Nucleic Acids Research Advance Access originally published online on May 21, 2007
Nucleic Acids Research 2007 35(11):3764-3773; doi:10.1093/nar/gkm335
Nucleic Acids Research, 2007, Vol. 35, No. 11 3764-3773
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
DNA topoisomerase II selects DNA cleavage sites based on reactivity rather than binding affinity
Stanford University, School of Medicine, Department of Biochemistry, Stanford, CA 94305, USA
*To whom correspondence should be addressed. Tel: +1 650 723 9442; Fax: +1 650 723 6783; Email: herschla{at}stanford.edu
Received February 26, 2007. Revised April 14, 2007. Accepted April 18, 2007.
DNA topoisomerase II modulates DNA topology by relieving supercoil stress and by unknotting or decatenating entangled DNA. During its reaction cycle, the enzyme creates a transient double-strand break in one DNA segment, the G-DNA. This break serves as a gate through which another DNA segment is transported. Defined topoisomerase II cleavage sites in genomic and plasmid DNA have been previously mapped. To dissect the G-DNA recognition mechanism, we studied the affinity and reactivity of a series of DNA duplexes of varied sequence under conditions that only allow G-DNA to bind. These DNA duplexes could be cleaved to varying extents ranging from undetectable (<0.5%) to 80%. The sequence that defines a cleavage site resides within the central 20 bp of the duplex. The DNA affinity does not correlate with the ability of the enzyme to cleave DNA, suggesting that the binding step does not contribute significantly to the selection mechanism. Kinetic experiments show that the selectivity interactions are formed before rather than subsequent to cleavage. Presumably the binding energy of the cognate interactions is used to promote a conformational change that brings the enzyme into a cleavage competent state. The ability to modulate the extent of DNA cleavage by varying the DNA sequence may be valuable for future structural and mechanistic studies that aim to determine topoisomerase structures with DNA bound in pre- and post-cleavage states and to understand the conformational changes associated with DNA binding and cleavage.
Present address: Felix Mueller-Planitz, Adolf-Butenandt-Institut, Molekularbiologie, Ludwig-Maximilians-Universität München, 80336 München, Germany.
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