DNA topoisomerase II is an ATP-operated c
lamp that effects topo
logica
l changes by capturinga doub
le-stranded DNA segment and transporting it through another dup
lex. Surface p
lasmon resonance(SPR) was used to characterize interactions of human topoisomerase II
lpha.gif" BORDER=0> with different topo
logica
l formsof DNA. Using a
linear fragment of pUC18 DNA, the equi
librium binding constant of topoisomerase II
lpha.gif" BORDER=0> was determined to be 0.16 nM. The affinity was not affected by the absence of ATP or the presenceof the bisdioxopiperazine cata
lytic inhibitor ICRF-187. Besides, simi
lar affinities were found for severa
lbisdioxopiperazine-resistant mutant enzymes. These resu
lts suggest that the mechanism of topoisomeraseII
lpha.gif" BORDER=0> inhibition by ICRF-187 and its resistance does not direct
ly invo
lve the interaction of DNA with theenzyme. SPR was a
lso adapted to measure
leve
ls of the c
losed c
lamp form of topoisomerase II presenton DNA. As expected, a stab
le c
losed c
lamp form of the enzyme was detectab
le on circu
lar DNA but noton
linear DNA. Detection of the c
losed c
lamp required the presence of ATP and a bisdioxopiperazine, ora non-hydro
lyzab
le ana
logue of ATP. In the presence of ATP and ICRF-187, severa
l bisdioxopiperazine-resistant mutant enzymes fai
led to form detectab
le
leve
ls of stab
le c
losed c
lamp. Interesting
ly, a mutantof human topoisomerase II
lpha.gif" BORDER=0> with an a
ltered active site tyrosine showed
lower
leve
ls of c
losed c
lampformation. In conc
lusion, SPR is ab
le to (1) determine the kinetics of topoisomerase II with its DNAsubstrate and (2) quantify the enzyme's c
losed c
lamp formation under varying circumstances.