Abstract
The binding of DNA topoisomerase III (Topo III) to a single-stranded DNA substrate containing a strong cleavage site has been examined. The minimal substrate requirement for Topo III-catalyzed cleavage has been determined to consist of 7 bases; 6 bases 5' to the cleavage site and only 1 base 3' to the site. Nuclease P1 protection experiments indicate that the enzyme also binds to its substrate asymmetrically, protecting approximately 12 bases 5' to the cleavage site and only 2 bases 3' to the cleavage site. A catalytically inactive mutant of Topo III shows the same protection pattern as the active polypeptide, indicating that Topo III is a site-specific binding protein as well as a topoisomerase. Consistent with this view, an oligonucleotide containing a cleavage site is a more effective inhibitor and is bound more efficiently by Topo III than an oligonucleotide without a cleavage site.
Highlights
Escherichia coli has been shown to possess four DNA topoisomerase activities
The oligonucleotides were designed to vary the length of DNA sequence both 5Ј and 3Ј of a strong Topo III cleavage site [16]
Substrates as short as 7 bases were capable of being cleaved by Topo III; the length of sequence 5Ј to the cleavage was critical in determining whether the enzyme could productively interact with the substrate
Summary
Escherichia coli has been shown to possess four DNA topoisomerase activities. Two type I enzymes, DNA topoisomerase I (Topo I)1 [1] and DNA topoisomerase III [2,3,4], have been purified and characterized. Topo IV [9, 10] and Topo III [4, 11] have been shown to be potent decatenases in vitro, and it has been proposed that these enzymes are involved in the separation of nascent daughter chromosomes during the terminal stages of DNA replication. A model termed “sign inversion” [13] has been proposed that describes a unified mechanism for both type I and type II bacterial topoisomerases In this model, a topoisomerase binds to its substrate (either single-stranded DNA for a type I enzyme or double-stranded DNA for a type II enzyme), catalyzes a strand break (either single (type I) or double (type II)) and ʈ To whom correspondence should be addressed. The binding of Topo III to its substrate is examined, and a model for Topo III-catalyzed cleavage of DNA is proposed
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