本征的性质,需要去除外切酶活性。首先构建质粒,其可以表达消除外切酶的DNA聚合酶突变体,然后用IPTG诱导表达,利用镍柱纯化带有组氨酸标签的gp90及其突变体。(2)DNA聚合酶参与DNA复制以及通过8-oxoG损伤的动力学机制研究。通过稳态动力学方法,研究全长延伸、单个核苷酸插入和下一位碱基延伸中8-oxoG对DNA复制效率和保真度的影响;采用稳态前动力学方法研究DNA复制通过8-oxoG损伤时单点插入效率;采用生物物理相互作用研究方法,研究8-oxoG对DNA聚合酶与DNA相互作用的影响。(3)DNA聚合酶通过O~6-MeG的动力学机制研究。通过稳态动力学方法,研究全长延伸、单个核苷酸插入以及下一位碱基延伸中O~6-MeG对DNA复制效率和保真度的影响;利用稳态前动力学方法,研究单个核苷酸插入的效率;采用生物物理相互作用研究方法,研究O~6-MeG对聚合酶和DNA相互作用的影响;采用stopped-flow Auto SF-120快速荧光动力学方法研究O~6-MeG对聚合酶构象改变的影响机制。研究结果:(1)根据生物信息学分析预测外切酶活性位点,通过点突变试剂盒将gp90第60位谷氨酸、第137位天冬氨酸和第234位天冬氨酸分别突变成丙氨酸。然后低温诱导蛋白表达并用镍柱纯化。用32P同位素标记技术检测外切酶和聚合酶活性。结果发现只有gp90 D234A既消除了外切酶活性又保留相当的聚合酶活性,称为gp90 exo-。(2)DNA聚合酶gp90 exo-进行DNA复制时,与正常模板G相比,DNA聚合酶通过8-oxoG和O~6-MeG时,延伸产物明显减少,并伴随着中间产物生成,这说明DNA氧化损伤和甲基化损伤都会部分抑制gp90 exo-聚合反应。(3)DNA聚合酶在正常模板G上插入dNTP时,采用稳态动力学方法,相对于正配dCTP,发现单个核苷酸错配插入的催化效率kcat基本没变,而Km值增加了103倍,导致错配率在10-4-10-5之间。对于8-oxoG,DNA聚合酶插入四种核苷酸的效率都降低,但错配率依然保持在10-4-10-5之间,错配时Km增加且kcat值减小。通过O~6-MeG损伤时,DNA聚合酶插入四种核苷酸的效率降低,错配率也增加,尤其是更倾向于错误插入dTTP,其插入效率是正配dCTP的67倍,原因是Km减小且kcat值增加。(4)对于错配与正配对下一位碱基延伸反应的影响,相对于G:C配对,发现G:A配对导致下一位碱基延伸效率降低8倍,G:T配对效率降低50倍,其中Km基本没有改变,kcat值降低;对于8-oxoG,不管错配与否,对下一位碱基插入基本没有任何影响;O~6-MeG错误插入dTTP,反而促进下一位碱基的延伸。(5)DNA聚合酶进行单个dNTP插入的稳态前动力学研究,相对于其它错配dNTP,正配dCTP优先插入到G的对位,并显示出快速相,说明dCTP插入速率快于DNA聚合酶从DNA上解离的速率。在G的对位插入dATP、dTTP和dGTP,其延伸产物随着时间呈线性关系。dCTP和dATP优先插入8-oxoG的对位并产生快速相,而插入dTTP和dGTP时,其延伸产物随着时间呈线性关系;dCTP和dTTP优先插入O~6-MeG的对位并产生快速相,而插入dATP和dGTP时,其延伸产物随着时间呈线性关系。(6)DNA聚合酶与DNA的解离常数(Kd,DNA)用SPR方法测定,研究发现8-oxoG和O~6-MeG损伤不影响gp90 exo-与DNA的相互作用;但是存在dNTP和Mg2+时有助于DNA聚合酶与DNA的结合,而与正常G相比,8-oxoG和O~6-MeG都会削弱DNA聚合酶和DNA的结合;而错配会进一步削弱这种结合能力。(7)对于在O~6-MeG对位优先插入dTTP,采用SF-120检测DNA聚合酶的构象改变,发现插入dTTP时,gp90 exo--DNA-dTTP三元复合物中DNA聚合酶的构象改变很快,从而加快了DNA的复制速度。
[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.

【学位授予单位】：重庆理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R915

【参考文献】