Leroy F. Liu

Transcription generates positively and negatively supercoiled domains in the template.

We show that transcription of a DNA molecule inside a bacterium is accompanied by local and temporal supercoiling of the DNA template: as transcription proceeds, DNA in front of the transcription ensemble becomes positively supercoiled, and DNA behind the ensemble becomes negatively supercoiled. Because bacterial gyrase and topoisomerase I act differently on positively and negatively supercoiled DNA, the formation of twin supercoiled domains during transcription is manifested by a large increase or decrease in the linking number of an intracellular plasmid when bacterial DNA gyrase or topoisomerase I, respectively, is inhibited. Such changes in linking number are strongly dependent on transcription of the plasmid in cis and on the relative orientations of transcription units on the plasmid. These results indicate that the state of supercoiling of bacterial DNA is strongly modulated by transcription, and that DNA topoisomerases are normally involved in the elongation step of transcription.

Topoisomerase-targeting antitumor drugs.

Much has been learned about the unusual type of DNA damage produced by the topoisomerases. The mechanism by which these lesions trigger cell death, however, remains unclear, but it appears that DNA metabolic machinery transforms reversible single-strand cleavable complexes to overt strand breaks which may be an initial event in the cytotoxic pathway. For the topoisomerase I poisons, they produce breaks at replication forks that appear to be the equivalent of a break in duplex DNA. Indicating that this may be an important cytotoxic lesion is the hypersensitivity to camptothecin of the yeast mutant rad52, which is deficient in double-strand-break-repair. The topoisomerase poisons preferentially kill proliferating cells. In the case of the topoisomerase I poison camptothecin, dramatic S-phase-specific cytotoxicity can explain its preferential action on proliferating cells. For the topoisomerase II poisons, high levels of the enzyme in proliferating cells, and very low levels in quiescent cells appear to explain the resistance of quiescent cells to the drugs cytotoxic effects. Thus, the topoisomerase poisons convert essential enzymes into intracellular, proliferating-cell toxins. The identification of both topoisomerase I and II as the specific targets of cancer chemotherapeutic drugs now provides a rational basis for the development of topoisomerase I poisons for possible clinical use. Knowledge of the molecular mechanisms of cell killing may lead to the identification of new therapies for treating cancer. The topoisomerase poisons appear to be a good tool for studying cell killing mechanisms as they produce highly specific and reversible lesions.

Transcription-driven supercoiling of DNA: Direct biochemical evidence from in vitro studies

The translocation of an RNA polymerase elongation complex along double helical DNA has been proposed to generate positive supercoiling waves ahead of and negative supercoiling waves behind the transcription ensemble. This twin supercoiled domain model has been tested in vitro. In the presence of prokaryotic DNA topoisomerase I, which selectively removes negative supercoils, transcription from a single promoter results in rapid and extensive positive supercoiling of the DNA template. The accumulation of positive supercoils in the DNA template requires continued movement of the elongation complex as well as sizable nascent RNA chains. These in vitro results provide direct biochemical evidence supporting the twin supercoiled domain model of transcription. Furthermore, the magnitute of DNA supercoiling (torsional) waves generated by transcription is much greater than previously expected, suggesting that transcription is one of the principal factors affecting intracellular DNA supercoiling.

DNA Topoisomerases as Anticancer Drug Targets

Publisher Summary This chapter discusses the DNA topoisomerases as anticancer drug targets. Much progress has been made in recent years in understanding the mechanism of action of antitumor drugs that target topoisomerases. However, while it is now well established that these drugs interact with the cleavable complex, molecular details of the protein-DNA-drug interaction are only just starting to emerge. The large body of information available from the study of drug analogs has provided some detailed information on the structural requirements for drugs to interact successfully with the cleavable complex. Little is known about the actual cell killing mechanism. This question, which involves events beyond cleavable complex formation and its interaction with drugs, is becoming more and more important. Knowledge of the signals and events involved in cell killing, in particular the early signals induced by drug-mediated DNA damage, might eventually lead to the discovery and identification of new targets for antitumor drugs.

DNA looping alters local DNA conformation during transcription

The effect of protein-mediated DNA looping on local DNA conformation during active transcription was studied using the lac repressor-operator system. Our results suggest that lac repressor-mediated DNA looping within a plasmid DNA molecule containing two lac repressor binding sequences in vivo effectively separates plasmid DNA into two topological domains. Supercoils generated by transcription within each topological domain can be rapidly removed by DNA topoisomerase I.

Chapter 24. DNA Topoisomerases as Therapeutic Targets in Cancer Chemotherapy

Publisher Summary Recent findings have shown that a large number or clinically important anticancer drugs affect the breakage-rejoining reaction of mammalian DNA topoisomerase II by trapping a covalent enzyme-DNA cleavable complex to the mechanism of cell killing by anticancer drugs. DNA topoisomerase may be a key component in the regulation of chromatin conformation and cell proliferation. DNA topoisomerases are enzymes that control the topology of DNA. They are involved in DNA replication, RNA transcription and recombination. Based on their mechanism of action, DNA topoisomerases have been categorized into two types. Type I and type II DNA topoisomerases catalyze the topological changes in DNA by transiently breaking one strand or two strands of the DNA helix, respectively. The most characteristic reaction of a type I DNA topoisomerase is the relaxation of super-helical DNA, The type II topoisomerases can catalyze the passing of two DNA segments those can lead to a number of topoisomerization reactions of DNA such as super-coiling/relaxation, knotting/unknotting, and catenation/decatenation. Recent studies suggest that mammalian DNA topoisomerases is highly regulated by the growth state of cells. The antitumor activity of topoisomerase II —targeting anticancer drugs may well be determined at least in part by the differential regulation of topoisomerase II in neoplastic cells. This chapter discusses protein-linked DNA breaks induced by a number of antitumor drugs. Adriamycin, an anthracycline, has been one of the most important anticancer drugs to use as because of its mode of DNA binding, adriamycin interferes with many DNA functions such as replication and transcription. The chapter also discusses evidence that DNA Tonoisomerase II is the drug target responsible for protein-linked DNA breaks, drug-induced DNA cleavage on cellular chromatin in cultured mammalian cells and mechanism of cell killing.

Interlocking of plasmid DNAs due to lac repressor-operator interaction☆

The presence of a single lac repressor binding sequence on plasmid DNAs is shown to mediate the formation of interlocked dimers in E. coli. The presence of both homo- and hetero-interlocked dimers suggests that the lac repressor complex can bring together randomly two plasmid DNA molecules to facilitate gyrase-mediated interlocking. The exclusive formation of multiply intertwined dimers also suggest that the lac repressor complex may bind simultaneously to a pair of replicated daughter plasmid molecules prior to their segregation. The formation of interlocked plasmid DNAs can be indicative of interaction between two DNA bound proteins in vivo.

DNA STRAND PASSING AND THE FUNCTION OF TYPE II DNA TOPOISOMERASES1

ABSTRACT A DNA topoisomerase from bacteriophage T4-infected E. coli cells has recently been isolated and characterized. The T4 DNA topoisomerase catalyzes the relaxation of superhelical DNAs (whether positively or negatively coiled) in a reaction requiring ATP hydrolysis. The purified enzyme contains multiple subunits, which are apparently coded for by T4 genes 39, 52 and 60. Genetic and biochemical studies of mutants in these genes indicate that the T4 DNA topoisomerase is essential for phage T4 DNA replication and is most likely involved in the initiation of DNA replication forks. Mechanistic studies of this enzyme have clearly established that T4 DNA topoisomerase (as well as other type II DNA topo-isomerases) catalyzes the so-called “DNA strand passing reaction,”presumably via mechanisms that involve a transient double-chain scission on one of the two crossing DNA double helices. The passage of a second double-stranded DNA segment through this transient double-strand break results in a variety of DNA topoisomerization reactions, including knotting:unknotting; relaxation: supercoiling and catenation:decatenation. Using various assays specific for the type II DNA topoisomerases (such as unknotting, catenation and decatenation), an ATP dependent enzyme activity has been detected in all eukaryotic organisms tested thus far and highly purified from HeLa cells and calf thymus. The highly purified eukaryotic type II DNA topoisomerase (designated as topo II) is strikingly similar to the T4 DNA topoisomerase. The possible roles of such type II enzymes in a variety of biological functions will be discussed. A new eukaryotic type I DNA topoisomerase (designated as topo I) has also been purified to near homogeneity from HeLa cells. This type I DNA topoisomerase is a major nuclear protein and is most likely ubiquitous. Its possible relationship to the eukaryotic “nicking-closing” enzyme is discussed.