CHARACTERIZATION OF TOPOISOMERASE II-DNA INTERACTION AND IDENTIFICATION OF A DNA-BINDING DOMAIN BY ULTRAVIOLET-LASER CROSS-LINKING

We have used ultraviolet laser crosslinking to characterize the DNA-bi nding properties of highly purified yeast topoisomerase II in the abse nce of ATP. A single 5 ns, 20 mJ pulse of 266 nm light produced optima l crosslinking to a short DNA duplex, with an efficiency of 0.25%. An equilibrium binding constant (K-eq) of 1.2 +/- 0.5 x 10(8) M(-1) was d etermined from kinetic analysis, Topoisomerase II showed highest affin ity for supercoiled DNA. Limited proteolysis of crosslinked topoisomer ase II-DNA complexes showed a site of crosslinking to be within a 29-k Da fragment with Leu-681 at its amino-terminal end, This region contai ns the active Tyr-783 and is homologous to the amino-terminal region o f the DNA-binding bacterial gyrase GyrA subunit, suggesting a conserve d DNA-binding mechanism.