Interaction of human DNA topoisomerase II α with DNA:: Quantification by surface plasmon resonance
Interaction of human DNA topoisomerase II α with DNA:: Quantification by surface plasmon resonance
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DOI：
10.1021/bi0263614
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发表时间：
2002-11-12
期刊：
影响因子：
2.9
通讯作者：
Sehested, M
中科院分区：
文献类型：
--
作者：
Renodon-Cornière, A;Jensen, LH;Sehested, M
DNA拓扑异构酶II是一种atp操作钳，通过捕获双链DNA片段并将其通过另一个双链传输来影响拓扑变化。利用表面等离子体共振（SPR）表征了人类拓扑异构酶II α与不同拓扑形式DNA的相互作用。利用pUC18 DNA的线性片段，确定拓扑异构酶II α的平衡结合常数为0.16 nM。亲和力不受ATP或双氧哌嗪催化抑制剂ICRF-187存在的影响。此外，几个双氧哌嗪抗性突变酶也发现了相似的亲和力。这些结果表明，ICRF-187抑制拓扑异构酶II α及其抗性的机制并不直接涉及DNA与酶的相互作用。SPR也适用于测量DNA上拓扑异构酶II的闭合钳形水平。正如预期的那样，在圆形DNA上可以检测到稳定的闭钳形式的酶，但在线性DNA上却没有。检测闭合钳需要ATP和双氧哌嗪，或ATP的不可水解类似物的存在。在ATP和ICRF-187存在的情况下，几种双氧哌嗪抗性突变酶无法形成可检测水平的稳定闭合钳。有趣的是，具有改变活性位点酪氨酸的人类拓扑异构酶II α突变体显示出较低水平的闭合钳形成。综上所述，SPR能够(1)确定拓扑异构酶II与其DNA底物的动力学，(2)量化酶在不同情况下的闭合钳形成。
DNA topoisomerase II is an ATP-operated clamp that effects topological changes by capturing a double-stranded DNA segment and transporting it through another duplex. Surface plasmon resonance (SPR) was used to characterize interactions of human topoisomerase II alpha with different topological forms of DNA. Using a linear fragment of pUC18 DNA, the equilibrium binding constant of topoisomerase II alpha was determined to be 0.16 nM. The affinity was not affected by the absence of ATP or the presence of the bisdioxopiperazine catalytic inhibitor ICRF-187. Besides, similar affinities were found for several bisdioxopiperazine-resistant mutant enzymes. These results suggest that the mechanism of topoisomerase II alpha inhibition by ICRF-187 and its resistance does not directly involve the interaction of DNA with the enzyme. SPR was also adapted to measure levels of the closed clamp form of topoisomerase II present on DNA. As expected, a stable closed clamp form of the enzyme was detectable on circular DNA but not on linear DNA. Detection of the closed clamp required the presence of ATP and a bisdioxopiperazine, or a non-hydrolyzable analogue of ATP. In the presence of ATP and ICRF-187, several bisdioxopiperazine-resistant mutant enzymes failed to form detectable levels of stable closed clamp. Interestingly, a mutant of human topoisomerase II alpha with an altered active site tyrosine showed lower levels of closed clamp formation. In conclusion, SPR is able to (1) determine the kinetics of topoisomerase II with its DNA substrate and (2) quantify the enzyme's closed clamp formation under varying circumstances.