Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I

Camptothecin, a cytotoxic drug, is a strong inhibitor of nucleic acid synthesis in mammalian cells and a potent inducer of strand breaks in chromosomal DNA. Neither the equilibrium dialysis nor the unwinding measurement indicates any interaction between camptothecin and purified DNA. However, camptothecin induces extensive single strand DNA breaks in reactions containing purified mammalian DNA topoisomerase I. DNA breakage in vitro is immediate and reversible. Analyses of camptothecin-induced DNA breaks show that topoisomerase I is covalently linked to the 3' end of the broken DNA. In addition, camptothecin inhibits the catalytic activity of mammalian DNA topoisomerase I. We propose that camptothecin blocks the rejoining step of the breakage-reunion reaction of mammalian DNA topoisomerase I. This blockage results in the accumulation of a cleavable complex which resembles the transient intermediate proposed for eukaryotic DNA topoisomerase I. The inhibition of nucleic acid synthesis and the induction of DNA strand breaks observed in vivo may be related to the formation of this drug-induced cleavable complex.     

Homogeneous high-throughput screening assays for HIV-1 integrase 3beta-processing and strand transfer activities

HIV-1 integrase (HIV-IN) is a well-validated antiviral drug target catalyzing a multistep reaction to incorporate the HIV-1 provirus into the genome of the host cell. Small molecule inhibitors of HIV-1 integrase that specifically target the strand transfer step have demonstrated efficacy in the suppression of virus propagation. However, only few specific strand transfer inhibitors have been identified to date, and the need to screen for novel compound scaffolds persists. Here, the authors describe 2 homogeneous time-resolved fluorescent resonance energy transfer-based assays for the measurement of HIV-1 integrase 3'-processing and strand transfer activities. Both assays were optimized for high-throughput screening formats, and a diverse library containing more than 1 million compounds was screened in 1536-well plates for HIV-IN strand transfer inhibitors. As a result, compounds were found that selectively affect the enzymatic strand transfer reaction over 3beta processing. Moreover, several bioactive molecules were identified that inhibited HIV-1 reporter virus infection in cellular model systems. In conclusion, the assays presented herein have proven their utility for the identification of mechanistically interesting and biologically active inhibitors of HIV-1 integrase that hold potential for further development into potent antiviral drugs.     

Cleavage of DNA by mammalian DNA topoisomerase II

Using the P4 unknotting assay, DNA topoisomerase II has been purified from several mammalian cells. Similar to prokaryotic DNA gyrase, mammalian DNA topoisomerase II can cleave double-stranded DNA and be trapped as a covalent protein-DNA complex. This cleavage reaction requires protein denaturant treatment of the topoisomerase II-DNA complex and is reversible with respect to salt and temperature. The product after reversal of the cleavage reaction remains supertwisted, suggesting that the two ends of the putatively broken DNA are held tightly by the topoisomerase. Alternatively, the enzyme-DNA interaction is noncovalent, and the covalent linking of topoisomerase to DNA is induced by the protein denaturant. Detailed characterization of the cleavage products has revealed that topoisomerase II cuts DNA with a four-base stagger and is covalently linked to the protruding 5'-phosphoryl ends of each broken DNA strand. Calf thymus DNA topoisomerase II cuts SV40 DNA at multiple and specific sites. However, no sequence homology has been found among the cleavage sites as determined by direct nucleotide-sequencing studies.     

Recombinogenic flap ligation mediated by human topoisomerase I

Aberration of eukaryotic topoisomerase I catalysis leads to potentially recombinogenic pathways by allowing the joining of heterologous DNA strands. Recently, a new ligation pathway (flap ligation) was presented for vaccinia virus topoisomerase I, in which blunt end cleavage complexes ligate the recessed end of duplex acceptors having a single-stranded 3'-tail. This reaction was suggested to play an important role in the repair of topoisomerase I-induced DNA double-strand breaks. Here, we characterize flap ligation mediated by human topoisomerase I. We demonstrate that cleavage complexes containing the enzyme at a blunt end allow invasion of a 3'-acceptor tail matching the scissile strand of the donor, which facilitates ligation of the recessed 5'-hydroxyl end. However, the reaction was strictly dependent on the length of double-stranded DNA of the donor complexes, and longer stretches of base-pairing inhibited strand invasion. The stabilization of the DNA helix was most probably provided by the covalently bound enzyme itself, since deleting the N-terminal domain of human topoisomerase I stimulated flap ligation. We suggest that stabilization of the DNA duplex upon enzyme binding may play an important role during normal topoisomerase I catalysis by preventing undesired strand transfer reactions. For flap ligation to function in a repair pathway, factors other than topoisomerase I, such as helicases, would be necessary to unwind the DNA duplex and allow strand invasion.     

Inhibition of Zn(II) Binding Type IA Topoisomerases by Organomercury Compounds and Hg(II)

Type IA topoisomerase activities are essential for resolving DNA topological barriers via an enzyme-mediated transient single strand DNA break. Accumulation of topoisomerase DNA cleavage product can lead to cell death or genomic rearrangement. Many antibacterial and anticancer drugs act as topoisomerase poison inhibitors that form stabilized ternary complexes with the topoisomerase covalent intermediate, so it is desirable to identify such inhibitors for type IA topoisomerases. Here we report that organomercury compounds were identified during a fluorescence based screening of the NIH diversity set of small molecules for topoisomerase inhibitors that can increase the DNA cleavage product of Yersinia pestis topoisomerase I. Inhibition of relaxation activity and accumulation of DNA cleavage product were confirmed for these organomercury compounds in gel based assays of Escherichia coli topoisomerase I. Hg(II), but not As(III), could also target the cysteines that form the multiple Zn(II) binding tetra-cysteine motifs found in the C-terminal domains of these bacterial topoisomerase I for relaxation activity inhibition. Mycobacterium tuberculosis topoisomerase I activity is not sensitive to Hg(II) or the organomercury compounds due to the absence of the Zn(II) binding cysteines. It is significant that the type IA topoisomerases with Zn(II) binding domains can still cleave DNA when interfered by Hg(II) or organomercury compounds. The Zn(II) binding domains found in human Top3α and Top3β may be potential targets of toxic metals and organometallic complexes, with potential consequence on genomic stability and development.     

Single strand DNA cleavage reaction of duplex DNA by Drosophila topoisomerase II

A unique reaction for type II DNA topoisomerase is its cleavage of a pair of DNA strands in concert. We show however, that in a reaction mixture containing a molar excess of EDTA over Mg2+, or when Mg2+ is substituted by Ca2+, Mn2+, or Co2+, the enzyme cleaves only one rather than both strands. These results suggest that the divalent cations may play an important role in coordinating the two subunits of DNA topoisomerase II during the strand cleavage reaction. The single strand and the double strand cleavage reactions are similar in the following aspects: both require the addition of a protein denaturant, can be reversed by low temperature or high salt, and a topoisomerase II molecule is attached covalently to the 5' phosphoryl end of each broken DNA strand. Furthermore, the single strand cleavage sites share a similar sequence preference with double strand cleavage sites. There is, however, a strand bias for the single strand cleavage reaction. We show also that under single strand cleavage conditions, topoisomerase II still possesses a low level of double strand passage activity: it can introduce topological knots into both covalently closed or nicked DNA rings, and change the linking number of a plasmid DNA by steps of two. The implication of this observation on the sequential cleavage of the two strands of the DNA duplex during the normal DNA double strand passage process catalyzed by type II DNA topoisomerases is discussed.     

HIV-1整合酶链转移反应抑制剂的荧光筛选方法

整合酶被认为是抗HIV-1药物研究的理想靶点之一。为了建立便捷高效的整合酶链转移反应抑制剂筛选方法,首先将HIV-1整合酶原核表达载体pNL-IN转化入大肠杆菌感受态细胞BL21(DE3)进行原核表达,并用镍琼脂糖凝胶进行亲和纯化,获得了纯度和活性均较高的整合酶重组蛋白;然后设计了生物素标记的供体DNA和FITC标记的靶DNA,用链霉亲和素磁珠捕获反应体系中的DNA产物;最后用荧光分析仪检测DNA产物的荧光信号,并计算待测样品的抑制率。用已知整合酶抑制剂S-1360和MK-0518对筛选方法进行了验证,测定结果与已有实验数据相当,表明本筛选方法能够有效应用于HIV-1整合酶链转移反应抑制剂的筛选。与现有的整合酶链转移反应抑制剂筛选方法相比,本筛选方法步骤更为简化、耗时更短、成本更低。     

Flavonoids in Helichrysum pamphylicum inhibit mammalian type I DNA topoisomerase

DNA topoisomerases are important targets for cancer chemotherapy. We investigated the effects of a methanolic extract of Helichrysum pamphylicum on mammalian DNA topoisomerase I via in vitro plasmid supercoil relaxation assays. The extracts manifested a considerable inhibition of the enzyme's activity in a dose-dependent manner. We also performed a HPLC analysis to identify the flavonoid content of the H. pamphylicum extract and tested the identified flavonoids; luteolin, luteolin-4-glucoside, naringenin, helichrysinA and isoquercitrin, on DNA topoisomerase I activity. The measurement of the total antioxidant capacity of the flavonoid standards suggested that the topoisomerase inhibition might be correlated with the antioxidant capacity of the plant.     

Inhibition of DNA topoisomerase II may trigger illegitimate recombination in living cells: experiments with a model system

We have developed a plasmid test system to study recombination in vitro and in mammalian cells in vivo, and to analyze the possible role of DNA topoisomerase II. The system is based on a plasmid construct containing an inducible marker gene ccdB ("killer" (KIL) gene) whose product is lethal for bacterial cells, flanked by two different potentially recombinogenic elements. The plasmids were subjected to recombinogenic conditions in vitro or in vivo after transient transfection into COS-1 cells, and subsequently transformed into E. coli which was then grown in the presence of the ccdB gene inducer. Hence, all viable colonies contained recombinant plasmids since only recombination between the flanking regions could remove the KIL gene. Thus, it was possible to detect recombination events and to estimate their frequency. We found that the frequency of topoisomerase II-mediated recombination in vivo is significantly higher than in a minimal in vitro system. The presence of VM-26, an inhibitor of the religation step of the topoisomerase II reaction, increased the recombination frequency by 60%. We propose that cleavable complexes of topoisomerase II are either not religated, triggering error-prone repair of the DNA breaks, or are incorrectly religated resulting in strand exchange. We also studied the influence of sequences known to contain preferential breakpoints for recombination in vivo after chemotherapy with topoisomerase II-targeting drugs, but no preferential stimulation of recombination by these sequences was detected in this non-chromosomal context.     

The role of DNA bending in type IIA topoisomerase function

Type IIA topoisomerases control DNA supercoiling and separate newly replicated chromosomes using a complex DNA strand cleavage and passage mechanism. Structural and biochemical studies have shown that these enzymes sharply bend DNA by as much as 150°; an invariant isoleucine, which has been seen structurally to intercalate between two base pairs outside of the DNA cleavage site, has been suggested to promote deformation. To test this assumption, we examined the role of isoleucine on DNA binding, bending and catalytic activity for a bacterial type IIA topoisomerase, Escherichia coli topoisomerase IV (topo IV), using a combination of site-directed mutagenesis and biochemical assays. Our data show that alteration of the isoleucine (Ile₁₇₂) did not affect the basal ATPase activity of topo IV or its affinity for DNA. However, the amino acid was important for DNA bending, DNA cleavage and supercoil relaxation. Moreover, an ability to bend DNA correlated with efficacy with which nucleic acid substrates stimulate ATP hydrolysis. These data show that DNA binding and bending by topo IV can be uncoupled, and indicate that the stabilization of a highly curved DNA geometry is critical to the type IIA topoisomerase catalytic cycle.     

Nonintercalative antitumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II

Many intercalative antitumor drugs have been shown to cleave DNA indirectly through their specific effect on the stabilization of a cleavable complex formed between mammalian DNA topoisomerase II and DNA (Nelson, E.M., Tewey, K.M., and Liu, L.F. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1361-1365). Antitumor epipodophyllotoxins (VP-16 and VM-26) which do not intercalate DNA can similarly induce protein-linked DNA breaks in cultured mammalian cells. In vitro studies using purified mammalian DNA topoisomerase II show that epipodophyllotoxins interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II by stabilizing a cleavable complex. Treatment of this stabilized cleavable complex with protein denaturants results in DNA strand breaks and the covalent linking of a topoisomerase subunit to the 5'-end of the broken DNA. Furthermore, epipodophyllotoxins also inhibit the strand-passing activity of mammalian DNA topoisomerase II, presumably as a result of drug-enzyme interaction. The agreement between the in vivo and in vitro studies suggests that mammalian DNA topoisomerase II is a drug target in vivo. The similarity between the effect of epipodophyllotoxins on mammalian DNA topoisomerase II and the effect of nalidixic acid on Escherichia coli DNA gyrase suggests that the cytotoxic action of epipodophyllotoxins may be analogous to the bactericidal action of nalidixic acid.     

High-throughput fluorescence flow-injection topoisomerase II inhibition assay

A high-throughput fluorescence flow-injection assay is described, suitable for determining the catalytic inhibition of DNA topoisomerase II. The method, which separates high molecular mass trypanosome kinetoplast DNA from its decatenated product by centrifugation, should be useful for the rapid and accurate screening of potential anticancer topoisomerase II inhibitors and the determination of their inhibition constants. Advantages of the flow-injection method over agarose gel electrophoresis and radioactive centrifugation assays are that it is faster, more sensitive, highly linear in its response to product formation, and does not require the production of radioactive trypanosome kinetoplast DNA substrate.     

DNA strand transfer reactions catalyzed by vaccinia topoisomerase: hydrolysis and glycerololysis of the covalent protein-DNA intermediate.

Vaccinia topoisomerase forms a covalent protein-DNA intermediate at sites containing the sequence 5'-CCCTT. The T nucleotide is linked via a 3'-phosphodiester bond to Tyr-274 of the enzyme. Here, we report that the enzyme catalyzes hydrolysis of the covalent intermediate, resulting in formation of a 3'-phosphate-terminated DNA cleavage product. The hydrolysis reaction is pH-dependent (optimum pH = 9.5) and is slower, by a factor of 10(-5), than the rate of topoisomerase-catalyzed strand transfer to a 5'-OH terminated DNA acceptor strand. Mutants of vaccinia topoisomerase containing serine or threonine in lieu of the active site Tyr-274 form no detectable covalent intermediate and catalyze no detectable DNA hydrolysis. This suggests that hydrolysis occurs subsequent to formation of the covalent protein-DNA adduct and not via direct attack by water on DNA. Vaccinia topoisomerase also catalyzes glycerololysis of the covalent intermediate. The rate of glycerololysis is proportional to glycerol concentration and is optimal at pH 9.5.     

Vaccinia topoisomerase and Cre recombinase catalyze direct ligation of activated DNA substrates containing a 3'-para-nitrophenyl phosphate ester

DNA topoisomerases and DNA site-specific recombinases are involved in a diverse set of cellular processes but both function by making transient breaks in DNA. Type IB topoisomerases and tyrosine recombinases cleave DNA by transesterification of an active site tyrosine to generate a DNA–3′-phosphotyrosyl–enzyme adduct and a free 5′-hydroxyl (5′-OH). Strand ligation results when the 5′-OH attacks the covalent complex and displaces the enzyme. We describe the synthesis of 3′-phospho-(para-nitrophenyl) oligonucleotides (3′-pNP DNAs), which mimic the natural 3′-phosphotyrosyl intermediate, and demonstrate that such pre-activated strands are substrates for DNA ligation by vaccinia topoisomerase and Cre recombinase. Ligation occurs by direct attack of a 5′-OH strand on the 3′-pNP DNA (i.e., without a covalent protein–DNA intermediate) and generates free para-nitrophenol as a product. The chromogenic DNA substrate allows ligation to be studied in real-time and in the absence of competing cleavage reactions and can be exploited for high-throughput screening of topoisomerase/recombinase inhibitors.     

A solid phase assay for topoisomerase I interfacial poisons and catalytic inhibitors

We report a mechanism-based screening technique to rapidly identify eukaryotic topoisomerase I targeting agents. The method is based on genetic tagging of topoisomerase I to immobilize the enzyme on a solid surface in a microtiter well format. DNA is added to the wells, and retained DNA is detected by Pico Green fluorescence. Compounds that result in an increase in Pico Green staining represent potential topoisomerase interfacial poisons, whereas those that reduce fluorescence report catalytic inhibitors; therefore, the solid phase assay represents a "bimodal" readout that reveals mechanisms of action. The method has been demonstrated to work with known interfacial poisons and catalytic inhibitors. This method is rapid, robust, economical, and scalable for large library screens.     

Ribonuclease activity of vaccinia DNA topoisomerase IB: kinetic and high-throughput inhibition studies using a robust continuous fluorescence assay

Vaccinia type I DNA topoisomerase exhibits a strong site-specific ribonuclease activity when provided a DNA substrate that contains a single uridine ribonucleotide within a duplex DNA containing the sequence 5' CCCTU 3'. The reaction involves two steps: attack of the active site tyrosine nucleophile of topo I at the 3' phosphodiester of the uridine nucleotide to generate a covalent enzyme-DNA adduct, followed by nucleophilic attack of the uridine 2'-hydroxyl to release the covalently tethered enzyme. Here we report the first continuous spectroscopic assay for topoisomerase that allows monitoring of the ribonuclease reaction under multiple-turnover conditions. The assay is especially robust for high-throughput screening applications because sensitive molecular beacon technology is utilized, and the topoisomerase is released during the reaction to allow turnover of multiple substrate molecules by a single molecule of enzyme. Direct computer simulation of the fluorescence time courses was used to obtain the rate constants for substrate binding and release, covalent complex formation, and formation of the 2',3'-cyclic phosphodiester product of the ribonuclease reaction. The assay allowed rapid screening of a 500 member chemical library from which several new inhibitors of topo I were identified with IC(50) values in the range of 2-100 microM. Three of the most potent hits from the high-throughput screening were also found to inhibit plasmid supercoil relaxation by the enzyme, establishing the utility of the assay in identifying inhibitors of the biologically relevant DNA relaxation reaction. One of the most potent inhibitors of the vaccinia enzyme, 3-benzo[1,3]dioxol-5-yl-2-oxoproprionic acid, did not inhibit the closely related human enzyme. The inhibitory mechanism of this compound is unique and involves a step required for recycling the enzyme for steady-state turnover.     

Correlation of enzyme-induced cleavage sites on negatively superhelical DNA between prokaryotic topoisomerase I and S1 nuclease

Negatively superhelical pNS1 DNA with a molecular weight of 2.55 MDa (4 kbp) was found to contain 13 specific, unbasepaired sites that are sensitive to a single-strand-specific S₁ nuclease cleavage. The S₁-cleavage occurred once at these sites. In the absence of added Mg²⁺, the topoisomerase I purified from Haemophilus gallinarum formed a complex with the superhelical pNS1 DNA which has a hidden strand cleavage. Extensive proteinase K digestion of the complex led to cleavage of the DNA chain. Then the proteinase K-cleaved product was digested with S₁, which can cut the opposite strand at the preexisting strand cleavage to generate unit-length linear DNA. Restriction endonuclease analysis of the linear DNA shows that the topoisomerase-induced cleavage occurred once at ten specific sites on the DNA. The topoisomerase caused mainly single-strand cleavage at these sites, but infrequently also caused double-strand cleavage at the same sites. Of interest is the fact that these sites considerably coincide with the S₁-cleavable, unbasepaired sites.     

Recombinogenic flap ligation pathway for intrinsic repair of topoisomerase IB-induced double-strand breaks

Topoisomerase IB catalyzes recombinogenic DNA strand transfer reactions in vitro and in vivo. Here we characterize a new pathway of topoisomerase-mediated DNA ligation in vitro (flap ligation) in which vaccinia virus topoisomerase bound to a blunt-end DNA joins the covalently held strand to a 5' resected end of a duplex DNA containing a 3' tail. The joining reaction occurs with high efficiency when the sequence of the 3' tail is complementary to that of the scissile strand immediately 5' of the cleavage site. A 6-nucleotide segment of complementarity suffices for efficient flap ligation. Invasion of the flap into the duplex apparently occurs while topoisomerase remains bound to DNA, thereby implying a conformational flexibility of the topoisomerase clamp around the DNA target site. The 3' flap acceptor DNA mimics a processed end in the double-strand-break-repair recombination pathway. Our findings suggest that topoisomerase-induced breaks may be rectified by flap ligation, with ensuing genomic deletions or translocations.