Interaction of Human DNA Topoisomerase II with DNA: Quantification by Surface Plasmon Resonance
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- 作者：Axelle Renodon-Corniè ; re ; Lars H. Jensen ; John L. Nitiss ; Peter B. Jensen ; and Maxwell Sehested
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：November 12, 2002
- 年：2002
- 卷：41
- 期：45
- 页码：13395 - 13402
- 全文大小：100K
- 年卷期：v.41,no.45(November 12, 2002)
- ISSN：1520-4995
文摘
DNA topoisomerase II is an ATP-operated clamp that effects topological changes by capturinga double-stranded DNA segment and transporting it through another duplex. Surface plasmon resonance(SPR) was used to characterize interactions of human topoisomerase II 
with different topological formsof DNA. Using a linear fragment of pUC18 DNA, the equilibrium binding constant of topoisomerase II was determined to be 0.16 nM. The affinity was not affected by the absence of ATP or the presenceof the bisdioxopiperazine catalytic inhibitor ICRF-187. Besides, similar affinities were found for severalbisdioxopiperazine-resistant mutant enzymes. These results suggest that the mechanism of topoisomeraseII inhibition by ICRF-187 and its resistance does not directly involve the interaction of DNA with theenzyme. SPR was also adapted to measure levels of the closed clamp form of topoisomerase II presenton DNA. As expected, a stable closed clamp form of the enzyme was detectable on circular DNA but noton linear DNA. Detection of the closed clamp required the presence of ATP and a bisdioxopiperazine, ora 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 mutantof human topoisomerase II with an altered active site tyrosine showed lower levels of closed clampformation. In conclusion, SPR is able to (1) determine the kinetics of topoisomerase II with its DNAsubstrate and (2) quantify the enzyme's closed clamp formation under varying circumstances.
with different topological formsof DNA. Using a linear fragment of pUC18 DNA, the equilibrium binding constant of topoisomerase II was determined to be 0.16 nM. The affinity was not affected by the absence of ATP or the presenceof the bisdioxopiperazine catalytic inhibitor ICRF-187. Besides, similar affinities were found for severalbisdioxopiperazine-resistant mutant enzymes. These results suggest that the mechanism of topoisomeraseII inhibition by ICRF-187 and its resistance does not directly involve the interaction of DNA with theenzyme. SPR was also adapted to measure levels of the closed clamp form of topoisomerase II presenton DNA. As expected, a stable closed clamp form of the enzyme was detectable on circular DNA but noton linear DNA. Detection of the closed clamp required the presence of ATP and a bisdioxopiperazine, ora 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 mutantof human topoisomerase II with an altered active site tyrosine showed lower levels of closed clampformation. In conclusion, SPR is able to (1) determine the kinetics of topoisomerase II with its DNAsubstrate and (2) quantify the enzyme's closed clamp formation under varying circumstances.