Interdomain communication in DNA topoisomerase II. DNA binding and enzyme activation

Topoisomerase II catalyzes the ATP-dependent transport of a DNA segment (T-DNA) through a transient double strand break in another DNA segment (G-DNA). A fundamental mechanistic question is how the individual steps in this process are coordinated. We probed communication between the DNA binding sites and the individual enzymatic activities, ATP hydrolysis, and DNA cleavage. We employed short DNA duplexes to control occupancy at the two binding sites of wild-type enzyme and a variant with a G-DNA site mutation. The DNA concentration dependence of ATP hydrolysis and a fluorescence anisotropy assay provided thermodynamic information about DNA binding. The results suggest that G-DNA binds with higher affinity than T-DNA. Enzyme with only G-DNA bound is competent to cleave DNA, indicating that T-DNA is dispensable for DNA cleavage. The ATPase activity of enzyme bound solely to G-DNA is partially stimulated. Full stimulation requires binding of T-DNA. Both DNA binding sites therefore signal to the ATPase domains. The results support and extend current mechanistic models for topoisomerase II-catalyzed DNA transport and provide a framework for future mechanistic dissection.     

Mechanism of ATP inhibition of mammalian type I DNA topoisomerase: DNA binding, cleavage, and rejoining are insensitive to ATP

A general, unrefined mechanism of type I DNA topoisomerase action involves several steps including DNA binding, single-strand scission, strand passage resealing, and, possibly, readoption of an active enzyme conformation. None of these steps requires an energy cofactor; however, we have shown previously that several mammalian type I topoisomerases are, in fact, inhibited by ATP. In this study, we wanted to examine which steps in the gross topoisomerase mechanism were sensitive or insensitive to ATP. Nitrocellulose filter binding experiments showed that ATP did not interfere with the binding of DNA by the enzyme and that ATP binding by topoisomerase was 5-fold greater in the presence of DNA than in its absence. Agarose gel electrophoresis in the presence or absence of ethidium bromide indicated that resealing was unaffected by added ATP. The addition of the adenine nucleotide did not alter the pattern of camptothecin-stimulated cleavage of DNA, indicating that strand scission was not the point of inhibition. To test whether strand passage or the readoption of an active conformation was an inhibited step, we used a unique DNA topoisomer as substrate. The results argued against readoption of an active enzyme conformation as an ATP-sensitive process.     

Bacterial DNA topoisomerase I can relax positively supercoiled DNA containing a single-stranded loop

Using heteroduplex molecules formed from a pair of plasmids, one of which contains a small deletion relative to the other, it is shown that bacterial topoisomerase I can relax a positively supercoiled DNA if a short single-stranded loop is placed in the DNA. This result supports the postulate that the specificity of bacterial DNA topoisomerase I for negatively supercoiled DNA in its relaxation reaction derives from the requirement of a short single-stranded DNA segment in the active enzyme-substrate complex. Nucleolytic and chemical probing of complexes between bacterial DNA topoisomerase I and heteroduplex DNA molecules containing single-stranded loops ranging from 13 to 27 nucleotides in length suggests that the enzyme binds specifically to the region containing a single-stranded loop; the site of DNA cleavage by the topoisomerase appears to lie within the single-stranded loop, with the enzyme interacting with nucleotides on both sides of the point of cleavage.     

Inhibition of HeLa cell DNA topoisomerase I by ATP and phosphate.

The relaxation activity of DNA topoisomerase I from HeLa cell nuclei is strongly inhibited by a variety of purine nucleotides in the presence but not absence of 1 mM potassium phosphate. For ATP, 3-4 mM causes nearly complete inhibition. The 2'-and 3'-AMP isomer are active as well in the presence of 1 mM phosphate, but the 5'-AMP isomer and adenosine are inert. At 3 mM ATP, the titration curve for phosphate is sigmoidal with inhibition beginning abruptly at about 0.5 mM. The negatively-supercoiled DNA isolated from an "inhibited" reaction is relaxed as well as the standard DNA template in the absence of ATP and phosphate suggesting that inhibition does not result from an alteration of the template which protects against its relaxation. Relaxation of positively-supercoiled DNA is also inhibited. Catalysis by E. coli DNA topoisomerase I and HeLa DNA topoisomerase II is not inhibited at concentrations of ATP and phosphate sufficient to cause 80-90% inhibition of HeLa type 1 enzyme.     

The catalytic activities of DNA topoisomerase II are most closely associated with the DNA cleavage/religation steps.

DNA topoisomerase II regulates the three-dimensional organisation of DNA and is the principal target of many important anticancer and antimicrobial agents. These drugs usually act on the DNA cleavage/religation steps of the catalytic cycle resulting in accumulation of covalent DNA-topoisomerase II complexes. We have studied the different steps of the catalytic cycle as a function of salt concentration, which is a classical way to evaluate the biochemical properties of proteins. The results show that the catalytic activity of topoisomerase II follows a bell-shaped curve with optimum between 100 and 225 mM KCl. No straight-forward correlation exists between DNA binding and catalytic activity. The highest levels of drug-induced covalent DNA-topoisomerase II complexes are observed between 100 and 150 mM KCl. Remarkably, at salt concentrations between 150 mM and 225 mM KCl, topoisomerase II is converted into a drug-resistant form with greatly reduced levels of drug-induced DNA-topoisomerase II complexes. This is due to efficient religation rather than to absence of DNA cleavage as witnessed by relaxation of the supercoiled DNA substrate. In the absence of DNA, ATP hydrolysis is strongest at low salt concentrations. Unexpectedly, the addition of DNA stimulates ATP hydrolysis at 100 and 150 mM KCl, but has little or no effect below 100 mM KCl in spite of strong non-covalent DNA binding at these salt concentrations. Therefore, DNA-stimulated ATP hydrolysis appears to be associated with covalent rather than non-covalent binding of DNA to topoisomerase II. Taken together, the results suggest that it is the DNA cleavage/religation steps that are most closely associated with the catalytic activities of topoisomerase II providing a unifying theme for the biological and pharmacological modulation of this enzyme.     

Mode of action of topoisomerase II-targeting agents at a specific DNA sequence. Uncoupling the DNA binding, cleavage and religation events.

Methods of uncoupling the DNA binding, cleavage and religation reactions of topoisomerase II were employed to investigate the influence of topoisomerase II-directed drugs on the individual steps in the enzyme's catalytic cycle. A special DNA substrate containing a major topoisomerase II interaction site, which can be cleaved by the enzyme in the absence of any concomitant religation, was used to examine the effect of topoisomerase II-directed agents upon the DNA cleavage reaction. The experiment demonstrated that the topoisomerase II targeting agent Ro 15-0216 stimulates the DNA cleavage reaction extensively, whereas the traditional topoisomerase II inhibitor, mAMSA, has only a minor effect on this reaction. Topoisomerase II trapped in the cleavage complexes can religate to the 3' hydroxyl end of another DNA strand. Using this religation assay, it was demonstrated that the major effect of mAMSA is an inhibition of the enzyme's religation reaction, whereas Ro 15-0216 has no effect on this reaction. Recently, considerable attention has been given to drugs preventing topoisomerase II from introducing DNA cleavages. In the present paper the initial non-covalent DNA binding reaction of topoisomerase II was investigated under conditions excluding enzyme-mediated DNA cleavage. This demonstrated that the anthracycline, aclarubicin, prevents topoisomerase II from performing its initial non-covalent DNA binding reaction and thereby abolishes the DNA cleavage reaction of the enzyme. The results presented here demonstrate that profound differences exist in the mode of action of different agents targeting topoisomerase II, and that the enzyme can be affected by such agents at both its DNA binding, cleavage and religation subreactions.     

Reconstitution of DNA topoisomerase VI of the thermophilic archaeon Sulfolobus shibatae from subunits separately overexpressed in Escherichia coli.

DNA topoisomerase VI from the hyperthermophilic archaeon Sulfolobus shibatae is the prototype of a novel family of type II DNA topoisomerases that share little sequence similarity with other type II enzymes, including bacterial and eukaryal type II DNA topoisomerases and archaeal DNA gyrases. DNA topoisomerase VI relaxes both negatively and positively supercoiled DNA in the presence of ATP and has no DNA supercoiling activity. The native enzyme is a heterotetramer composed of two subunits, A and B, with apparent molecular masses of 47 and 60 kDa, respectively. Here wereport the overexpression in Escherichia coli and the purification of each subunit. The A subunit exhibits clusters of arginines encoded by rare codons in E.coli . The expression of this protein thus requires the co-expression of the minor E.coli arginyl tRNA which reads AGG and AGA codons. The A subunit expressed in E.coli was obtained from inclusion bodies after denaturation and renaturation. The B subunit was overexpressed in E.coli and purified in soluble form. When purified B subunit was added to the renatured A subunit, ATP-dependent relaxation and decatenation activities of the hyperthermophilic DNA topoisomerase were reconstituted. The reconstituted recombinant enzyme exhibits a specific activity similar to the enzyme purified from S.shibatae . It catalyzes transient double-strand cleavage of DNA and becomes covalently attached to the ends of the cleaved DNA. This cleavage is detected only in the presence of both subunits and in the presence of ATP or its non-hydrolyzable analog AMPPNP.     

Nuclease protection by Drosophila DNA topoisomerase II. Enzyme/DNA contacts at the strong topoisomerase II cleavage sites

A DNA consensus sequence for topoisomerase II cleavage sites was derived previously based on a statistical analysis of the nucleotide sequences around 16 sites that can be efficiently cleaved by Drosophila topoisomerase II (Sander, M., and Hsieh, T. (1985) Nucleic Acids Res. 13, 1057-1072). A synthetic 21-mer DNA sequence containing this cleavage consensus sequence was cloned into a plasmid vector, and DNA topoisomerase II can cleave this sequence at the position predicted by the cleavage consensus sequence. DNase I footprint analysis showed that topoisomerase II can protect a region of approximately 25 nucleotides in both strands of the duplex DNA, with the cleavage site located near the center of the protected region. Similar correlation between the DNase I footprints and strong topoisomerase II cleavage sites has been observed in the intergenic region of the divergent HSP70 genes. This analysis therefore suggests that the strong DNA cleavage sites of Drosophila topoisomerase II likely correspond to specific DNA-binding sites of this enzyme. Furthermore, the extent of DNA contacts made by this enzyme suggests that eucaryotic topoisomerase II, in contrast to bacterial DNA bacterial DNA gyrase, cannot form a complex with extensive DNA wrapping around the enzyme. The absence of DNA wrapping is probably the mechanistic basis for the lack of DNA supercoiling action for eucaryotic topoisomerase II.     

Studies of the topoisomerase II-mediated cleavage and religation reactions by use of a suicidal double-stranded DNA substrate.

The cleavage and religation reactions of eukaryotic topoisomerase II were studied by use of a 5'-recessed DNA substrate containing a strong recognition sequence for the enzyme. Cleavage of the DNA substrate was suicidal, that is the enzyme was unable to religate the cleaved DNA due to a release of DNA 5' to the cleavage position. With this substrate cleavage products accumulated with time in the absence of protein-denaturing agents, and the cleavage reaction was not reversible with salt. The suicide cleavage complexes contained a kinetically competent topoisomerase II enzyme as determined by the enzyme's ability to perform intermolecular ligation of the cleaved DNA to a free 3'-hydroxyl end on another DNA strand. The efficiency of the religation reaction depended on the ability of the religation substrate to base pair to the DNA in the cleaved enzyme-DNA complex. Higher levels of religation were obtained with dinucleotides than with long DNA substrates. Mononucleotides also were efficiently religated, indicating an ability of the enzyme to mediate religation without making contacts to a long stretch of nucleotides 5' to the cleavage position.     

Mapping of eukaryotic DNA topoisomerase I catalyzed cleavage without concomitant religation in the vicinity of DNA structural anomalies

Sensitive sites for covalent trapping of eukaryotic topoisomerase I at DNA structural anomalies were mapped by a new method using purified enzyme and defined DNA substrates. To insure that the obtained topoisomerase I trapping patterns were not influenced by DNA sequence variations, a single DNA imperfection was placed centrally within a homonucleotide track. Mapping of topoisomerase I-mediated irreversible cleavage sites on homopolymeric DNA substrates containing mismatches showed trapping of the enzyme in several positions in close vicinity of the DNA imperfection, with a strong preference for the 5' junction between the duplex DNA and the base-pairing anomaly. On homopolymeric DNA substrates containing a nick, sites of topoisomerase I-mediated cleavage on the intact strand were located just opposite to the nick and from one to ten nucleotides 5' to the nick. Sites of enzyme-mediated cleavage next to a nick and an immobile single-stranded branch were located 5' to the strand interruption in distances of two to six nucleotides and two to ten nucleotides, respectively. Taken together these findings suggest that covalent trapping of topoisomerase I proceeds at positions adjacent to mismatches, nicks and single-stranded branches, where the cleavage reaction is allowed and the ensuing ligation reaction prevented. In principle, the developed interference method might be of general utility to define topoisomerase-DNA interactions relative to different types of structural anomalies.     

DNA topoisomerase VI generates ATP-dependent double-strand breaks with two-nucleotide overhangs

A key step in the DNA transport by type II DNA topoisomerase is the formation of a double-strand break with the enzyme being covalently linked to the broken DNA ends (referred to as the cleavage complex). In the present study, we have analyzed the formation and structure of the cleavage complex catalyzed by Sufolobus shibatae DNA topoisomerase VI (topoVI), a member of the recently described type IIB DNA topoisomerase family. A purification procedure of a fully soluble recombinant topoVI was developed by expressing both subunits simultaneously in Escherichia coli. Using this recombinant enzyme, we observed that the formation of the double-strand breaks on supercoiled or linear DNA is strictly dependent on the presence of ATP or AMP-PNP. This result suggests that ATP binding is required to stabilize an enzyme conformation able to cleave the DNA backbone. The structure of cleavage complexes on a linear DNA fragment have been analyzed at the nucleotide level. Similarly to other type II DNA topoisomerases, topoVI is covalently attached to the 5'-ends of the broken DNA. However, sequence analysis of the double-strand breaks revealed that they are all characterized by staggered two-nucleotide long 5' overhangs, contrasting with the four-base staggered double-strand breaks catalyzed by type IIA DNA topoisomerases. While no clear consensus sequences surrounding the cleavage sites could be described, interestingly A and T nucleotides are highly represented on the 5' extensions, giving a first insight on the preferred sequences recognized by this type II DNA topoisomerase.     

Interaction of human DNA topoisomerase II alpha with DNA: quantification by surface plasmon resonance

DNA topoisomerase II is an ATP-operated clamp that effects topological changes by capturing a double-stranded DNA segment and transporting it through another duplex. Surface plasmon resonance (SPR) was used to characterize interactions of human topoisomerase II alpha with different topological forms of DNA. Using a linear fragment of pUC18 DNA, the equilibrium binding constant of topoisomerase II alpha was determined to be 0.16 nM. The affinity was not affected by the absence of ATP or the presence of the bisdioxopiperazine catalytic inhibitor ICRF-187. Besides, similar affinities were found for several bisdioxopiperazine-resistant mutant enzymes. These results suggest that the mechanism of topoisomerase II alpha inhibition by ICRF-187 and its resistance does not directly involve the interaction of DNA with the enzyme. SPR was also adapted to measure levels of the closed clamp form of topoisomerase II present on DNA. As expected, a stable closed clamp form of the enzyme was detectable on circular DNA but not on linear DNA. Detection of the closed clamp required the presence of ATP and a bisdioxopiperazine, or a non-hydrolyzable analogue of ATP. In the presence of ATP and ICRF-187, several bisdioxopiperazine-resistant mutant enzymes failed to form detectable levels of stable closed clamp. Interestingly, a mutant of human topoisomerase II alpha with an altered active site tyrosine showed lower levels of closed clamp formation. In conclusion, SPR is able to (1) determine the kinetics of topoisomerase II with its DNA substrate and (2) quantify the enzyme's closed clamp formation under varying circumstances.     

ATP-dependent DNA topoisomerase from D. melanogaster reversibly catenates duplex DNA rings

Extracts of Drosophila embryos contain an enzymatic activity that converts circular DNAs into huge networks of catenated rings in an ATP-dependent fashion. The catenation activity is resolved into two protein components during purification. One component is a novel DNA topoisomerase that requires the presence of ATP in order to relax supercoiled DNA. We have shown that the ATP-dependent DNA topoisomerase relaxes DNA by a mechanism distinct from that of nicking-closing enzymes. The Drosophila ATP-dependent topoisomerase allows one segment of a circular DNA to pass through transient breaks in both strands at another site on the DNA circle without any relative rotation between the ends at the transient break. This mechanism can convert negative supertwists to positive twists and vice versa until a relaxed equilibrium state is reached. The formation of catenated rings is mediated by an analogous bimolecular reaction which can occur between two nonhomologous DNA circles. The catenation reaction is fully reversible: in the presence of the second protein component, circular DNA is converted quantitatively into catenated forms; in its absence, the ATP-dependent topoisomerase resolves catenated networks back into monomer circles. The Drosophila ATP-dependent topoisomerase appears to be closely related to E. coli DNA gyrase in that both use a similar mechanism to change the topology of DNA, both require ATP and both are inhibited by the antibiotic novobiocin. The presence of an enzyme that allows one DNA helix to pass freely through another could not only be useful in relaxation of topological constraints, but also may be involved in the folding and unfolding of eucaryotic chromosomes.     

DNA topoisomerase II from Drosophila melanogaster. Relaxation of supercoiled DNA

In order to study the double-strand DNA passage reaction of eukaryotic type II topoisomerases, a quantitative assay to monitor the enzymic conversion of supercoiled circular DNA to relaxed circular DNA was developed. Under conditions of maximal activity, relaxation catalyzed by the Drosophila melanogaster topoisomerase II was processive and the energy of activation was 14.3 kcal . mol-1. Removal of supercoils was accompanied by the hydrolysis of either ATP or dATP to inorganic phosphate and the corresponding nucleoside diphosphate. Apparent Km values were 200 microM for pBR322 plasmid DNA, 140 microM for SV40 viral DNA, 280 microM for ATP, and 630 microM for dATP. The turnover number for the Drosophila enzyme was at least 200 supercoils of DNA relaxed/min/molecule of topoisomerase II. The enzyme interacts preferentially with negatively supercoiled DNA over relaxed molecules, is capable of removing positive superhelical twists, and was found to be strongly inhibited by single-stranded DNA. Kinetic and inhibition studies indicated that the beta and gamma phosphate groups, the 2'-OH of the ribose sugar, and the C6-NH2 of the adenine ring are important for the interaction of ATP with the enzyme. While the binding of ATP to Drosophila topoisomerase II was sufficient to induce a DNA strand passage event, hydrolysis was required for enzyme turnover. The ATPase activity of the topoisomerase was stimulated 17-fold by the presence of negatively supercoiled DNA and approximately 4 molecules of ATP were hydrolyzed/supercoil removed. Finally, a kinetic model describing the switch from a processive to a distributive relaxation reaction is presented.     

Negative supercoiling of DNA by eukaryotic DNA topoisomerase II and dextran sulfate.

In the presence of a molar excess of eukaryotic DNA topoisomerase II and an appropriate concentration of dextran sulfate, relaxed closed circular DNA is converted to a negatively supercoiled form. The reaction is dependent on ATP. Neither adenosine 5'-[beta,gamma-imido]-triphosphate nor adenosine 5'-[gamma-thio]triphosphate can substitute for ATP. The negative supercoils formed are relaxed by subsequent addition of DNA topoisomerase I to the supercoiling reaction mixture. Covalent closure of a nicked circular DNA in the presence of DNA topoisomerase II and dextran sulfate but in the absence of ATP causes a small decrease in the linking number. These results suggest that when an appropriate concentration of dextran sulfate is present, the binding of a molar excess of eukaryotic DNA topoisomerase II constrains a small number of negative supercoils in DNA, which in turn generate unconstrained negative supercoils at the expense of ATP.     

Communication between the ATPase and cleavage/religation domains of human topoisomerase IIalpha

The DNA strand passage activity of eukaryotic topoisomerase II relies on a cascade of conformational changes triggered by ATP binding to the N-terminal domain of the enzyme. To investigate the interdomain communication between the ATPase and cleavage/religation domains of human topoisomerase IIalpha, we characterized a mutant enzyme that contains a deletion at the interface between the two domains, covering amino acids 350-407. The ATPase domain retained full activity with a rate of ATP hydrolysis that was severalfold higher than normal, but the ATPase activity was unaffected by DNA. The cleavage and religation activities of the enzyme were comparable with those of the wild-type enzyme both in the absence and presence of cancer chemotherapeutic agents. However, neither ATP nor a nonhydrolyzable ATP analog stimulated cleavage complex formation. Although both conserved domains retained full activity, the mutant enzyme was unable to coordinate these activities into strand passage. Our findings suggest that the normal conformational transitions occurring in the enzyme upon ATP binding are hampered or lacking in the mutant enzyme. Consistent with this hypothesis, the enzyme displayed an abnormal clamp closing activity. In summary, the region covering amino acids 350-407 in human topoisomerase IIalpha seems to be essential for correct interdomain communication and probably is involved in signaling ATP binding to the rest of the enzyme.     

Type II DNA topoisomerases: enzymes that can unknot a topologically knotted DNA molecule via a reversible double-strand break

The T4 DNA topoisomerase is a recently discovered multisubunit protein that appears to have an essential role in the initiation of T4 bacteriophage DND replication. Treatment of double-stranded circular DNA with large amounts of this topoisomerase in the absence of ATP yields new DNA species which are knotted topological isomers of the double-stranded DNA circle. These knotted DNA circles, whether covalently closed or nicked, are converted to unknotted circles by treatment with trace amounts of the T4 topoisomerase in the presence of ATP. Very similar ATP-dependent enzyme activities capable of unknotting DNA are present in extracts of Drosophila eggs. Xenopus laevis eggs and mammalian tissue culture cells. The procaryotic enzyme, DNA gyrase, is also capable of unknotting DNA. We propose that these unknotting enzymes constitute a new general class of DNA topoisomerases (type II DNA topoisomerases). These enzymes must act via mechanisms that involve the concerted cleavage and rejoining of two opposite DNA strands, such that the DNA double helix is transiently broken. The passage of a second double-stranded DNA segment through this reversible double-strand break results in a variety of DNA topoisomerization reactions, including relaxation:super-coiling; knotting:unknotting and catenation:decatenation. In support of this type of mechanism, we demonstrate that the T4 DNA topoisomerase changes the linking number of a covalently closed double-stranded circular DNA molecule only by multiples of two. We discuss the possible roles of such enzymes in a variety of biological functions, along with their probable molecular mechanisms.     

Effect of casein kinase II-mediated phosphorylation on the catalytic cycle of topoisomerase II. Regulation of enzyme activity by enhancement of ATP hydrolysis.

The catalytic activity of topoisomerase II is stimulated approximately 2-3-fold following phosphorylation by casein kinase II (Ackerman, P., Glover, C. V. C., and Osheroff, N. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3164-3168). In order to delineate the mechanism by which the activity of the enzyme is enhanced, the effects of casein kinase II-mediated phosphorylation on the individual steps of the catalytic cycle of Drosophila topoisomerase II were characterized. Phosphorylation did not affect reaction steps that preceded hydrolysis of the enzyme's high energy ATP cofactor. This included enzyme-DNA binding, pre-strand passage DNA cleavage/religation, the double-stranded DNA passage event, and post-strand passage DNA cleavage/religation. In contrast, the rate of topoisomerase II-mediated ATP hydrolysis was stimulated 2.7-fold following phosphorylation by casein kinase II. Since ATP hydrolysis is a prerequisite for enzyme turnover, it is concluded that phosphorylation modulates the overall catalytic activity of topoisomerase II by stimulating the enzyme's ATPase activity.     

DNA topoisomerases as targets for the anticancer drug TAS-103: primary cellular target and DNA cleavage enhancement

TAS-103 is a novel antineoplastic agent that is active against in vivo tumor models [Utsugi, T., et al. (1997) Jpn. J. Cancer Res. 88, 992-1002]. This drug is believed to be a dual topoisomerase I/II-targeted agent, because it enhances both topoisomerase I- and topoisomerase II-mediated DNA cleavage in treated cells. However, the relative importance of these two enzymes for the cytotoxic actions of TAS-103 is not known. Therefore, the primary cellular target of the drug and its mode of action were determined. TAS-103 stimulated DNA cleavage mediated by mammalian topoisomerase I and human topoisomerase IIalpha and beta in vitro. The drug was less active than camptothecin against the type I enzyme but was equipotent to etoposide against topoisomerase IIalpha. A yeast genetic system that allowed manipulation of topoisomerase activity and drug sensitivity was used to determine the contributions of topoisomerase I and II to drug cytotoxicity. Results indicate that topoisomerase II is the primary cellular target of TAS-103. In addition, TAS-103 binds to human topoisomerase IIalpha in the absence of DNA, suggesting that enzyme-drug interactions play a role in formation of the ternary topoisomerase II.drug.DNA complex. TAS-103 induced topoisomerase II-mediated DNA cleavage at sites similar to those observed in the presence of etoposide. Like etoposide, it enhanced cleavage primarily by inhibiting the religation reaction of the enzyme. Based on these findings, it is suggested that TAS-103 be classified as a topoisomerase II-targeted drug.     

Kinetic mechanism of human DNA ligase I reveals magnesium-dependent changes in the rate-limiting step that compromise ligation efficiency

DNA ligase I (LIG1) catalyzes the ligation of single-strand breaks to complete DNA replication and repair. The energy of ATP is used to form a new phosphodiester bond in DNA via a reaction mechanism that involves three distinct chemical steps: enzyme adenylylation, adenylyl transfer to DNA, and nick sealing. We used steady state and pre-steady state kinetics to characterize the minimal mechanism for DNA ligation catalyzed by human LIG1. The ATP dependence of the reaction indicates that LIG1 requires multiple Mg(2+) ions for catalysis and that an essential Mg(2+) ion binds more tightly to ATP than to the enzyme. Further dissection of the magnesium ion dependence of individual reaction steps revealed that the affinity for Mg(2+) changes along the reaction coordinate. At saturating concentrations of ATP and Mg(2+) ions, the three chemical steps occur at similar rates, and the efficiency of ligation is high. However, under conditions of limiting Mg(2+), the nick-sealing step becomes rate-limiting, and the adenylylated DNA intermediate is prematurely released into solution. Subsequent adenylylation of enzyme prevents rebinding to the adenylylated DNA intermediate comprising an Achilles' heel of LIG1. These ligase-generated 5'-adenylylated nicks constitute persistent breaks that are a threat to genomic stability if they are not repaired. The kinetic and thermodynamic framework that we have determined for LIG1 provides a starting point for understanding the mechanism and specificity of mammalian DNA ligases.