Structure/function analysis of Escherichia coli DNA topoisomerase III

Abstract

The generalized nucleic acid-binding domain of Escherichia coli DNA topoisomerase III (Topo III) has been identified using a selection procedure designated to isolate inactive Topo III polypeptides. Deletion of this binding domain, contained within the carboxyl terminal 50 amino acid residues of Topo III, results in a drastic reduction in the ability of the enzyme to bind to single-stranded DNA and RNA substrates. Successive truncation of the enzyme within this region results in the gradual loss of nucleic binding activity and in a gradual change in the mechanism of Topo III-catalyzed relaxation of negatively supercoiled DNA. Truncation of the enzyme to 605 amino acids (Topo III contains 653 amino acids) reduced DNA relaxation activity and binding activity to 1-2% the activity of the full length polypeptide. The reduction of nucleic acid binding activity of the truncated polypeptides does not result in a loss of cleavage site specificity for the enzyme, suggesting other amino acids are involved in the positioning of the nucleic acid within the nicking/closing site of the topoisomerase. Topo III binds asymmetrically to its substrate relative to its cleavage site. Topo III protects 14 nucleotides (12 nucleotides upstream of the cleavage site and 2 nucleotides downstream) from nuclease P1 digestion. The minimum substrate requirement for Topo III-catalyzed cleavage of DNA is only 7 nucleotides; however, this minimum substrate exhibits the same asymmetric sequence preference (6 nucleotides upstream of the cleavage site and 1 nucleotide downstream). In addition, a substrate that contained a Topo III cleavage/binding site formed a more stable complex with the enzyme than a substrate that did not contain a Topo III cleavage/binding site. An oligonucleotide with a Topo III cleavage/binding site was a better competitor for Topo III than an oligonucleotide without the site. Based on these results, a model is proposed in which Topo III binds to DNA and diffuses along the substrate until a cleavage site is located. When a cleavage site is found, Topo III halts and stably binds to the cleavage site. A hybrid of molecule consisting of Topo III cleavage domain and the Topo I binding domain has been used to demonstrate that the domains for cleavage and binding could be separated. The hybrid was created by fusing the first 604 amino acids of Topo III with the carboxyl-terminal 313 amino acids of Topo I. Addition of the C-terminal amino acids of Topo I restored both DNA relaxation activity and DNA binding activity to the enzyme; however, the ability of the enzyme to segregate newly synthesized replication molecules was not drastically enhanced. This suggests that C-terminal domain of Topo III may specifically contribute to its ability to decatenate newly replicated chromosomes.