REQUIREMENTS FOR NONCOVALENT BINDING OF VACCINIA TOPOISOMERASE-I TO DUPLEX DNA

Vaccinia DNA topoisomerase binds duplex DNA and forms a covalent adduc t at sites containing a conserved sequence element 5'(C/T)CCTTdown arr ow in the scissile strand. Distinctive aspects of noncovalent versus c ovalent interaction emerge from analysis of the binding properties of Topo(Phe-274), a mutated protein which is unable to cleave DNA, but wh ich binds DNA noncovalently. Whereas DNA cleavage by wild type enzyme is most efficient with 'suicide' substrates containing fewer than 10 b ase pairs distal to the scissile bond, optimal noncovalent binding by Topo(Phe-274) requires at least 10-bp of DNA 3' of the cleavage site. Thus, the region of DNA flanking the pentamer motif serves to stabiliz e the noncovalent topoisomerase-DNA complex. This result is consistent with the downstream dimensions of the DNA binding site deduced from n uclease footprinting. Topo(Phe-274) binds to duplex DNA lacking the co nsensus pentamer with 7-10-fold lower affinity than to CCCTT-containin g DNA.