Crystal structure of a covalent intermediate in DNA cleavage and rejoining by Escherichia coli DNA topoisomerase I

Abstract

DNA topoisomerases control DNA topology by breaking and rejoining DNA strands via covalent complexes with cleaved DNA substrate as catalytic intermediates. Here we report the structure of Escherichia coli topoisomerase I catalytic domain (residues 2-695) in covalent complex with a cleaved single-stranded oligonucleotide substrate, refined to 2.3-Å resolution. The enzyme-substrate intermediate formed after strand cleavage was captured due to the presence of the D111N mutation. This structure of the covalent topoisomerase-DNA intermediate, previously elusive for type IA topoisomerases, shows distinct conformational changes from the structure of the enzyme without bound DNA and provides detailed understanding of the covalent catalysis required for strand cleavage to take place. The portion of cleaved DNA 5′ to the site of cleavage is anchored tightly with extensive noncovalent protein-DNA interactions as predicted by the "enzyme-bridged" model. Distortion of the scissile strand at the −4 position 5′ to the cleavage site allows specific selectivity of a cytosine base in the binding pocket. Many antibacterial and anticancer drugs initiate cell killing by trapping the covalent complexes formed by topoisomerases. We have demonstrated in previous mutagenesis studies that accumulation of the covalent complex of bacterial topoisomerase I is bactericidal. This structure of the covalent intermediate provides the basis for the design of novel antibiotics that can trap the enzyme after formation of the covalent complex.protein-DNA complex | topA | DNA relaxation | cytosine recognition A hallmark in reactions of all DNA topoisomerases is the formation of a covalent intermediate, in which an enzyme tyrosyl residue is covalently linked to a DNA phosphoryl group (1). These enzymes play a vital role in modulating DNA supercoiling and in untangling intracellular DNA during various cellular transactions (2). Structural information on the covalent intermediates between members of the topoisomerase family and their DNA substrates is important not only in understanding how these enzymes manage the passage of DNA strands or double helices through one another, but also in the design of antimicrobial and antitumor agents that target the DNA topoisomerases. Compounds that result in the accumulation of the covalent complexes formed by type IIA and type IB topoisomerases, referred collectively as topoisomerase poisons, have been utilized extensively in antibacterial and anticancer therapy (3-5). All bacteria contain at least one type IA topoisomerase, usually topoisomerase I, along with at least one type IIA topoisomerase, usually DNA gyrase (6). We have shown in previous genetic studies that trapping of bacterial topoisomerase I covalent complex intermediate due to mutations inhibiting DNA rejoining can result in rapid bacterial cell death (7, 8) in a mechanism involving reactive oxygen species (9). Bacterial topoisomerase I is thus a validated target that could be utilized for discovery of leads for antibacterial drugs that stabilize its c...

Synthesis and dissolution of hemicatenanes by type IA DNA topoisomerases

“…Recent work showed that three highly conserved arginine and aromatic residues in domain I (R169, R173, and Y177 in E. coli Top1) can account for DNA cleavage site selection of Top1, known as "−4C specificity," the fourth residue 3′ to the cleavage being a cytosine (23). These residues are absent in both eukaryotic and prokaryotic Top3.…”

Biochemical Characterization of the Topoisomerase Domain of Methanopyrus kandleri Topoisomerase V

“…All type IA topoisomerases utilize an enzyme-bridged strand passage mechanism to change the topology of DNA and alter the linking number strictly in steps of one (6). In the crystal structures of type IA topoisomerase-DNA complexes, four conserved residues (Tyr, Lys, Arg, and Glu) form contacts at or near the scissile phosphate group (13)(14)(15). Roles for each amino acid have been proposed; Tyr is the nucleophile, Arg and Lys stabilize the penta-* This work was supported, in whole or in part, by National Institutes of Health Grant R01GM51350 (to A. M.).…”

“…1 To whom correspondence should be addressed: Dept. valent protein-DNA complex, and Glu is the proton donor for the DNA cleavage reaction (13)(14)(15). A conserved nearby histidine plays a role in proton transfer, whereas three conserved acidic residues are involved in Mg 2ϩ binding (16 -20).…”

DNA topoisomerases and their functions in a cell

“…The tyrosine residue of the active site covalently binds with the 5' phosphate, while the 3' end of the broken strand interacts with the enzyme noncovalently [10]. Magnesium ions, which are essential for catalysis, help to keep the 3' end of the cleaved strand in a proper position in the catalytic site [10,11]. After the complementary strand is passed through the resulting cleft to relax one supercoil, the enzyme cavity is closed, and the broken strand is ligated.…”