Russell J. DiGate

RecQ helicase and topoisomerase III comprise a novel DNA strand passage function: a conserved mechanism for control of DNA recombination.

E. coli RecQ protein is a multifunctional helicase with homologs that include the S. cerevisiae Sgs1 helicase and the H. sapiens Wrn and Blm helicases. Here we show that RecQ helicase unwinds a covalently closed double-stranded DNA (dsDNA) substrate and that this activity specifically stimulates E. coli topoisomerase III (Topo III) to fully catenate dsDNA molecules. We propose that these proteins functionally interact and that their shared activity is responsible for control of DNA recombination. RecQ helicase has a comparable effect on the Topo III homolog of S. cerevisiae, consistent with other RecQ and Topo III homologs acting together in a similar capacity. These findings highlight a novel, conserved activity that offers insight into the function of the other RecQ-like helicases.

Crystal structure of a complex of a type IA DNA topoisomerase with a single-stranded DNA molecule.

A variety of cellular processes, including DNA replication, transcription, and chromosome condensation, require enzymes that can regulate the ensuing topological changes occurring in DNA. Such enzymes—DNA topoisomerases—alter DNA topology by catalysing the cleavage of single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), the passage of DNA through the resulting break, and the rejoining of the broken phosphodiester backbone. DNA topoisomerase III from Escherichia coli belongs to the type IA family of DNA topoisomerases, which transiently cleave ssDNA via formation of a covalent 5′ phosphotyrosine intermediate. Here we report the crystal structure, at 2.05 Å resolution, of an inactive mutant of E. coli DNA topoisomerase III in a non-covalent complex with an 8-base ssDNA molecule. The enzyme undergoes a conformational change that allows the oligonucleotide to bind within a groove leading to the active site. We note that the ssDNA molecule adopts a conformation like that of B-DNA while bound to the enzyme. The position of the DNA within the realigned active site provides insight into the role of several highly conserved residues during catalysis. These findings confirm various aspects of the type IA topoisomerase mechanism while suggesting functional implications for other topoisomerases and proteins that perform DNA rearrangements.

The structure of Escherichia coli DNA topoisomerase III

BACKGROUND DNA topoisomerases are enzymes that change the topology of DNA. Type IA topoisomerases transiently cleave one DNA strand in order to pass another strand or strands through the break. In this manner, they can relax negatively supercoiled DNA and catenate and decatenate DNA molecules. Structural information on Escherichia coli DNA topoisomerase III is important for understanding the mechanism of this type of enzyme and for studying the mechanistic differences among different members of the same subfamily. RESULTS The structure of the intact and fully active E. coli DNA topoisomerase III has been solved to 3.0 A resolution. The structure shows the characteristic fold of the type IA topoisomerases that is formed by four domains, creating a toroidal protein. There is remarkable structural similarity to the 67 kDa N-terminal fragment of E. coli DNA topoisomerase I, although the relative arrangement of the four domains is significantly different. A major difference is the presence of a 17 amino acid insertion in topoisomerase III that protrudes from the side of the central hole and could be involved in the catenation and decatenation reactions. The active site is formed by highly conserved amino acids, but the structural information and existing biochemical and mutagenesis data are still insufficient to assign specific roles to most of them. The presence of a groove in one side of the protein is suggestive of a single-stranded DNA (ssDNA)-binding region. CONCLUSIONS The structure of E. coli DNA topoisomerase III resembles the structure of E. coli DNA topoisomerase I except for the presence of a positively charged loop that may be involved in catenation and decatenation. A groove on the side of the protein leads to the active site and is likely to be involved in DNA binding. The structure helps to establish the overall mechanism for the type IA subfamily of topoisomerases with greater confidence and expands the structural basis for understanding these proteins.

A role for topoisomerase III in a recombination pathway alternative to RuvABC

The physiological role of topoisomerase III is unclear for any organism. We show here that the removal of topoisomerase III in temperature sensitive topoisomerase IV mutants in Escherichia coli results in inviability at the permissive temperature. The removal of topoisomerase III has no effect on the accumulation of catenated intermediates of DNA replication, even when topoisomerase IV activity is removed. Either recQ or recA null mutations, but not helD null or lexA3, partially rescued the synthetic lethality of the double topoisomerase III/IV mutant, indicating a role for topoisomerase III in recombination. We find a bias against deleting the gene encoding topoisomerase III in ruvC53 or ΔruvABC backgrounds compared with the isogenic wild‐type strains. The topoisomerase III RuvC double mutants that can be constructed are five‐ to 10‐fold more sensitive to UV irradiation and mitomycin C treatment and are twofold less efficient in transduction efficiency than ruvC53 mutants. The overexpression of ruvABC allows the construction of the topoisomerase III/IV double mutant. These data are consistent with a role for topoisomerase III in disentangling recombination intermediates as an alternative to RuvABC to maintain the stability of the genome.

The mechanism of type IA topoisomerase-mediated DNA topological transformations.

Type IA DNA topoisomerases possess several domains forming a toroidal molecule with a central hole large enough to accommodate single- or double-stranded DNA. The sign inversion model predicts several protein-DNA intermediates, including those in which DNA is trapped within the hole. Opposing cysteine residues were incorporated into two independent domains surrounding the putative DNA binding cavity of E. coli topoisomerase III, creating a molecule that can be covalently closed or opened by oxidizing or reducing the disulfide bond. The formation of the disulfide bond allowed the trapping of single- and double-stranded DNA within the cavity of the enzyme and the identification of other intermediates proposed by the sign inversion model.

The traE Gene of Plasmid RP4 Encodes a Homologue of Escherichia coli DNA Topoisomerase III

The polypeptide encoded by the plasmid RP4traE gene shows extensive protein sequence similarity toEscherichia coli topB, the gene encoding DNA topoisomerase III (Topo III). The traE gene product has been cloned into a bacteriophage T7-based transient expression system, and the polypeptide has been expressed and purified. The TraE protein exhibits topoisomerase activity similar to that of Topo III. Relaxation is stimulated by high temperature and low concentrations of Mg2+. In addition, similar to E. coli Topo III, the TraE protein is a potent decatenase and can substitute for Topo III activity in vivo. The biochemical properties of the TraE protein in vitro suggest that the protein may be involved in the resolution of plasmid DNA replication intermediates either during vegetative replication or in conjugative DNA transfer. Putative homologues of Topo III have been found to be encoded by other broad host range, conjugative plasmids isolated from both Gram-negative and Gram-positive organisms, suggesting that Topo III-like polypeptides may have an essential role in the propagation of many promiscuous plasmids.

Pseudocontact shifts used in the restraint of the solution structures of electron transfer complexes.

The geometry of the ferricytochrome b5-ferricytochrome c complex has been analysed using long-range interprotein paramagnetic dipolar shifts. Heteronuclear filtered NMR spectra of samples containing 15N-labelled cytochrome b5 in complex with unlabelled cytochrome c allowed unambiguous assessment of pseudocontact shifts relative to diamagnetic reference states. Because pseudocontact shifts can be observed for protons as much as 20 Å from the paramagnetic centre, this approach allows study of electron transfer proteins in fast exchange. Our findings provide the first physical evidence confirming hypotheses presented in previous theoretical studies. The absence of certain predicted shifts that are expected based on the best fit to a static model of the complex suggests that cytochrome b5 is more dynamic in solution than in the crystal, in agreement with molecular dynamics simulations.

Identification of a unique domain essential for Escherichia coli DNA topoisomerase III‐catalysed decatenation of replication intermediates

A 17‐amino‐acid residue domain has been identified in Escherichia coli DNA topoisomerase III (Topo III) that is essential for Topo III‐mediated resolution of DNA replication intermediates in vitro. Deletion of this domain reduced Topo III‐catalysed resolution of DNA replication intermediates and decatenation of multiply linked plasmid DNA dimers by four orders of magnitude, whereas reducing Topo III‐catalysed relaxation of negatively supercoiled DNA substrates only 20‐fold. The presence of this domain has been detected in multiple plasmid‐encoded topoisomerases, raising the possibility that these enzymes may also be decatenases.

Escherichia coli DNA Topoisomerase III Is a Site-specific DNA Binding Protein That Binds Asymmetrically to Its Cleavage Site

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 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.

The Role of the Carboxyl-terminal Amino Acid Residues in Escherichia coli DNA Topoisomerase III-mediated Catalysis

The role that the carboxyl-terminal amino acids of Escherichia coli DNA topoisomerase I (Topo I) and III (Topo III) play in catalysis was examined by comparing the properties of Topo III with those of a truncated enzyme lacking the generalized DNA binding domain of Topo III, Topo I, and a hybrid topoisomerase polypeptide containing the amino-terminal 605 amino acids of Topo III and the putative generalized DNA binding domain of Topo I. The deletion of the carboxyl-terminal 49 amino acids of Topo III decreases the affinity of the enzyme for its substrate, single-stranded DNA, by approximately 2 orders of magnitude and reduces Topo III-catalyzed relaxation of supercoiled DNA and Topo III-catalyzed resolution of DNA replication intermediates to a similar extent. Fusion of the carboxyl-terminal 312 amino acid residues of Topo I onto the truncated molecule stimulates topoisomerase-catalyzed relaxation 15-20-fold, to a level comparable with that of full-length Topo III. However, topoisomerase-catalyzed resolution of DNA replication intermediates was only stimulated 2-3-fold. Therefore, the carboxyl-terminal amino acids of these topoisomerases constitute a distinct and separable domain, and this domain is intimately involved in determining the catalytic properties of these polypeptides.