Structure and function of dna topoisomerases, antibiotic and herbicide development. Most of the 1,000 different mitochondrial proteins are synthesized as precursors in the cytosol and are imported into mitochondria by five transport. Here, we show that a eukaryoticlike topoib is present in the recently sequenced genomes of two archaea of the newly proposed phylum thaumarchaeota. There is, however, no homology between the cterminal dna binding domains of escherichia coli and m. How to grow oyster mushrooms from used coffee grounds cheap and easy part 1 duration. Topoisomerases are enzymes that act to relax supercoiling, a form of builtup strain in dna.
Topoisomerase inhibitors are important antitumour drugs, thought to act by stabilizing a covalent. Structure and mechanism of action of type ia dna topoisomerases. In addition to topoisomerase i and dna gyrase topoisomerase ii, e. Dna topoisomerases are classified according to their mechanism of action. The cterminal region of the prokaryotic topoisomerases has been solved for multiple species. Pdf on dec 1, 1994, p m watt and others published structure and function of type ii dna topoisomerases find, read and cite all the. Crystal structure of an intact type ii dna topoisomerase. To investigate the structure function of mttop1 and to target its activity for development of new tb therapy, it is desirable to have a rapid genetic assay for its catalytic. Dna topoisomerases are a diverse set of essential enzymes responsible for maintaining chromosomes in an appropriate topological state. The structural themes common to all topoisomerases include hinged clamps that open and close to bind dna, the presence of dna binding cavities for temporary storage of dna segments, and the coupling of protein conformational changes to dna rotation or dna movement. A nice clip i found on the mechanisms of action of topoisomerase 1 and 2. Jan, 2010 poxviruses encode their own type ib topoisomerases topibs, which release superhelical tension generated by replication and transcription of their genomes. Structural basis for gatedna recognition and bending by type iia topoisomerases ken c.
To investigate the reaction catalyzed by viral topibs, we have determined the structure of a variola virus topoisomerase dna complex trapped as a vanadate transition state mimic. This backbone tracing was made from protein data bank file 1jsq 120. Although dna topoisomerases have been studied extensively, steadystate kinetics has not been systematically investigated because of the lack of an appropriate assay. Dna topoisomerase iii and iv have similar functions. N2 the crystal structure of a large fragment of yeast type ii dna topoisomerase reveals a heartshaped dimeric protein with a large central hole.
These ubiquitous enzymes are essential for cell viability and are highly potent targets for the development of antibacterial and antitumoral drugs. Dna topoisomerases solve the topological problems associated with dna replication, transcription, recombination, and chromatin remodeling by introducing temporary single or doublestrand breaks in the dna. Topoisomerase iii is incapable of relaxing positive supercoils, but it works to support replication fork movement on plasmid dna in vitro. A shorter version was then found in viruses and later on in several bacteria, but not in archaea. Although a general framework exists for type iia topoisomerase function, the architecture of the fulllength enzyme has remained undefined. Dna topoisomerases structure, function, and mechanism. The crystal structure of a large fragment of yeast type ii dna topoisomerase reveals a heartshaped dimeric protein with a large central hole. For the type ii topoisomerases, the binding and hydrolysis of atp further. Type ia topoisomerases change the linking number of a circular dna strand by units of strictly 1. Topoisomerase iv is a type ii enzyme with a high degree of sequence similarity to dna gyrase, which can. Dna topoisomerases and their functions in a cell springerlink. Full text get a printable copy pdf file of the complete article 1.
Molecular mechanism of dna replication article khan. Structure and mechanism of dna topoisomerase ii johns. Dna replication california state university, northridge. Type ia topoisomerases have a toroidal protein structure that bears a superficial resemblance to a padlock 3. During transcription and dna replication, the dna needs to be unwound in order for the. Read mechanisms of camptothecin resistance by human topoisomerase i mutations, journal of molecular biology on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. The protein can be divided into three different functional domains, the toprim.
Topoisomerase i is capable of nicking a single strand of dna, while topoisomerase ii is able to nick the doublestranded structure in its entirety. A double helix separate into two single strands and each strand serves as a template on which complementary strand is synthesized. Dna topoisomerase ib topoib was thought for a long time to be a eukaryotic specific enzyme. Structural basis for gate dna recognition and bending by type iia topoisomerases ken c. We combine protein signatures from a number of member databases into a single searchable resource, capitalising on their individual strengths to produce a powerful integrated database and diagnostic tool. Abstract dna topoisomerases solve the topological problems associated with dna replication, transcription, recombination, and chromatin remodeling. Type ii enzymes catalyze the transfer of a dna duplex through another one in an atpdependent mechanism. Champoux department of microbiology, school of medicine, university of washington, seattle. Dec 08, 2011 how to grow oyster mushrooms from used coffee grounds cheap and easy part 1 duration.
The mechanism of crisprcas9mediated genome engineering. The camptothecin cpt top1 topoisomerase i inhibitors exert their anticancer activity by reversibly trapping top1 dna cleavage complexes top1ccs and inducing replicationassociated dna doublestrand breaks dsbs. In the eukaryotic cell, the topological structure of dna is modulated by two groups of ubiquitous enzymes known as type i and type i1 topo isomerases. New mechanistic and functional insights into dna topoisomerases. Dna topoisomerases are the magicians of the dna world by allowing dna strands or double helices to pass through each other, they can solve all of the topological problems of dna in replication, transcription and other cellular transactions. Here we present two crystal structures of the atpase domain of human dna topoisomerase iialpha in different nucleotidebound states. Interpro provides functional analysis of proteins by classifying them into families and predicting domains and important sites. The dna topoisomerases i and ii present in cells act through scission of the dna backbone on one or two strands, respectively, followed by relief of torsional stress and then relegation of the broken dna backbone. Antibiotics targeting dna gyrase have been a clinical success story for the past halfcentury, and the emergence of bacterial resistance has fueled the search for new gyrase inhibitors. Dna topoisomerase ii, genotoxicity, and cancer europe. They accomplish this feat via a mechanism involving the breakage and reformation of phosphodiester bonds in the dna backbone 17. The bacterial and human topoisomerases are having similar mechanisms in nature. Author summary dna topoisomerases are essential for transcription, dna repair and dna replication due to their function to break and change the topological state of dna molecules. On the other hand topoisomerase ii cuts both strands in dna and needs atp for their function or activity.
Each unpaired nucleotide will attract a complementary nucleotide from the medium. Effects of yeast dna topoisomerase iii on telomere. Dna topoisomerases top are a group of isomerase enzymes responsible for controlling the topological problems caused by dna double helix in the cell during the processes of replication, transcription and recombination. A wealth of new data concerning the structure and functions of topoisomerases was published recently. In the strand passage mechanism, the cleavage of dna is key to allow the tsegment to transfer through the gsegment. In this section we describe the two different classes of topoisomerases and their role in dna replication. Dna gyrase is a prokaryotic type ii topoisomerase that utilises atp to introduce twist in the bacterial chromosome.
Structure and mechanism of dna topoisomerase ii nature. Type ii topoisomerases function in numerous dna processes and are required for recombination, the separation of daughter chromosomes, and proper chromosome structure, condensation, and decondensation 57,1216. To date, there is limited structural information on type ii enzymes. Wang dna topoisomerases are the magicians of the dna world by allowing dna strands or double helices to pass through each other, they can solve all of the topological problems of dna in replication, transcription and other cellular transactions. Ribosome structure and function, mechanism of protein synthesis, singlemolecule biophysics max gottesman, columbia university, new york, new york, united states. This is the difference between topoisomerase i and ii. Although they vary considerably in structure and mechanism, the partnership between topoisomerases and dna has engendered commonalities in how these enzymes engage nucleic acid substrates and control dna strand manipulations. The enzymes alter the dna linking number, which is the. The key enzyme that regulates dna super structure satoshi kato and akihiko kikuchi laboratory ofmedical mycology, res. We conclude that topoisomerase ii is the main modulator of dna topology in chromatin fibers. Each gene has been mutated with a nonpolar insertion, and the structures of lpss from the resulti. While it is structurally unique from type ia and ib topoisomerases, it shares a similar mechanism with type ib topoisomerase. Antitumour drugs impede dna uncoiling by topoisomerase i. Definition dna supercoiling refers to the over or under winding of a dna strand, and is an expression of the strain on that strand.
The nonessential topoisomerase i then assists dna relaxation where chromatin structure impairs dna. A wealth of new data concerning the structure and functions of topois. Later these cuts in dna backbone are resealed again. Abstract dna topoisomerases solve the topological problems associated with dna replication, transcription. Type i topoisomerases relax dna by nicking and then closing one strand of.
Dna gyrase, or simply gyrase, is an enzyme within the class of topoisomerase and is a subclass of type ii topoisomerases that reduces topological strain in an atp dependent manner while doublestranded dna is being unwound by elongating rnapolymerase or. Structure of a topoisomerase iidnanucleotide complex. Pdf structure and function of type ii dna topoisomerases. Jun 11, 2009 mycobacterium tuberculosis dna topoisomerase i is an attractive target for discovery of novel tb drugs that act by enhancing the accumulation of the topoisomerase dna cleavage product. Mycobacterium tuberculosis topoisomerase i mttop1 and escherichia coli topoisomerase i have highly homologous transesterification domains, but the two enzymes have distinctly different cterminal domains.
Dna topoisomerases are marvelous molecular machines that manage the topological state of the dna in the cell. Gyrase belongs to a class of enzymes known as topoisomerases that are involved in the control of topological transitions of dna. Nucleic acids and chromatin dna topoisomerases topoisomerases create temporary strand breaks in dna, thereby allowing the dna to swivel around the helical axis and releasing torsional strain within the area before resealing the break. Insights from the structure of a smallpox virus topoisomerase. The mechanism by which type2a topoisomerases transport one dna duplex through a transient doublestrand break produced in another exhibits fascinating traits. Phylogenetic analyses suggest that a topoib was present. They use the hydrolysis of atp, unlike type i topoisomerase. As type iib topo vi shares most of the functional domains with type iia enzymes, the proposed mechanism of dna duplex transfer can be generalized to all type ii dna topoisomerases. Structural insights into the gating of dna passage by the.
Pdf human dna topoisomerase i is a 765aa monomeric enzyme composed. Topoisomerase ii, not topoisomerase i, is the proficient. The pdbfiles were retrieved from the protein data bank 4 and processed using the swisspdbviewer 38. Topoisomerase iii is another type ia enzyme that is more active as a decatenating enzyme than as a dna relaxing enzyme 7. The structure of the cat alytic domain is very similar to core subdomain iii and a 19 amino acid region encompassing the active site of human topoisomerase i. Topoisomerase can both cleave dna at a desired replication site and also ligate the. It provides a molecular model of the enzyme as an atp. Dna topoisomerases topos are molecular machines that regulate the topology of dna in the cell by allowing dna strands or double helices to pass through one another. It shares a common transesterification domain with other type ia dna topoisomerases. Mitochondria are essential organelles with numerous functions in cellular metabolism and homeostasis. The structure and mechanism of the action of typeib dna. The mechanism by which gyrase is able to influence the topological state of dna molecules is of inherent interest from an enzymological standpoint. Mar 11, 2008 dna topoisomerases resolve dna topological problems created during transcription, replication, and recombination.
Begins with the unwinding of the double helix to expose the bases in each strand of dna. In this paper we demonstrate that a new class of gyrase inhibitors, the gyramides, are bacteriostatic agents that competitively inhibit the atpase activity of escherichia coli gyrase and produce supercoiled dna. The results concerning the thermodynamic, structural, and kinetic aspects of the. For the many basic scientists and clinicians who want better to. Type ib topoisomerases form a covalent intermediate with the 3 end of dna. Cocrystal structures of top1 inhibitors illustrate the interfacial inhibition paradigm by which a.
Recent developments in dna topoisomerase ii structure and. More recently, several structures of the dna bound structure have been solved in an attempt to understand both the chemical mechanism for dna cleavage and the structural basis for inhibition of topoisomerase by antibacterial poisons. May 28, 2010 dna topoisomerases responsible for the superspiralization of genomic dna participate in almost all vitally important cell processes, including replication, transcription, and recombination, and are essential for normal cell functioning. Gyramides prevent bacterial growth by inhibiting dna gyrase. T1 structure and mechanism of dna topoisomerase ii. For the type ii topoisomerases, the binding and hydrolysis of atp further modulate conformational changes in the enzymes to effect changes in dna topology. These results suggest that yeast dna topoisomerase iii might affect telomere stability, and plausible mechanisms are discussed. Structural studies of type i topoisomerases nucleic acids research. Topoisomerases role of topoisomerases in supercoiling. Dna topoisomerases are enzymes responsible for regulation of genomic dna supercoiling.
Dna topoisomerases are among the most conserved proteins 1,2,3 explained by their key function in relieving torsional stress of dna during fundamental cellular processes such as replication. In this process, these enzymes change the linking number of circular dna by 2. The overall process is orchestrated by the opening and closing of molecular gates in the topoisomerase structure, which is regulated by atp binding, hydrolysis, and release of adp and inorganic phosphate. Topoisomerase is an enzyme which participates in the unwinding of dna helix. It will not be possible to completely understand dna function without having a complete knowledge of the roles of topoisomerases. Consequently, these enzymes are able to remove superhelical twists from dna and resolve knotted or tangled duplex molecules.
In addition, these enzymes finetune the steadystate level of dna supercoiling both to facilitate protein interactions with the dna and to prevent excessive supercoiling that is. The synthetic sgrna or crrnatracrrna structure directs a cas9 endonuclease to almost arbitrary dna sequence in the genome through a. Two tyrosine amino acids, shown in red, cleave the dna strands and form a covalent bond with them, holding them tightly until the dna can be restored. Dna topoisomerases 397 catalytic domain amino acids 814 of the enzyme. In molecular biology type i topoisomerases are enzymes that cut one of the two strands of doublestranded dna, relax the strand, and reanneal the strand. Dna topoiaomerase topoisomerase i and ii mechanism. Relaxation of dna supercoiling is a key function of top1 enzymes.
The present structure of intact sstopo vi and its comparison to the recently published structure of mmtopo vi provide new insights into the general mechanism for. These enzymes effect topological changes in dna through a strand passage mechanism4,5, in which a single dna strand is cleaved and physically opened, and a second dna strand is navigated through the gap. Enzymology and drugs provides, for the first time, a comprehensive description of the methods and experimental systems needed to investigate fully the catalytic mechanisms of dna topoisomerases from bacterial, viral, and eukaryotic sources. Analysis of the eukaryotic topoisomerase ii dna gate. Difference between topoisomerase i and ii key difference. Dna topoisomerases unravel twists in dna that occur as a result of dna transcription and replication. Kinetic study of dna topoisomerases by supercoilingdependent.
Type ii topoisomerases cut both strands of the dna helix simultaneously in order to manage dna tangles and supercoils. Here, we identify a target of bns22 by proteomic profiling analysis, which suggests that bns22 belongs to the same cluster as icrf193, a dna topoisomerase ii top2 catalytic inhibitor. The structural themes common to all topoisomerases include hinged clamps that open and close to bind dna, the presence of dna binding cavities for temporary. Copying genetic information for transmission to the next generation. A dna topoisomerase ib in thaumarchaeota testifies for the. Bns22, a chemically synthesized derivative of the natural plant product gut70, has antiproliferative activity against human cancer cells, the mechanism of which is unknown. Jan 20, 2009 dna topoisomerases topos are molecular machines that regulate the topology of dna in the cell by allowing dna strands or double helices to pass through one another. They are further subdivided into two structurally and mechanistically distinct topoisomerases. Interestingly, these enzymes have been known since long to be key molecular machines in several cellular processes. The topoisomerase is thought to be a highly dynamic structure, with several gates for entry of dna into the two dna sized holes.
Deoxyribonucleic acid dna topoisomerases are essential for removing the supercoiling that normally builds up ahead of replication forks. Structure of a topoisomerase iidnanucleotide complex reveals a new control mechanism for atpase activity. Mus81mediated dna cleavage resolves replication forks. Identification of a smallmolecule inhibitor of dna. One of them is the fine coupling between interdomainal movements and atp usage. Dna topoisomerases are essential enzymes for all living organisms and important targets for anticancer drugs and antibiotics. A mechanism is required to separate the strands locally for replication. Type iia topoisomerases control dna supercoiling and disentangle chromosomes through a complex atpdependent strandpassage mechanism. Type iia topoisomerases top2s manipulate the handedness of dna crossovers by introducing a transient and proteinlinked doublestrand break in one dna duplex, termed the dna gate, whose opening. Because supercoiling of dna is an important property of dna and affects almost every aspect of dna function, the roles of topoisomerases in transcription are expectedly complex. Topoisomerase is a valuable enzyme for untangling supercoils and making space for new dna strands to be created. Dna topoisomerases are sophisticated enzymes that control the dna topology in a cell. Analysis of dna relaxation and cleavage activities of.
It can decatenate the winding that is happening behind the replication fork by focusing on nicks in the dna. Type ic topoisomerase also called topoisomerase v has been identified. A mechanism is required to release the strain created by local unwinding. The mechanism of dna cleavage by type iia topoisomerases has recently been the focus of many biochemical and structural biology studies.