Analysis of DNA relaxation and cleavage activities of recombinant Mycobacterium tuberculosis DNA topoisomerase I from a new expression and purification protocol.
Analysis of DNA relaxation and cleavage activities of recombinant Mycobacterium tuberculosis DNA topoisomerase I from a new expression and purification protocol.
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DOI：
10.1186/1471-2091-10-18
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发表时间：
2009-06-11
期刊：
影响因子：
--
通讯作者：
Tse-Dinh YC
中科院分区：
文献类型：
--
作者：
Annamalai T;Dani N;Cheng B;Tse-Dinh YC
结核分枝杆菌 DNA 拓扑异构酶 I 是发现新型结核病药物的一个有吸引力的靶标，这些药物通过增强拓扑异构酶-DNA 裂解产物的积累来发挥作用。它与其他 IA 型 DNA 拓扑异构酶共享一个共同的酯交换结构域。然而，大肠杆菌和结核分枝杆菌 DNA 拓扑异构酶 I 蛋白的 C 端 DNA 结合域之间没有同源性。一种用于表达和纯化重组结核分枝杆菌 DNA 拓扑异构酶 I (MtTOP) 的新方案已被开发出来，以产生比先前表征的重组酶具有更高比活性的酶。研究发现，在从部分松弛的 DNA 中去除剩余的负超螺旋方面，MtTOP 的效率低于大肠杆菌 DNA 拓扑异构酶 I (EcTOP)。首次表征了 MtTOP 对 DNA 的切割。 DNA 切割位点选择性与 EcTOP 的比较表明切割位点偏好存在差异，但两种酶的首选位点在 -4 位都有一个 C 核苷酸。重组结核分枝杆菌 DNA 拓扑异构酶 I 可以表达为可溶性蛋白质，并通过新方案从大肠杆菌宿主中高产量纯化。用结核分枝杆菌 DNA 底物进行的 DNA 切割分析表明，优选的 DNA 切割位点在 -4 位有一个 C 核苷酸。
Mycobacterium tuberculosis DNA topoisomerase I is an attractive target for discovery of novel TB drugs that act by enhancing the accumulation of the topoisomerase-DNA cleavage product. It shares a common transesterification domain with other type IA DNA topoisomerases. There is, however, no homology between the C-terminal DNA binding domains of Escherichia coli and M. tuberculosis DNA topoisomerase I proteins. A new protocol for expression and purification of recombinant M. tuberculosis DNA topoisomerase I (MtTOP) has been developed to produce enzyme of much higher specific activity than previously characterized recombinant enzyme. MtTOP was found to be less efficient than E. coli DNA topoisomerase I (EcTOP) in removal of remaining negative supercoils from partially relaxed DNA. DNA cleavage by MtTOP was characterized for the first time. Comparison of DNA cleavage site selectivity with EcTOP showed differences in cleavage site preferences, but the preferred sites of both enzymes have a C nucleotide in the -4 position. Recombinant M. tuberculosis DNA topoisomerase I can be expressed as a soluble protein and purified in high yield from E. coli host with a new protocol. Analysis of DNA cleavage with M. tuberculosis DNA substrate showed that the preferred DNA cleavage sites have a C nucleotide in the -4 position.
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影响因子：
14.9
作者：
Goulaouic, H;Roulon, T;Riou, JF
通讯作者：
Riou, JF
影响因子：
4.8
作者：
Viard, T;Lamour, V;de la Tour, CB
通讯作者：
de la Tour, CB
影响因子：
11.8
作者：
Hooper, DC
通讯作者：
Hooper, DC
DOI：
10.1046/j.1432-1327.1999.00128.x
发表时间：
1999-02-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
Jaxel, C;Duguet, M;Nadal, M
通讯作者：
Nadal, M
影响因子：
5.6
作者：
WANG, JC
通讯作者：
WANG, JC