Characterization of topoisomerase II-DNA interaction and identification of a DNA-binding domain by ultraviolet laser crosslinking.
We have used ultraviolet laser crosslinking to characterize the DNA-binding properties of highly purified yeast topoisomerase II in the absence of ATP. A single 5 ns, 20 mJ pulse of 266 nm light produced optimal crosslinking to a short DNA duplex, with an efficiency of 0.25%. An equilibrium binding constant (Keq) of 1.2 +/- 0.5 x 10(8) M(-1) was determined from kinetic analysis. Topoisomerase II showed highest affinity for supercoiled DNA. Limited proteolysis of crosslinked topoisomerase II-DNA complexes showed a site of crosslinking to be within a 29-kDa fragment with Leu-681 at its amino-terminal end. This region contains the active Tyr-783 and is homologous to the amino-terminal region of the DNA-binding bacterial gyrase GyrA subunit, suggesting a conserved DNA-binding mechanism.
Hung
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Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
Luo
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Sauvé
D M
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Muller
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Roberge
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D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D004250:DNA Topoisomerases, Type II / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000528:radiation effects; D004278:DNA, Superhelical / Q000737:chemistry / Q000378:metabolism; D007834:Lasers; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D004278:DNA, Superhelical; D004247:DNA; D004250:DNA Topoisomerases, Type II