Characterization of topoisomerase II-DNA interaction and identification of a DNA-binding domain by ultraviolet laser crosslinking

FEBS Letters
F HungM Roberge

Abstract

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.

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Citations

Sep 28, 1998·Biochimica Et Biophysica Acta·J M Berger
Mar 31, 1998·Current Opinion in Structural Biology·J M Berger
Feb 2, 2000·The EMBO Journal·A E de la BarreS Dimitrov
Sep 16, 2017·Science·Matthew J SchellenbergR Scott Williams
Sep 3, 1999·Nucleic Acids Research·S LejnineJ P Langmore

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