The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Neil Osheroff - One of the best experts on this subject based on the ideXlab platform.
-
the DNA Cleavage reaction of topoisomerase ii wolf in sheep s clothing
Nucleic Acids Research, 2009Co-Authors: Joseph E Deweese, Neil OsheroffAbstract:Topoisomerase II is an essential enzyme that is required for virtually every process that requires movement of DNA within the nucleus or the opening of the double helix. This enzyme helps to regulate DNA under- and overwinding and removes knots and tangles from the genetic material. In order to carry out its critical physiological functions, topoisomerase II generates transient double-stranded breaks in DNA. Consequently, while necessary for cell survival, the enzyme also has the capacity to fragment the genome. The DNA Cleavage/ligation reaction of topoisomerase II is the target for some of the most successful anticancer drugs currently in clinical use. However, this same reaction also is believed to trigger chromosomal translocations that are associated with specific types of leukemia. This article will familiarize the reader with the DNA Cleavage/ligation reaction of topoisomerase II and other aspects of its catalytic cycle. In addition, it will discuss the interaction of the enzyme with anticancer drugs and the mechanisms by which these agents increase levels of topoisomerase II-generated DNA strand breaks. Finally, it will describe dietary and environmental agents that enhance DNA Cleavage mediated by the enzyme.
-
dietary polyphenols as topoisomerase ii poisons b ring and c ring substituents determine the mechanism of enzyme mediated DNA Cleavage enhancement
Chemical Research in Toxicology, 2008Co-Authors: Omari J Bandele, Sara J Clawson, Neil OsheroffAbstract:Dietary polyphenols are a diverse and complex group of compounds that are linked to human health. Many of their effects have been attributed to the ability to poison (i.e., enhance DNA Cleavage by) topoisomerase II. Polyphenols act against the enzyme by at least two different mechanisms. Some compounds are traditional, redox-independent topoisomerase II poisons, interacting with the enzyme in a noncovalent manner. Conversely, others enhance DNA Cleavage in a redox-dependent manner that requires covalent adduction to topoisomerase II. Unfortunately, the structural elements that dictate the mechanism by which polyphenols poison topoisomerase II have not been identified. To resolve this issue, the activities of two classes of polyphenols against human topoisomerase IIα were examined. The first class was a catechin series, including (−)-epigallocatechin gallate (EGCG), (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECG), and (−)-epicatechin (EC). The second was a flavonol series, including myricetin, qu...
-
chlorella virus marburg topoisomerase ii high DNA Cleavage activity as a characteristic of chlorella virus type ii enzymes
Biochemistry, 2005Co-Authors: Jennifer S Dickey, Taejin Choi, James L Van Etten, Neil OsheroffAbstract:Although the formation of a covalent enzyme-cleaved DNA complex is a prerequisite for the essential functions of topoisomerase II, this reaction intermediate has the potential to destabilize the genome. Consequently, all known eukaryotic type II enzymes maintain this complex at a low steady-state level. Recently, however, a novel topoisomerase II was discovered in Paramecium bursaria chlorella virus-1 (PBCV-1) that has an exceptionally high DNA Cleavage activity [Fortune et al. (2001) J. Biol. Chem. 276, 24401-24408]. If robust DNA Cleavage is critical to the physiological functions of chlorella virus topoisomerase II, then this remarkable characteristic should be conserved throughout the viral family. Therefore, topoisomerase II from Chlorella virus Marburg-1 (CVM-1), a distant family member, was expressed in yeast, isolated, and characterized. CVM-1 topoisomerase II is 1058 amino acids in length, making it the smallest known type II enzyme. The viral topoisomerase II displayed a high DNA strand passage activity and a DNA Cleavage activity that was approximately 50-fold greater than that of human topoisomerase IIalpha. High DNA Cleavage appeared to result from a greater rate of scission rather than promiscuous DNA site utilization, inordinately tight DNA binding, or diminished religation rates. Despite the fact that CVM-1 and PBCV-1 topoisomerase II share approximately 67% amino acid sequence identity, the two enzymes displayed clear differences in their DNA Cleavage specificity/site utilization. These findings suggest that robust DNA Cleavage is intrinsic to the viral enzyme and imply that chlorella virus topoisomerase II plays a physiological role beyond the control of DNA topology.
-
cobalt enhances DNA Cleavage mediated by human topoisomerase ii alpha in vitro and in cultured cells
Biochemistry, 2004Co-Authors: Erin L Baldwin, Neil OsheroffAbstract:: Although cobalt is an essential trace element for humans, the metal is genotoxic and mutagenic at higher concentrations. Treatment of cells with cobalt generates DNA strand breaks and covalent protein-DNA complexes. However, the basis for these effects is not well understood. Since the toxic events induced by cobalt resemble those of topoisomerase II poisons, the effect of the metal on human topoisomerase IIalpha was examined. The level of enzyme-mediated DNA scission increased 6-13-fold when cobalt(II) replaced magnesium(II) in Cleavage reactions. Cobalt(II) stimulated Cleavage at all DNA sites observed in the presence of magnesium(II), and the enzyme cut DNA at several "cobalt-specific" sites. The increased level of DNA Cleavage in the presence of cobalt(II) was partially due to a decrease in the rate of enzyme-mediated religation. Topoisomerase IIalpha retained many of its catalytic properties in reactions that included cobalt(II), including sensitivity to the anticancer drug etoposide and the ability to relax and decatenate DNA. Finally, cobalt(II) stimulated topoisomerase IIalpha-mediated DNA Cleavage in the presence of magnesium(II) in purified systems and in human MCF-7 cells. These findings demonstrate that cobalt(II) is a topoisomerase II poison in vitro and in cultured cells and suggest that at least some of the genotoxic effects of the metal are mediated through topoisomerase IIalpha.
-
DNA Cleavage and religation by human topoisomerase ii alpha at high temperature
Biochemistry, 2001Co-Authors: Kenneth D Bromberg, Neil OsheroffAbstract:A common DNA religation assay for topoisomerase II takes advantage of the fact that the enzyme can rejoin cleaved nucleic acids but cannot mediate DNA scission at suboptimal temperatures (either high or low). Although temperature-induced DNA religation assays have provided valuable mechanistic information for several type II enzymes, high-temperature shifts have not been examined for human topoisomerase IIα. Therefore, the effects of temperature on the DNA Cleavage/religation activity of the enzyme were characterized. Human topoisomerase IIα undergoes two distinct transitions at high temperatures. The first transition occurs between 45 and 55 °C and is accompanied by a 6-fold increase in the level of DNA Cleavage at 60 °C. It also leads to a loss of DNA strand passage activity, due primarily to an inability of ATP to convert the enzyme to a protein clamp. The enzyme alterations that accompany the first transition appear to be stable and do not revert at lower temperature. The second transition in human to...
Keith R Jerome - One of the best experts on this subject based on the ideXlab platform.
-
DNA Cleavage enzymes for treatment of persistent viral infections recent advances and the pathway forward
Virology, 2014Co-Authors: Nicholas D Weber, Martine Aubert, Chung H Dang, Daniel Stone, Keith R JeromeAbstract:Treatment for most persistent viral infections consists of palliative drug options rather than curative approaches. This is often because long-lasting viral DNA in infected cells is not affected by current antivirals, providing a source for viral persistence and reactivation. Targeting latent viral DNA itself could therefore provide a basis for novel curative strategies. DNA Cleavage enzymes can be used to induce targeted mutagenesis of specific genes, including those of exogenous viruses. Although initial in vitro and even in vivo studies have been carried out using DNA Cleavage enzymes targeting various viruses, many questions still remain concerning the feasibility of these strategies as they transition into preclinical research. Here, we review the most recent findings on DNA Cleavage enzymes for human viral infections, consider the most relevant animal models for several human viral infections, and address issues regarding safety and enzyme delivery. Results from well-designed in vivo studies will ideally provide answers to the most urgent remaining questions, and allow continued progress toward clinical application.
-
predictors of hepatitis b cure using gene therapy to deliver DNA Cleavage enzymes a mathematical modeling approach
PLOS Computational Biology, 2013Co-Authors: Joshua T Schiffer, Daniel Stone, D A Swan, Keith R JeromeAbstract:Most chronic viral infections are managed with small molecule therapies that inhibit replication but are not curative because non-replicating viral forms can persist despite decades of suppressive treatment. There are therefore numerous strategies in development to eradicate all non-replicating viruses from the body. We are currently engineering DNA Cleavage enzymes that specifically target hepatitis B virus covalently closed circular DNA (HBV cccDNA), the episomal form of the virus that persists despite potent antiviral therapies. DNA Cleavage enzymes, including homing endonucleases or meganucleases, zinc-finger nucleases (ZFNs), TAL effector nucleases (TALENs), and CRISPR-associated system 9 (Cas9) proteins, can disrupt specific regions of viral DNA. Because DNA repair is error prone, the virus can be neutralized after repeated Cleavage events when a target sequence becomes mutated. DNA Cleavage enzymes will be delivered as genes within viral vectors that enter hepatocytes. Here we develop mathematical models that describe the delivery and intracellular activity of DNA Cleavage enzymes. Model simulations predict that high vector to target cell ratio, limited removal of delivery vectors by humoral immunity, and avid binding between enzyme and its DNA target will promote the highest level of cccDNA disruption. Development of de novo resistance to Cleavage enzymes may occur if DNA Cleavage and error prone repair does not render the viral episome replication incompetent: our model predicts that concurrent delivery of multiple enzymes which target different vital cccDNA regions, or sequential delivery of different enzymes, are both potentially useful strategies for avoiding multi-enzyme resistance. The underlying dynamics of cccDNA persistence are unlikely to impact the probability of cure provided that antiviral therapy is given concurrently during eradication trials. We conclude by describing experiments that can be used to validate the model, which will in turn provide vital information for dose selection for potential curative trials in animals and ultimately humans.
Giovanni Capranico - One of the best experts on this subject based on the ideXlab platform.
-
the topoisomerase ii poison clerocidin alkylates non paired guanines of DNA implications for irreversible stimulation of DNA Cleavage
Nucleic Acids Research, 2001Co-Authors: Barbara Gatto, Giovanni Capranico, Sara N Richter, Stefano Moro, Manlio PalumboAbstract:Clerocidin, a diterpenoid with antibacterial and antitumor activity, stimulates in vitro DNA Cleavage mediated by mammalian and bacterial topoisomerase (topo) II. Different from the classical topoisomerase poisons, clerocidin-stimulated breaks at guanines immediately preceding the sites of DNA Cleavage are not resealed upon heat or salt treatment. To understand the mechanism of irreversible trapping of the topo II-cleavable complex, we have investigated the reactivity of clerocidin per se towards DNA. We show here that the drug is able to nick negatively supercoiled plasmids. DNA Cleavage by clerocidin in enzyme-free medium is due to the ability of the drug to form covalent adducts with guanines. Indeed, clerocidin was able to specifically react with short oligonucleotides when the guanines were unpaired and exposed as in bulges or in the single-strand form. The clerocidin epoxy group attacks the nitrogen at position 7 of guanines, leading to strand scission at the modified site. Our findings also demonstrate that trapping of topoisomerases by clerocidin is specific for type II enzymes. The guanine-alkylating ability of clerocidin suggests an unprecedented mechanism of topo II poisoning, according to which the enzyme renders the drug reactive toward DNA by distorting the double-helical structure of the nucleic acid at the Cleavage site.
-
amsacrine promoted DNA Cleavage site determinants for the two human DNA topoisomerase ii isoforms α and β
Biochemical Pharmacology, 1996Co-Authors: Katherine L Marsh, Giovanni Capranico, Stella Tinelli, Mark L Fisher, Elaine Willmore, Mariagrazia Cornarotti, Emma L Meczes, Caroline A AustinAbstract:Abstract Site-specific DNA Cleavage by topoisomerase II (EC 5.99.1.3) is induced by many antitumour drugs. Although human cells express two genetically distinct topoisomerase II isoforms, thus far the role and determinants of drug-induced DNA Cleavage have been examined only for a. Here we report the first high-resolution study of amsacrine (mAMSA) induced DNA breakage by human topoisomerase I1β (overexpressed and purified from yeast) and a direct comparison with the recombinant α isoform. DNA Cleavage in plasmid pBR322 and SV40 DNA was induced by α or β in the absence or presence of the antitumour agent mAMSA, and sites were mapped using sequencing gel methodology. Low-resolution studies indicated that recombinant human α promoted DNA breakage at sites akin to those of β, although some sites were only cleaved by one enzyme and different intensities were observed at some sites. However, statistical analysis of 70 drug-induced sites for β and 70 sites for α revealed that both isoforms share the same base preferences at 13 positions relative to the enzyme Cleavage site, including a very strong preference for A at +1. The result for recombinant α isoform is in agreement with previous studies using α purified from human cell lines. Thus, α and β proteins apparently form similar ternary complexes with mAMSA and DNA. Previous studies have emphasized the importance of DNA topoisomerase II α; the results presented here demonstrate that β is an in vitro target with similar site determinants, strongly suggesting that β should also be considered a target of mAMSA in vivo .
-
unique sequence specificity of topoisomerase ii DNA Cleavage stimulation and DNA binding mode of streptonigrin
Journal of Biological Chemistry, 1994Co-Authors: Giovanni Capranico, Stella Tinelli, Manlio Palumbo, Franco ZuninoAbstract:: Streptonigrin stimulated unique intensity patterns of topoisomerase II-mediated DNA Cleavage in agarose and sequencing gels with no similarity to those of doxorubicin, VM-26,4'(9-acridinylamino)-methanesulfon-m-anisidide, genistein, and mitoxantrone. Surprisingly, a statistical analysis of 60 sites stimulated by streptonigrin in SV40 and pBR322 DNAs showed that the drug required the dinucleotide 5'-TA-3' from 2- to 3-positions at the DNA Cleavage site. Streptonigrin did not intercalate into the double helix; however, a positive value of the reduced linear dichroism indicated that indeed the drug interacted with the DNA. An angle of 45 degrees was found between the major drug and local DNA axes, suggesting a minor groove binding mode. Moreover, a DNA winding assay showed that streptonigrin may tighten the helical twist of DNA, similar to the known minor groove binder distamycin. Drug competition for receptor site binding was then evaluated by drug combination in the Cleavage reaction. DNA Cleavage intensity patterns were altered only with the streptonigrin/mitoxantrone combination, suggesting that the two compounds may compete for ternary complex formation. The results indicate that streptonigrin may bind to the DNA in a manner similar to that of minor groove binders and that its pharmacophore, possibly different from other topoisomerase II inhibitors, may be an important determinant of its unique sequence position specificity.
Yves Pommier - One of the best experts on this subject based on the ideXlab platform.
-
mus81 mediated DNA Cleavage resolves replication forks stalled by topoisomerase i DNA complexes
Journal of Cell Biology, 2011Co-Authors: Marie Regairaz, Yongwei Zhang, Keli Agama, Nalini Tata, Surbhi Agrawal, Mirit I Aladjem, Yves PommierAbstract:Deoxyribonucleic acid (DNA) topoisomerases are essential for removing the supercoiling that normally builds up ahead of replication forks. The camptothecin (CPT) Top1 (topoisomerase I) inhibitors exert their anticancer activity by reversibly trapping Top1–DNA Cleavage complexes (Top1cc’s) and inducing replication-associated DNA double-strand breaks (DSBs). In this paper, we propose a new mechanism by which cells avoid Top1-induced replication-dependent DNA damage. We show that the structure-specific endonuclease Mus81-Eme1 is responsible for generating DSBs in response to Top1 inhibition and for allowing cell survival. We provide evidence that Mus81 cleaves replication forks rather than excises Top1cc’s. DNA combing demonstrated that Mus81 also allows efficient replication fork progression after CPT treatment. We propose that Mus81 cleaves stalled replication forks, which allows dissipation of the excessive supercoiling resulting from Top1 inhibition, spontaneous reversal of Top1cc, and replication fork progression.
-
DNA Cleavage assay for the identification of topoisomerase i inhibitors
Nature Protocols, 2008Co-Authors: Thomas S Dexheimer, Yves PommierAbstract:The inhibition of DNA topoisomerase I (Top1) has proven to be a successful approach in the design of anticancer agents. However, despite the clinical successes of the camptothecin derivatives, a significant need for less toxic and more chemically stable Top1 inhibitors still persists. Here, we describe one of the most frequently used protocols to identify novel Top1 inhibitors. These methods use uniquely 3'-radiolabeled DNA substrates and denaturing polyacrylamide gel electrophoresis to provide evidence for the Top1-mediated DNA cleaving activity of potential Top1 inhibitors. These assays allow comparison of the effectiveness of different drugs in stabilizing the Top1-DNA intermediate or Cleavage (cleavable) complex. A variation on these assays is also presented, which provides a suitable system for determining whether the inhibitor blocks the forward Cleavage or religation reactions by measuring the reversibility of the drug-induced Top1-DNA Cleavage complexes. This entire protocol can be completed in approximately 2 d.
-
importance of the fourth alpha helix within the cap homology domain of type ii topoisomerase for DNA Cleavage site recognition and quinolone action
Antimicrobial Agents and Chemotherapy, 2002Co-Authors: D Strumberg, John L. Nitiss, Jiaowang Dong, Jerrylaine Walker, Marc C Nicklaus, Kurt W Kohn, Jonathan G Heddle, Anthony Maxwell, Siegfried Seeber, Yves PommierAbstract:We report that point mutations causing alteration of the fourth alpha-helix (α4-helix) of the CAP homology domain of eukaryotic (Saccharomyces cerevisiae) type II topoisomerases (Ser740Trp, Gln743Pro, and Thr744Pro) change the selection of type II topoisomerase-mediated DNA Cleavage sites promoted by Ca2+ or produced by etoposide, the fluoroquinolone CP-115,953, or mitoxantrone. By contrast, Thr744Ala substitution had minimal effect on Ca2+- and drug-stimulated DNA Cleavage sites, indicating the selectivity of single amino acid substitutions within the α4-helix on type II topoisomerase-mediated DNA Cleavage. The equivalent mutation in the gene for Escherichia coli gyrase causing Ser83Trp also changed the DNA Cleavage pattern generated by Ca2+ or quinolones. Finally, Thr744Pro substitution in the yeast type II topoisomerase rendered the enzyme sensitive to antibacterial quinolones. This study shows that the α4-helix within the conserved CAP homology domain of type II topoisomerases is critical for selecting the sites of DNA Cleavage. It also demonstrates that selective amino acid residues in the α4-helix are important in determining the activity and possibly the binding of quinolones to the topoisomerase II-DNA complexes.
Alfred Pingoud - One of the best experts on this subject based on the ideXlab platform.
-
on the divalent metal ion dependence of DNA Cleavage by restriction endonucleases of the ecori family
Journal of Molecular Biology, 2009Co-Authors: Vera Pingoud, Albert Jeltsch, Jürgen Alves, Wolfgang Wende, Peter Friedhoff, Monika Reuter, Letif Mones, Monika Fuxreiter, Alfred PingoudAbstract:Restriction endonucleases of the PD...D/EXK family need Mg(2+) for DNA Cleavage. Whereas Mg(2+) (or Mn(2+)) promotes catalysis, Ca(2+) (without Mg(2+)) only supports DNA binding. The role of Mg(2+) in DNA Cleavage by restriction endonucleases has elicited many hypotheses, differing mainly in the number of Mg(2+) involved in catalysis. To address this problem, we measured the Mg(2+) and Mn(2+) concentration dependence of DNA Cleavage by BamHI, BglII, Cfr10I, EcoRI, EcoRII (catalytic domain), MboI, NgoMIV, PspGI, and SsoII, which were reported in co-crystal structure analyses to bind one (BglII and EcoRI) or two (BamHI and NgoMIV) Me(2+) per active site. DNA Cleavage experiments were carried out at various Mg(2+) and Mn(2+) concentrations at constant ionic strength. All enzymes show a qualitatively similar Mg(2+) and Mn(2+) concentration dependence. In general, the Mg(2+) concentration optimum (between approximately 1 and 10 mM) is higher than the Mn(2+) concentration optimum (between approximately 0.1 and 1 mM). At still higher Mg(2+) or Mn(2+) concentrations, the activities of all enzymes tested are reduced but can be reactivated by Ca(2+). Based on these results, we propose that one Mg(2+) or Mn(2+) is critical for restriction enzyme activation, and binding of a second Me(2+) plays a role in modulating the activity. Steady-state kinetics carried out with EcoRI and BamHI suggest that binding of a second Mg(2+) or Mn(2+) mainly leads to an increase in K(m), such that the inhibitory effect of excess Mg(2+) or Mn(2+) can be overcome by increasing the substrate concentration. Our conclusions are supported by molecular dynamics simulations and are consistent with the structural observations of both one and two Me(2+) binding to these enzymes.
-
on the divalent metal ion dependence of DNA Cleavage by restriction endonucleases of the ecori family
Journal of Molecular Biology, 2009Co-Authors: Vera Pingoud, Albert Jeltsch, Jürgen Alves, Wolfgang Wende, Peter Friedhoff, Monika Reuter, Letif Mones, Monika Fuxreiter, Alfred PingoudAbstract:Abstract Restriction endonucleases of the PD…D/EXK family need Mg2+ for DNA Cleavage. Whereas Mg2+ (or Mn2+) promotes catalysis, Ca2+ (without Mg2+) only supports DNA binding. The role of Mg2+ in DNA Cleavage by restriction endonucleases has elicited many hypotheses, differing mainly in the number of Mg2+ involved in catalysis. To address this problem, we measured the Mg2+ and Mn2+ concentration dependence of DNA Cleavage by BamHI, BglII, Cfr10I, EcoRI, EcoRII (catalytic domain), MboI, NgoMIV, PspGI, and SsoII, which were reported in co-crystal structure analyses to bind one (BglII and EcoRI) or two (BamHI and NgoMIV) Me2+ per active site. DNA Cleavage experiments were carried out at various Mg2+ and Mn2+ concentrations at constant ionic strength. All enzymes show a qualitatively similar Mg2+ and Mn2+ concentration dependence. In general, the Mg2+ concentration optimum (between ∼ 1 and 10 mM) is higher than the Mn2+ concentration optimum (between ∼ 0.1 and 1 mM). At still higher Mg2+ or Mn2+ concentrations, the activities of all enzymes tested are reduced but can be reactivated by Ca2+. Based on these results, we propose that one Mg2+ or Mn2+ is critical for restriction enzyme activation, and binding of a second Me2+ plays a role in modulating the activity. Steady-state kinetics carried out with EcoRI and BamHI suggest that binding of a second Mg2+ or Mn2+ mainly leads to an increase in Km, such that the inhibitory effect of excess Mg2+ or Mn2+ can be overcome by increasing the substrate concentration. Our conclusions are supported by molecular dynamics simulations and are consistent with the structural observations of both one and two Me2+ binding to these enzymes.
-
does the restriction endonuclease ecorv employ a two metal ion mechanism for DNA Cleavage
Biochemistry, 1997Co-Authors: Detlef H. Groll, Albert Jeltsch, Ursel Selent, Alfred PingoudAbstract:Two models for the catalytic mechanism of the restriction endonuclease EcoRV exist which differ in the number and function of metal ions proposed to be directly involved in catalysis. In one model, two metal ions bound by Glu45, Asp74, and Asp90 are assumed to have a direct catalytic function; in the other, only one metal ion bound by Asp74 and Asp90. We show here that in the presence of Mn2+, the catalytic activity of an EcoRV-E45A mutant is only slightly reduced (1.8-fold) as compared to wild type EcoRV and that the single-turnover rate constant of DNA Cleavage by E45A is reduced only 39-fold, whereas the D74A and D90A mutants are catalytically inactive under all conditions. These findings make an important catalytic function of Glu45, like binding of an essential divalent metal ion, unlikely. In addition, we have analyzed the dependence of the DNA Cleavage rate by EcoRV and EcoRV mutants on the concentration of Mg2+ and Mn2+. We found for the wild type enzyme a sigmoidal dependence of the rate of DNA c...