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...

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,...

Escherichia coli DNA topoisomerase I is the best characterized bacterial type I DNA topoisomerases (for review, see Refs. 1 and 2). Its major function in vivo is the removal of negative supercoils from DNA (3, 4). In vitro, such relaxation activity requires that Mg(II) be present in the reaction mixture (3, 5).When other divalent ions were tested (5), Ca(II) could partly replace the Mg(II) in the relaxation of negatively supercoiled DNA while the presence of other divalent ions, such as Mn(II), did not support the relaxation of negatively supercoiled DNA by the enzyme. Moreover, the presence of 2 mM Mn(II) had an inhibitory effect when co-incubated with the enzyme and 2 mM Mg(II) (5). These results suggested that there are specific interactions between Mg(II) and the enzyme or enzyme⅐DNA complex.The three-dimensional structure of the 67-kDa N-terminal fragment of E. coli DNA topoisomerase I has been determined by x-ray crystallography (6). It was noted (6) that near the active site nucleophile Tyr-319, there are three acidic residues, Asp-111, Asp-113, and Glu-115 arranged similarly to the three acidic residues known to coordinate two divalent ions in Klenow fragment (7). According to the models proposed for DNA polymerase mechanism, these coordinated divalent ions are essential for the nucleotidyl transfer catalytic activity (8, 9) of DNA polymerases. However, divalent ions were not present in the topoisomerase I crystal structure (6). It is also known that Mg(II) is not required for DNA cleavage by topoisomerase and formation of the covalent protein-DNA intermediate (10) although Mg(II) is required for intermolecular religation to be observed (11). It remains unclear if Mg(II) interacts directly with the enzyme.The basis for the requirement of Mg(II) for relaxation activity needs to be elucidated to fully understand the enzyme mechanism. There are several possible roles for Mg(II) in the relaxation of negatively supercoiled DNA by E. coli DNA topoisomerase I. For a direct role in catalysis, one or more Mg(II) bound at the active site may activate the Tyr-319 hydroxyl nucleophile and stabilize the DNA 3ЈOH-leaving group during the DNA strand cleavage step, and/or they may activate the DNA 3ЈOH as the attacking nucleophile and stabilze the Tyr-319 hydroxyl-leaving group during the DNA religation step. More indirectly, Mg(II) coordination may place the DNA phosphates and enzyme catalytic groups in the positions required for catalysis. In addition, binding of Mg(II) may allow the enzyme to undergo the conformational changes postulated to be required for the strand passage, DNA religation and substrate release steps in the proposed mechanism of reaction (6).The fluorescence of tryptophan residues is highly dependent on their local environment and enzyme conformation. We demonstrated here that significant changes in enzyme structure could be observed in the presence of 2 mM MgCl 2 , affecting the tryptophan emission intensity. This suggests that Mg(II) binding may allow the enzyme to assume a conformation necessary ...

Ser10 and Lys13 found near the active site tyrosine of Escherichia coli DNA topoisomerase I are conserved among the type IA topoisomerases. Site-directed mutagenesis of these two residues to Ala reduced the relaxation and DNA cleavage activity, with a more severe effect from the Lys13 mutation. Changing Ser10 to Thr or Lys13 to Arg also resulted in loss of DNA cleavage and relaxation activity of the enzyme. In simulations of the open form of the topoisomerase–DNA complex, Lys13 interacts directly with Glu9 (proposed to be important in the catalytic mechanism). This interaction is removed in the K13A mutant, suggesting the importance of lysine as either a proton donor or a stabilizing cation during strand cleavage, while the Lys to Arg mutation significantly distorts catalytic residues. Ser10 forms a direct hydrogen bond with a phosphate group near the active site and is involved in direct binding of the DNA substrate; this interaction is disturbed in the S10A and S10T mutants. This combination of a lysine and a serine residue conserved in the active site of type IA topoisomerases may be required for correct positioning of the scissile phosphate and coordination of catalytic residues relative to each other so that DNA cleavage and subsequent strand passage can take place.