Nucleic Acids Research, 1994, Vol. 22, No. 24 5360-5365
© 1994
Articles |
Requirements for noncovalent binding of vaccinia topoisomerase I to duplex DNA
Molecular Biology Program, Sloan-Kettering Institute New York, NY 10021, USA
*To whom correspondence should be addressed
Received August 12, 1994. Accepted November 3, 1994.
Vaccinia DNA topoisomerase binds duplex DNA and forms a covalent adduct at sites containing a conserved sequence element 5'(C/T)CCTT' In the scissile strand. Distinctive aspects of noncovalent versus covalent interaction emerge from analysis of the binding properties of Topo(Phe-274), a mutated protein which is unable to cleave DNA, but which binds DNA noncovalently. Whereas DNA cleavage by wild type enzyme is most efficient with ‘suicide’ substrates containing fewer than 10 base 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 stabilize the noncovalent topoisomerase - DNA complex. This result is consistent with the downstream dimensions of the DNA binding site deduced from nuclease footprintlng. Topo(Phe-274) binds to duplex DNA lacking the consensus pentamer with 7- 10-fold lower affinity than to CCCTT-containing DNA.
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