Dna Topoisomerase Ii Selects Dna Cleavage Sites Based on Reactivity Rather Than Binding Affinity.
From: Stanford University, School of Medicine, Department of Biochemistry, Stanford, CA 94305, USA.
Nucleic acids research
- Publish Date: 2007
- ISSN: 1362-4962
- Volume: 35
- Issue: 11
- Pages: 3764-73
- Medium: Internet
- Language: English
- Citation (JAMA): Mueller-Planitz Felix, Herschlag Daniel, et al. Dna Topoisomerase Ii Selects Dna Cleavage Sites Based on Reactivity Rather Than Binding Affinity.. Nucleic Acids Res. 2007;35:3764-73
Abstract
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.
Mesh Headings (Keywords): Binding Sites, DNA, DNA Topoisomerases, Type II, Eukaryotic, DNA-Binding Proteins, Kinetics, Protein Conformation, Substrate Specificity, Thermodynamics
Check for Full Text / PubMed Unique Identifier (PMID): 17517767
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