結(jié)核分枝桿菌螺旋酶GyrA與GyrB互作區(qū)域的缺失突變體對酶功能的影響
發(fā)布時間：2018-11-25 12:06
【摘要】：DNA螺旋酶是唯一一種可以引入DNA負(fù)超螺旋的type II拓?fù)洚悩?gòu)酶,它是原核細(xì)胞所必須的基本酶類,參與了細(xì)胞內(nèi)DNA的生理活動,如DNA復(fù)制、轉(zhuǎn)錄、基因重組、染色體重組等生理活動,因人體不存在DNA螺旋酶,螺旋酶被開發(fā)作為很多抗癌藥物、抗生素類藥物、多肽類蛋白的作用靶標(biāo),如喹喏T美嘁┪鎩被愣顧乩嘁┪鎩fpA Mt和QnrB4等。但是由于標(biāo)準(zhǔn)抗結(jié)核藥物已持續(xù)使用了幾十年,使得結(jié)核分枝桿菌這些藥物的耐藥性越來越大,產(chǎn)生了耐多藥結(jié)核病和廣泛耐藥結(jié)核病,給結(jié)核病的治療帶來了很大的困難,人們需要開發(fā)更有效、更有針對性的藥物。 DNA螺旋酶是由GyrA和GyrB兩個亞基組成的四聚體,單獨(dú)的GyrA或GyrB亞基不發(fā)揮其酶活特性,只有重組后才具有酶活功效。為了針對結(jié)核病的治療開發(fā)有效的藥物,我們需要藥物作用靶標(biāo)——DNA螺旋酶更有具體的信息。目前,對螺旋酶的GyrA和GyrB亞基的相互作用區(qū)域、結(jié)構(gòu)、位點(diǎn)等的研究并不是很深入,為了研究GyrB與GyrA亞基的相互作用位點(diǎn),對構(gòu)建的一系列GyrB亞基的突變體進(jìn)行了全酶酶活性的測定,以確定GyrB和GyrA亞基的相互作用區(qū)域。本課題研究結(jié)果如下： 1、野生型GyrA亞基、GyrB亞基、GyrB亞基一系列突變體(GyrB1-518、 GyrB1-531、GyrB△531-550、GyrB△548-564、GyrB-CTD、GyrB-NTD)的誘導(dǎo)表達(dá)、純化、濃縮、濃度測定。 2、確定本實(shí)驗(yàn)室條件下測定DNA螺旋酶松弛活性和切割活性的反應(yīng)條件和反應(yīng)體系的主要成分,松弛活性為37℃、30min,切割活性為37℃、20min,反應(yīng)體系中亞精胺、DTT、tRNA為必須成分,EDTA可以省略。 3、GyrB亞基C端是其與GyrA的相互作用的關(guān)鍵區(qū)域,531-550和548-564位氨基酸在松弛活性和切割活性中的重要作用,它們均位于GyrB亞基Toprim結(jié)構(gòu)域中,Toprim結(jié)構(gòu)域是GyrA和GyrB相互作用的關(guān)鍵結(jié)構(gòu)域。對其中的位點(diǎn)進(jìn)行缺失突變后發(fā)現(xiàn),531-550區(qū)域含有DxD的功能模式位點(diǎn),它的作用同時表現(xiàn)在對酶活的影響和結(jié)構(gòu)的支撐上；而548-564位氨基酸的功能更多的是表現(xiàn)在對結(jié)構(gòu)的支撐和構(gòu)造上。
[Abstract]:DNA helicase is the only type II topoisomerase that can introduce DNA negative superhelix. It is the basic enzyme necessary for prokaryotic cells and participates in the physiological activities of intracellular DNA, such as DNA replication, transcription, gene recombination. Chromosome recombination and other physiological activities, because the human body does not exist DNA helicase, helicase has been developed as a variety of anticancer drugs, antibiotics, polypeptide protein targets, such as quine T scalloping? By? FpA Mt, QnrB4, etc. However, because the standard anti-tuberculosis drugs have been used for decades, the drug resistance of Mycobacterium tuberculosis has become more and more serious, resulting in multi-drug-resistant tuberculosis and widely drug-resistant tuberculosis, which has brought great difficulties to the treatment of tuberculosis. People need to develop more effective and targeted drugs. DNA helicase is a tetramer composed of two subunits of GyrA and GyrB. The single GyrA or GyrB subunit does not play its enzyme activity characteristic, only after recombination has the enzyme activity function. In order to develop effective drugs for the treatment of tuberculosis, we need more specific information about the target of drug action-DNA helicase. In order to study the interaction sites between GyrB and GyrA subunits, the interaction regions, structures and loci of GyrA and GyrB subunits of helicases are not well studied. The total enzyme activity of a series of GyrB subunit mutants was determined to determine the interaction region between GyrB and GyrA subunits. The results are as follows: 1. A series of mutants (GyrB1-518, GyrB1-531,GyrB 531-550 GyrB 548-564GyrB-CTDGyrB-NTD) of wild type GyrA subunits, GyrB subunits and GyrB subunits were induced, purified and concentrated. Concentration determination. 2. The reaction conditions and main components of DNA helicase relaxation activity and cleavage activity were determined in our laboratory. The relaxation activity was 37 鈩，
本文編號：2356042
[Abstract]:DNA helicase is the only type II topoisomerase that can introduce DNA negative superhelix. It is the basic enzyme necessary for prokaryotic cells and participates in the physiological activities of intracellular DNA, such as DNA replication, transcription, gene recombination. Chromosome recombination and other physiological activities, because the human body does not exist DNA helicase, helicase has been developed as a variety of anticancer drugs, antibiotics, polypeptide protein targets, such as quine T scalloping? By? FpA Mt, QnrB4, etc. However, because the standard anti-tuberculosis drugs have been used for decades, the drug resistance of Mycobacterium tuberculosis has become more and more serious, resulting in multi-drug-resistant tuberculosis and widely drug-resistant tuberculosis, which has brought great difficulties to the treatment of tuberculosis. People need to develop more effective and targeted drugs. DNA helicase is a tetramer composed of two subunits of GyrA and GyrB. The single GyrA or GyrB subunit does not play its enzyme activity characteristic, only after recombination has the enzyme activity function. In order to develop effective drugs for the treatment of tuberculosis, we need more specific information about the target of drug action-DNA helicase. In order to study the interaction sites between GyrB and GyrA subunits, the interaction regions, structures and loci of GyrA and GyrB subunits of helicases are not well studied. The total enzyme activity of a series of GyrB subunit mutants was determined to determine the interaction region between GyrB and GyrA subunits. The results are as follows: 1. A series of mutants (GyrB1-518, GyrB1-531,GyrB 531-550 GyrB 548-564GyrB-CTDGyrB-NTD) of wild type GyrA subunits, GyrB subunits and GyrB subunits were induced, purified and concentrated. Concentration determination. 2. The reaction conditions and main components of DNA helicase relaxation activity and cleavage activity were determined in our laboratory. The relaxation activity was 37 鈩，
本文編號：2356042
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