Multiple bacterial topoisomerases: Specialization or redundancy?

In the past few years, two new DNA topoisomerases have been discovered in bacteria, bringing the total number of DNA topoisomerases in E. coli to four. Two classes of topoisomerases, type 1 and type 2, are distinguishable by their amino acid homology and their apparent reaction mechanism. Of the four E. coli topoisomerases, there are two type 1 and two type 2 enzymes. In eukaryotes, the existence of multiple type 1 and type 2 enzymes has also become apparent. The existence of these multiple enzymes provokes a question whose answer has both evolutionary and physiological implications: are these topoisomerases functionally redundant, or have they acquired sufficient specialization that they now perform unique biological reactions? In bacteria, there is evidence for both specialization and redundancy in the functions of topoisomerases.     

DNA substrate specificity of reverse gyrase from extremely thermophilic archaebacteria

It has been shown earlier that eukaryotic type I DNA topoisomerases act on duplex DNA regions, while eubacterial type I topoisomerases require single-stranded regions. The present paper demonstrates that the type I topoisomerase from extremely thermophilic archaebacteria, reverse gyrase, winds DNA by binding to single-stranded DNA regions. Thus, type I topoisomerases, both relaxing one in eubacteria and reverse gyrase in extremely thermophilic archaebacteria share a substrate specificity to melted DNA regions. The important consequence of this specificity is that the cellular DNA superhelical stress actively controlled by bacterial topoisomerases is confined to a narrow range characterized by a low stability of the double helix. Hence we suppose that bacterial topoisomerase systems control duplex stability near its minimum, for which purpose they create an appropriate negative superhelicity at moderate temperatures or a positive one at extremely high temperatures, the feedback being ensured by the aforesaid specificity of type I bacterial topoisomerases.     

Proteins of the origin recognition complex (ORC) and DNA topoisomerases on mammalian chromatin

Background  

The process of DNA replication requires the separation of complementary DNA strands. In this process, the unwinding of circularly closed or long DNA duplices leads to torsional tensions which must be released by topoisomerases. So topoisomerases play an important role in DNA replication. In order to provide more information about topoisomerases in the initiation of mammalian replication, we investigated whether topoisomerases occur close to ORC in the chromatin of cultured human HeLa cells.     

DNA topoisomerases.

DNA topoisomerases play an important role in regulating DNA structure, thus affecting many aspects of chromosome function inside cells. Recent progress in this field raises exciting questions regarding the distinct and critical functions of multiple topoisomerases, and the roles of DNA topoisomerases in the processes of chromosome condensation, decondensation, and segregation.     

Separation and functional analysis of eukaryotic DNA topoisomerases by chromatography and electrophoresis

DNA topoisomerases are enzymes that control DNA topology by cleaving and rejoining DNA strands and passing other DNA strands through the transient gaps. Consequently, these enzymes play a crucial role in the regulation of the physiological function of the genome. Beyond their normal functions, topoisomerases are important cellular targets in the treatment of human cancers. In this review we summarize current protocols for extracting and purifying DNA topoisomerases, and for separating subtypes and isoforms of these enzymes. Furthermore, we discuss methods for measuring the catalytic activity of topoisomerases and for monitoring the molecular effects of topoisomerase-directed antitumor drugs in cell-free assays.     

Phylogenomics of DNA topoisomerases: their origin and putative roles in the emergence of modern organisms

Topoisomerases are essential enzymes that solve topological problems arising from the double-helical structure of DNA. As a consequence, one should have naively expected to find homologous topoisomerases in all cellular organisms, dating back to their last common ancestor. However, as observed for other enzymes working with DNA, this is not the case. Phylogenomics analyses indicate that different sets of topoisomerases were present in the most recent common ancestors of each of the three cellular domains of life (some of them being common to two or three domains), whereas other topoisomerases families or subfamilies were acquired in a particular domain, or even a particular lineage, by horizontal gene transfers. Interestingly, two groups of viruses encode topoisomerases that are only distantly related to their cellular counterparts. To explain these observations, we suggest that topoisomerases originated in an ancestral virosphere, and that various subfamilies were later on transferred independently to different ancient cellular lineages. We also proposed that topoisomerases have played a critical role in the origin of modern genomes and in the emergence of the three cellular domains.     

Thirteen-exon-motif signature for vertebrate nuclear and mitochondrial type IB topoisomerases

DNA topoisomerases contribute to various cellular activities that involve DNA. We previously identified a human nuclear gene that encodes a mitochondrial DNA topoisomerase. Here we show that genes for mitochondrial DNA topoisomerases (type IB) exist only in vertebrates. A 13-exon topoisomerase motif was identified as a characteristic of genes for both nuclear and mitochondrial type IB topoisomerases. The presence of this signature motif is thus an indicator of the coexistence of nuclear and mitochondrial type IB DNA topoisomerases. We hypothesize that the prototype topoisomerase IB with the 13-exon structure formed first, and then duplicated. One topoisomerase specialized for nuclear DNA and the other for mitochondrial DNA.     

Gossypol Interferes with Both Type I and Type II Topoisomerase Activities Without Generating Strand Breaks

A considerable number of agents with chemotherapeutic potentials reported over the past years were shown to interfere with the reactions of DNA topoisomerases, the essential enzymes that regulate conformational changes in DNA topology. Gossypol, a naturally occurring bioactive phytochemical is a chemopreventive agent against various types of cancer cell growth with a reported activity on mammalian topoisomerase II. The compounds targeting topoisomerases vary in their mode of action; class I compounds act by stabilizing covalent topoisomerase-DNA complexes resulting in DNA strand breaks while class II compounds interfere with the catalytic function of topoisomerases without generating strand breaks. In this study, we report Gossypol as the interfering agent with type I topoisomerases as well. We also carried out an extensive set of assays to analyze the type of interference manifested by Gossypol on DNA topoisomerases. Our results strongly suggest that Gossypol is a potential class II inhibitor as it blocked DNA topoisomerase reactions with no consequently formed strand breaks.     

The twisted 'life' of DNA in the cell: bacterial topoisomerases

DNA topoisomerases are essential to the cell for the regulation of DNA supercoiling levels and for chromosome decatenation. The proposed mechanisms for these reactions are essentially the same, except that a change in supercoiling is due to an intramolecular event, while decatenation requires an intermolecular event. The characterized bacterial topoisomerases appear capable of both types of reaction in vitro. Four DNA topoisomerases have been identified in Escherichia coli. Topoisomerase I, gyrase, and topoisomerase IV normally appear to have distinct essential functions within the cell, Gyrase and topoisomerase I are responsible for the regulation of DNA supercoiling. Both gyrase and topoisomerase IV are necessary for chromosomal decatenation. Multiple topoisomerases with distinct functions may give the cell more precise control over DNA topology by allowing tighter regulation of the principal enzymatic activities of these different proteins.     

Unravelling the mechanisms of Type 1A topoisomerases using single-molecule approaches

Topoisomerases are essential enzymes that regulate DNA topology. Type 1A family topoisomerases are found in nearly all living organisms and are unique in that they require single-stranded (ss)DNA for activity. These enzymes are vital for maintaining supercoiling homeostasis and resolving DNA entanglements generated during DNA replication and repair. While the catalytic cycle of Type 1A topoisomerases has been long-known to involve an enzyme-bridged ssDNA gate that allows strand passage, a deeper mechanistic understanding of these enzymes has only recently begun to emerge. This knowledge has been greatly enhanced through the combination of biochemical studies and increasingly sophisticated single-molecule assays based on magnetic tweezers, optical tweezers, atomic force microscopy and Förster resonance energy transfer. In this review, we discuss how single-molecule assays have advanced our understanding of the gate opening dynamics and strand-passage mechanisms of Type 1A topoisomerases, as well as the interplay of Type 1A topoisomerases with partner proteins, such as RecQ-family helicases. We also highlight how these assays have shed new light on the likely functional roles of Type 1A topoisomerases in vivo and discuss recent developments in single-molecule technologies that could be applied to further enhance our understanding of these essential enzymes.     

Archaebacterial reverse gyrase cleavage-site specificity is similar to that of eubacterial DNA topoisomerases I.

ATP-dependent type I topoisomerases from extremely thermophilic archaebacteria--reverse gyrases--drive positive supercoiling of DNA. We showed that reverse gyrase from Desulfurococcus amylolyticus breaks the DNA at specific sites and covalently binds to the 5' end. In 30 out of 31 sites located in pBR322 DNA fragments, cleavage occurs at the sequence 5'---CNNN/---(N is any base). The same rule was previously shown to hold for single-stranded DNA breakage by eubacterial topoisomerases I. The relative cleavage frequencies at different sites depend on Mg2+ and temperature. We discuss the possible physiological and mechanistic role of the above specificity of the bacterial topoisomerases I.