The Snf2 family represents a functionally diverse class of ATPase sharing the ability to modify DNA structure. Here, we use a magnetic trap and an atomic force microscope to monitor the activity of a member of this class: the RSC complex. This enzyme caused transient shortenings in DNA length involving translocation of typically 400 bp within 2 s, resulting in the formation of a loop whose size depended on both the force applied to the DNA and the ATP concentration. The majority of loops then decrease in size within a time similar to that with which they are formed, suggesting that the motor has the ability to reverse its direction. Loop formation was also associated with the generation of negative DNA supercoils. These observations support the idea that the ATPase motors of the Snf2 family of proteins act as DNA translocases specialized to generate transient distortions in DNA structure.

Escherichia coli DNA topoisomerase I (encoded by the topA gene) is important for maintaining steady-state DNA supercoiling and has been shown to influence vital cellular processes including transcription. Topoisomerase I activity is also needed to remove hypernegative supercoiling generated on the DNA template by the progressing RNA polymerase complex during transcription elongation. The accumulation of hypernegative supercoiling in the absence of topoisomerase I can lead to R-loop formation by the nascent transcript and template strand, leading to suppression of transcription elongation. Here we show by affinity chromatography and overlay blotting that E. coli DNA topoisomerase I interacts directly with the RNA polymerase complex. The protein-protein interaction involves the ␤ subunit of RNA polymerase and the C-terminal domains of E. coli DNA topoisomerase I, which are homologous to the zinc ribbon domains in a number of transcription factors. This direct interaction can bring the topoisomerase I relaxing activity to the site of transcription where its activity is needed. The zinc ribbon C-terminal domains of other type IA topoisomerases, including mammalian topoisomerase III, may also help link the enzyme activities to their physiological functions, potentially including replication, transcription, recombination, and repair.DNA topoisomerases are ubiquitous enzymes that have functional roles in many vital cellular processes (1, 2). Among different classes of topoisomerases, type IA topoisomerases found in archea, prokaryotes, and eukaryotes share the mechanistic feature of cutting and rejoining a single strand of DNA via a 5Ј-phosphotyrosine linkage and homologous amino acid sequences (3). Escherichia coli DNA topoisomerase I (encoded by the topA gene) is the most extensively studied example of this class of enzyme. Its most apparent physiological role is the maintenance of steady-state DNA supercoiling (4, 5). During transcription, the movement of the RNA polymerase complex on the DNA template creates local transcription-driven supercoiling with negative supercoiling generated behind the RNA polymerase and positive supercoiling generated ahead of the RNA polymerase (6, 7). DNA gyrase is needed for removing the positive supercoils, and topoisomerase I is responsible for removing the excess negative supercoils. In the absence of topoisomerase I function due to mutation in the topA gene, the accumulation of hypernegative supercoiling can lead to R-loop formation by nascent transcription and template stranding with the consequent suppression of transcription elongation (8,9).In previous studies, Tn5 transposase was found to copurify with E. coli DNA topoisomerase I and inhibit the topoisomerase I activity (10). RNA polymerase was also found to copurify with Tn5 transposase, but the copurification was reduced in extracts from a topA mutant strain, suggesting that the interaction between RNA polymerase and DNA topoisomerase I was responsible for the copurification of RNA polymerase with Tn5 transposase (10). The p...

DNA topoisomerases are important clinical targets for antibacterial and anticancer therapy. At least one type IA DNA topoisomerase can be found in every bacterium, making it a logical target for antibacterial agents that can convert the enzyme into poison by trapping its covalent complex with DNA. However, it has not been possible previously to observe the consequence of having such a stabilized covalent complex of bacterial topoisomerase I in vivo. We isolated a mutant of recombinant Yersinia pestis topoisomerase I that forms a stabilized covalent complex with DNA by screening for the ability to induce the SOS response in Escherichia coli. Overexpression of this mutant topoisomerase I resulted in bacterial cell death. From sequence analysis and site-directed mutagenesis, it was determined that a single amino acid substitution in the TOPRIM domain changing a strictly conserved glycine residue to serine in either the Y. pestis or E. coli topoisomerase I can result in a mutant enzyme that has the SOS-inducing and cell-killing properties. Analysis of the purified mutant enzymes showed that they have no relaxation activity but retain the ability to cleave DNA and form a covalent complex. These results demonstrate that perturbation of the active site region of bacterial topoisomerase I can result in stabilization of the covalent intermediate, with the in vivo consequence of bacterial cell death. Small molecules that induce similar perturbation in the enzyme-DNA complex should be candidates as leads for novel antibacterial agents.DNA topoisomerases are ubiquitous enzymes that are needed either for control of DNA supercoiling or for overcoming topological barriers during replication, transcription, recombination, or repair (reviewed in Refs. 1-3). Type IB and type II DNA topoisomerases are well utilized targets of many clinically important anticancer and antibacterial drugs (4 -7). These drugs cause cell death by stabilizing the covalent intermediate formed between topoisomerase protein and cleaved DNA during the catalytic cycle of the enzyme. There is at least one type IA DNA topoisomerase present in every genome examined so far. It is likely to be essential for overcoming topological barriers requiring single-stranded DNA passage (2). It has been proposed that bacterial type IA DNA topoisomerases could be a useful therapeutic target if small molecules that stabilize the covalent intermediate of this class of topoisomerases can be identified (8). However, it has never been demonstrated that stabilization of the covalent intermediate formed between a type IA topoisomerase and the cleaved single DNA strand can lead to cell death. It therefore remains unclear whether bacterial topoisomerase I can indeed be the target of "poison" molecules that would be bactericidal.In this study, a mutant bacterial topoisomerase I that forms a stabilized covalent intermediate with cleaved DNA was identified via an SOS induction screen (9) in Escherichia coli. Overexpression of this mutant topoisomerase in E. coli led to extensive cell ki...

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...

Escherichia coli topoisomerase I has an essential function in preventing hypernegative supercoiling of DNA. A full length structure of E. coli topoisomerase I reported here shows how the C-terminal domains bind single-stranded DNA (ssDNA) to recognize the accumulation of negative supercoils in duplex DNA. These C-terminal domains of E. coli topoisomerase I are known to interact with RNA polymerase, and two flexible linkers within the C-terminal domains may assist in the movement of the ssDNA for the rapid removal of transcription driven negative supercoils. The structure has also unveiled for the first time how the 4-Cys zinc ribbon domain and zinc ribbon-like domain bind ssDNA with primarily π-stacking interactions. This novel structure, in combination with new biochemical data, provides important insights into the mechanism of genome regulation by type IA topoisomerases that is essential for life, as well as the structures of homologous type IA TOP3α and TOP3β from higher eukaryotes that also have multiple 4-Cys zinc ribbon domains required for their physiological functions.

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On the basis of recently reported abyssinone II and olympicin A, a series of chemically modified flavonoid phytochemicals were synthesized and evaluated against Mycobacterium tuberculosis and a panel of Gram-positive and -negative bacterial pathogens. Some of the synthesized compounds exhibited good antibacterial activities against Gram-positive pathogens including methicillin resistant Staphylococcus aureus with minimum inhibitory concentration as low as 0.39 μg/mL. SAR analysis revealed that the 2-hydrophobic substituent and the 4-hydrogen bond donor/acceptor of the 4-chromanone scaffold together with the hydroxy groups at 5- and 7-positions enhanced antibacterial activities; the 2′,4′-dihydroxylated A ring and the lipophilic substituted B ring of chalcone derivatives were pharmacophoric elements for antibacterial activities. Mode of action studies performed on selected compounds revealed that they dissipated the bacterial membrane potential, resulting in the inhibition of macromolecular biosynthesis; further studies showed that selected compounds inhibited DNA topoisomerase IV, suggesting complex mechanisms of actions for compounds in this series.

To catalyze relaxation of supercoiled DNA, DNA topoisomerases form a covalent enzyme-DNA intermediate via nucleophilic attack of a tyrosine hydroxyl group on the DNA phosphodiester backbone bond during the step of DNA cleavage. Strand passage then takes place to change the linking number. This is followed by DNA religation during which the displaced DNA hydroxyl group attacks the phosphotyrosine linkage to reform the DNA phosphodiester bond. Mg(II) is required for the relaxation activity of type IA and type II DNA topoisomerases. A number of conserved amino acids with acidic and basic side chains are present near Tyr-319 in the active site of the crystal structure of the 67-kDa N-terminal fragment of Escherichia coli DNA topoisomerase I. Their roles in enzyme catalysis were investigated by site-directed mutation to alanine. Mutation of Arg-136 abolished all the enzyme relaxation activity even though DNA cleavage activity was retained. The Glu-9, Asp-111, Asp-113, Glu-115, and Arg-321 mutants had partial loss of relaxation activity in vitro. All the mutants failed to complement chromosomal topA mutation in E. coli AS17 at 42°C, possibly accounting for the conservation of these residues in evolution.DNA topoisomerases (for review, see Refs. 1-7) catalyze the interconversion of different DNA topological isomers by first forming a covalent enzyme-DNA intermediate via nucleophilic attack of a tyrosine hydroxyl on the DNA phosphodiester linkage. After strand passage through the break, religation involving nucleophilic attack of the displaced DNA hydroxyl group on the phosphotyrosine linkage takes place. Type IA and type II DNA topoisomerases are linked to the 5Ј-phosphoryl end of the cleaved DNA while type IB DNA topoisomerases are linked to the 3Ј-phosphoryl end. Mg(II) is required for the relaxation activities of both type IA and type II DNA topoisomerases but not for the type IB enzymes. The detailed catalytic mechanism of DNA cleavage and religation by topoisomerases remains to be elucidated. The mechanism of the type IA and type II topoisomerase may share similarities with other enzymes that also require Mg(II) for nucleotidyl transfer activity.Tyr-319 of Escherichia coli DNA topoisomerase I is the catalytic residue that provides the hydroxyl group for forming the covalent intermediate with DNA. The three-dimensional structure of the 67-kDa N-terminal domain of this enzyme has been determined by x-ray crystallography (8). In this structure, Tyr-319 is present in the interface between domains I and III. It has been pointed out (8) that the spatial arrangement of the three acidic residues Asp-111, Asp-113, and Glu-115 in the active site region is similar to the acidic residues that coordinate two divalent cations in the exonuclease catalytic site of Klenow fragment (9). However, the structure observed has to undergo additional conformational changes before there is sufficient space in the active site region for DNA and possibly Mg(II) to bind. A number of residues found in the active site, including Glu-9, Asp-111,...