Mechanism of Type IA Topoisomerases.

Topoisomerases in the type IA subfamily can catalyze change in topology for both DNA and RNA substrates. A type IA topoisomerase may have been present in a last universal common ancestor (LUCA) with an RNA genome. Type IA topoisomerases have since evolved to catalyze the resolution of topological barriers encountered by genomes that require the passing of nucleic acid strand(s) through a break on a single DNA or RNA strand. Here, based on available structural and biochemical data, we discuss how a type IA topoisomerase may recognize and bind single-stranded DNA or RNA to initiate its required catalytic function. Active site residues assist in the nucleophilic attack of a phosphodiester bond between two nucleotides to form a covalent intermediate with a 5'-phosphotyrosine linkage to the cleaved nucleic acid. A divalent ion interaction helps to position the 3'-hydroxyl group at the precise location required for the cleaved phosphodiester bond to be rejoined following the passage of another nucleic acid strand through the break. In addition to type IA topoisomerase structures observed by X-ray crystallography, we now have evidence from biophysical studies for the dynamic conformations that are required for type IA topoisomerases to catalyze the change in the topology of the nucleic acid substrates.

Decoding the conformation-linked functional properties of nucleic acids by the use of computational tools.

DNA and RNA are large and flexible polymers selected by nature to transmit information. The most common DNA three-dimensional structure is represented by the double helix, but this biopolymer is extremely flexible and polymorphic, and can easily change its conformation to adapt to different interactions and purposes. DNA can also adopt singular topologies, giving rise, for instance, to supercoils, formed because of the limited free rotation of the DNA domain flanking a replication or transcription complex. Our understanding of the importance of these unusual or transient structures is growing, as recent studies of DNA topology, supercoiling, knotting and linking have shown that the geometric changes can drive, or strongly influence, the interactions between protein and DNA, so altering its own metabolism. On the other hand, the unique self-recognition properties of DNA, determined by the strict Watson-Crick rules of base pairing, make this material ideal for the creation of self-assembling, predesigned nanostructures. The construction of such structures is one of the main focuses of the thriving area of DNA nanotechnology, where several assembly strategies have been employed to build increasingly complex DNA nanostructures. DNA nanodevices can have direct applications in biomedicine, but also in the materials science field, requiring the immersion of DNA in an environment far from the physiological one. Crucial help in the understanding and planning of natural and artificial nanostructures is given by modern computer simulation techniques, which are able to provide a reliable structural and dynamic description of nucleic acids.

Local site preference rationalizes disentangling by DNA topoisomerases.

To rationalize the disentangling action of type II topoisomerases, an improved wormlike DNA model was used to delineate the degree of unknotting and decatenating achievable by selective segment passage at specific juxtaposition geometries and to determine how these activities were affected by DNA circle size and solution ionic strength. We found that segment passage at hooked geometries can reduce knot populations as dramatically as seen in experiments. Selective segment passage also provided theoretical underpinning for an intriguing empirical scaling relation between unknotting and decatenating potentials.

Imaging and studying human topoisomerase I on mica surfaces in air and in liquid by atomic force microscopy.

In this study, the topography of human topoisomerase I (TOPO I) on mica surfaces in air and in liquid has been studied by atomic force microscopy (AFM). The average height of TOPO I on mica surface in air measured by AFM was 2.59+/-0.32 nm. After adsorption of the 0.3 U/microl TOPO I on mica surfaces for 2 h, and then imaged in liquid by AFM, well-separated single TOPO I was observed. The average height of TOPO I on mica surfaces in liquid measured by AFM was 2.93+/-0.42 nm. After adsorption of the 4 U/microl TOPO I on mica surfaces for 1.5 h, TOPO I monolayer can be formed. The produced TOPO I monolayer on mica was flat and exhibited good stability.

Revealing the mode of action of DNA topoisomerase I and its inhibitors by atomic force microscopy.

In this study, we used, for the first time, atomic force microscope (AFM) images to investigate the mode of action of DNA topoisomerase I (topo I) in the presence and absence of its inhibitors: camptothecin (CPT) and tyrphostin AG-1387. The results revealed that in the absence of the inhibitors, the enzyme relaxed supercoiled DNA starting from a certain point in the DNA molecules and proceeded in one direction towards one of the edges of the DNA molecule. In addition, the relaxation of the supercoiled DNA is subsequently followed by a knotting event. In the presence of CPT, enzyme-supercoiled DNA complexes in which the enzyme is locked inside a relaxed region of the supercoiled DNA molecule were observed. Tyrphostin AG-1387 altered the DNA relaxation process of topo I producing unique shapes of DNA molecules. AFM images of the topo I protein provided a picture of the enzyme, which resembles its known crystallographic structure. Thus, AFM images provide new information on the mode of action of topo I in the absence and presence of its inhibitors.

Three-dimensional electron microscopy of the reverse gyrase from Sulfolobus tokodaii.

Reverse gyrase is a type IA topoisomerase, found in various hyperthermophiles and promotes ATP-dependent positive supercoiling of DNA. Electron microscopy combined with single particle analyses revealed the three-dimensional structure of the DNA-free Sulfolobus tokodaii reverse gyrase and two-dimensional average images of both the protein alone and that complexed with double-stranded DNA. The 23A resolution map exhibited a parallelogrammatic morphology of 110 x 87 x 43A, which is in good agreement with the crystal structure of the Archaeoglobus fulgidus reverse gyrase. The average image of the complex revealed that the monomeric enzyme binds DNA duplex. Together with this average image of the complex, the three-dimensional map implies that, at the beginning of the supercoiling reaction, DNA is bound within a 10-20A wide cleft in the helicase-like domain. We also speculate that DNA may pass through a 20A wide hole at the end of the cleft.

A catalytic domain of eukaryotic DNA topoisomerase I.

Eukaryotic type IB topoisomerases catalyze the cleavage and rejoining of DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate. The 314-amino acid vaccinia topoisomerase is the smallest member of this family and is distinguished from its cellular counterparts by its specificity for cleavage at the target sequence 5'-CCCTT downward arrow. Here we show that Topo-(81-314), a truncated derivative that lacks the N-terminal domain, performs the same repertoire of reactions as the full-sized topoisomerase: relaxation of supercoiled DNA, site-specific DNA transesterification, and DNA strand transfer. Elimination of the N-terminal domain slows the rate of single-turnover DNA cleavage by 10(-3.6), but has little effect on the rate of single-turnover DNA religation. DNA relaxation and strand cleavage by Topo-(81-314) are inhibited by salt and magnesium; these effects are indicative of reduced affinity in noncovalent DNA binding. We report that identical properties are displayed by a full-length mutant protein, Topo(Y70A/Y72A), which lacks two tyrosine side chains within the N-terminal domain that contact the DNA target site in the major groove. We speculate that Topo-(81-314) is fully competent for transesterification chemistry, but is compromised with respect to a rate-limiting precleavage conformational step that is contingent on DNA contacts made by Tyr-70 and Tyr-72.

Intramolecular synapsis of duplex DNA by vaccinia topoisomerase.

Complexes formed by vaccinia topoisomerase I on plasmid DNA were visualized by electron microscopy. The enzyme formed intramolecular loop structures in which non-contiguous DNA segments were synapsed within filamentous protein stems. At high enzyme concentrations the DNA appeared to be zipped up within the protein filaments such that the duplex was folded back on itself. Formation of loops and filaments was also observed with an active site mutant, Topo-Phe274. Binding of Topo-Phe274 to relaxed DNA circles in solution introduced torsional strain, which, after relaxation by catalytic amounts of wild-type topo-isomerase, resulted in acquisition of negative supercoils. We surmise that the topoisomerase-DNA complex is a plectonemic supercoil in which the two duplexes encompassed by the protein filaments are interwound in a right handed helix. We suggest that topoisomerase-mediated DNA synapsis plays a role in viral recombination and in packaging of the 200 kbp vaccinia genome during virus assembly.

Peptide sequencing and site-directed mutagenesis identify tyrosine-727 as the active site tyrosine of Saccharomyces cerevisiae DNA topoisomerase I.

Extensive digestion of the covalent intermediate between DNA and Saccharomyces cerevisiae DNA topoisomerase I with trypsin yields a 7-amino acid peptide covalently linked to DNA. Direct sequencing of the DNA-linked peptide identifies Tyr-727 as the active site tyrosine that forms an O4-phosphotyrosine bond with DNA when the enzyme cleaves a DNA phosphodiester bond. Site-directed mutagenesis of the cloned yeast TOP1 gene encoding the enzyme confirms the essentiality of Tyr-727 for the relaxation of supercoiled DNA by the enzyme. From amino acid sequence homology, Tyr-771 and -773 are readily identified as the active site tyrosines of Schizosaccharomyces pombe and human DNA topoisomerase I, respectively. Sequence comparison and site-directed mutagenesis also implicate Tyr-274 of vaccinia virus DNA topoisomerase as the active site residue. There appears to be a 70-amino acid domain near the carboxyl terminus of eukaryotic DNA topoisomerase I and vaccinia topoisomerase, within which the active site tyrosine resides.