Interaction of human DNA topoisomerase II alpha with DNA: quantification by surface plasmon resonance.
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.
Renodon-Cornière
Axelle
A
Department of Pathology, Laboratory and Finsen Centers, Rigshospitalet 5444, DK-2100 Copenhagen, Denmark.
Jensen
Lars H
LH
Nitiss
John L
JL
Jensen
Peter B
PB
Sehested
Maxwell
M
D000266:Adenylyl Imidodiphosphate / Q000737:chemistry; D000951:Antigens, Neoplasm; D001665:Binding Sites / Q000235:genetics; D004250:DNA Topoisomerases, Type II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D054659:Diketopiperazines; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010879:Piperazines / Q000037:antagonists & inhibitors / Q000494:pharmacology; D010957:Plasmids / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D011929:Razoxane / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D020349:Surface Plasmon Resonance / Q000379:methods; D059005:Topoisomerase II Inhibitors; D014443:Tyrosine / Q000235:genetics
D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D054659:Diketopiperazines; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D010879:Piperazines; D059005:Topoisomerase II Inhibitors; C070899:4,4'-(1,2-dimethyl-1,2-ethanediyl)bis-2,6-piperazinedione; D000266:Adenylyl Imidodiphosphate; D014443:Tyrosine; D011929:Razoxane; D004250:DNA Topoisomerases, Type II