Rejoining Of DNA Strands Research Articles

Mutations of human DNA topoisomerase I at poly(ADP-ribose) binding sites: modulation of camptothecin activity by ADP-ribose polymers.

BackgroundDNA topoisomerases are key enzymes that modulate the topological state of DNA through the breaking and rejoining of DNA strands. Human topoisomerase I belongs to the family of poly(ADP-ribose)-binding proteins and is the target of camptothecin derived anticancer drugs. Poly(ADP-ribosyl)ation occurs at specific sites of the enzyme inhibiting the cleavage and enhancing the religation steps during the catalytic cycle. Thus, ADP-ribose polymers antagonize the activity of topoisomerase I poisons, whereas PARP inhibitors increase their antitumor effects.MethodsUsing site-directed mutagenesis we have analyzed the interaction of human topoisomerase I and poly(ADP-ribose) through enzymatic activity and binding procedures.ResultsMutations of the human topoisomerase I hydrophobic or charged residues, located on the putative polymer binding sites, are not sufficient to abolish or reduce the binding of the poly(ADP-ribose) to the protein. These results suggest either the presence of additional binding sites or that the mutations are not enough perturbative to destroy the poly(ADP-ribose) interaction, although in one mutant they fully abolish the enzyme activity.ConclusionsIt can be concluded that mutations at the hydrophobic or charged residues of the putative polymer binding sites do not interfere with the ability of poly(ADP-ribose) to antagonize the antitumor activity of topoisomerase I poisons.

Mutations of human DNA topoisomerase I at poly(ADP-ribose) binding sites: modulation of camptothecin activity by ADP-ribose polymers

BackgroundDNA topoisomerases are key enzymes that modulate the topological state of DNA through the breaking and rejoining of DNA strands. Human topoisomerase I belongs to the family of poly(ADP-ribose)-binding proteins and is the target of camptothecin derived anticancer drugs. Poly(ADP-ribosyl)ation occurs at specific sites of the enzyme inhibiting the cleavage and enhancing the religation steps during the catalytic cycle. Thus, ADP-ribose polymers antagonize the activity of topoisomerase I poisons, whereas PARP inhibitors increase their antitumor effects.MethodsUsing site-directed mutagenesis we have analyzed the interaction of human topoisomerase I and poly(ADP-ribose) through enzymatic activity and binding procedures.ResultsMutations of the human topoisomerase I hydrophobic or charged residues, located on the putative polymer binding sites, are not sufficient to abolish or reduce the binding of the poly(ADP-ribose) to the protein. These results suggest either the presence of additional binding sites or that the mutations are not enough perturbative to destroy the poly(ADP-ribose) interaction, although in one mutant they fully abolish the enzyme activity.ConclusionsIt can be concluded that mutations at the hydrophobic or charged residues of the putative polymer binding sites do not interfere with the ability of poly(ADP-ribose) to antagonize the antitumor activity of topoisomerase I poisons.

Molecular mechanism of the camptothecin resistance of Glu710Gly topoisomerase IB mutant analyzed in vitro and in silico

BackgroundDNA topoisomerases are key enzymes that modulate the topological state of DNA through the breaking and rejoining of DNA strands. Human topoisomerase IB can be inhibited by several compounds that act through different mechanisms, including clinically used drugs, such as the derivatives of the natural compound camptothecin that reversibly bind the covalent topoisomerase-DNA complex, slowing down the religation of the cleaved DNA strand, thus inducing cell death. Three enzyme mutations, which confer resistance to irinotecan in an adenocarcinoma cell line, were recently identified but the molecular mechanism of resistance was unclear.MethodsThe three resistant mutants have been investigated in S. cerevisiae model system following their viability in presence of increasing amounts of camptothecin. A systematical analysis of the different catalytic steps has been made for one of these mutants (Glu710Gly) and has been correlated with its structural-dynamical properties studied by classical molecular dynamics simulation.ResultsThe three mutants display a different degree of camptothecin resistance in a yeast cell viability assay. Characterization of the different steps of the catalytic cycle of the Glu710Gly mutant indicated that its resistance is related to a high religation rate that is hardly affected by the presence of the drug. Analysis of the dynamic properties through simulation indicate that the mutant displays a much lower degree of correlation in the motion between the different protein domains and that the linker almost completely loses its correlation with the C-terminal domain, containing the active site tyrosine.ConclusionsThese results indicate that a fully functional linker is required to confer camptothecin sensitivity to topoisomerase I since the destabilization of its structural-dynamical properties is correlated to an increase of religation rate and drug resistance.

Intermediates in serine recombinase-mediated site-specific recombination

Site-specific recombinases are enzymes that promote precise rearrangements of DNA sequences. They do this by cutting and rejoining the DNA strands at specific positions within a pair of target sites recognized and bound by the recombinase. One group of these enzymes, the serine recombinases, initiates strand exchange by making double-strand breaks in the DNA of the two sites, in an intermediate built around a catalytic tetramer of recombinase subunits. However, these catalytic steps are only the culmination of a complex pathway that begins when recombinase subunits recognize and bind to their target sites as dimers. To form the tetramer-containing reaction intermediate, two dimer-bound sites are brought together by protein dimer-dimer interactions. During or after this initial synapsis step, the recombinase subunit and tetramer conformations change dramatically by repositioning of component subdomains, bringing about a transformation of the enzyme from an inactive to an active configuration. In natural serine recombinase systems, these steps are subject to elaborate regulatory mechanisms in order to ensure that cleavage and rejoining of DNA strands only happen when and where they should, but we and others have identified recombinase mutants that have lost dependence on this regulation, thus facilitating the study of the basic steps leading to catalysis. We describe how our studies on activated mutants of two serine recombinases, Tn3 resolvase and Sin, are providing us with insights into the structural changes that occur before catalysis of strand exchange, and how these steps in the reaction pathway are regulated.

Abstract 1688: Effect of Gefitinib on the reactions of mammalian type I and type II DNA topoisomerases

Abstract Developing small molecule inhibitors in anti-cancer drug research is an integral part of clinical oncology. Gefitinib (Iressa) is a promising small molecule inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase. It was shown to have the potential to improve the clinical effectiveness of EGFR-targeted therapies and gained a significant impact in treatment of non-small-cell lung cancer (NSCLC). Over the past years, several naturally occurring and synthetic compounds with anti-cancer activities were shown to exert their actions via interfering with normal DNA topoisomerase reactions. DNA topoisomerases are ubiquitous enzymes that regulate DNA topology by making transient breakages at nucleic acid backbone and rejoining of DNA strands. Topoisomerases carry out their reactions by two different mechanisms; Type I topoisomerases make a single-stranded break in a DNA duplex while type II topoisomerases create transient breaks in both strands of a duplex. In this study we aimed to examine the effects of Gefitinib on these enzymes to extend the evaluation of its biological activities by plasmid supercoil relaxation and kinetoplast decatenation assays for type I and type II topoisomerases, respectively. Our results are discussed in relation to the outcomes of Gefitinib reported in clinical trials, which together would contribute in identifying the mechanisms of Gefitinib-induced tumor cell-killing. Key Words; Gefitinib; DNA topoisomerases; anti-cancer drugs Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1688.

Secondary metabolites as DNA topoisomerase inhibitors: A new era towards designing of anticancer drugs.

A large number of secondary metabolites like alkaloids, terpenoids, polyphenols and quinones are produced by the plants. These metabolites can be utilized as natural medicines for the reason that they inhibit the activity of DNA topoisomerase which are the clinical targets for anticancer drugs. DNA topoisomerases are the cellular enzymes that change the topological state of DNA through the breaking and rejoining of DNA strands. Synthetic drugs as inhibitors of topoisomerases have been developed and used in the clinical trials but severe side effects are a serious problem for them therefore, there is a need for the development of novel plant-derived natural drugs and their analogs which may serve as appropriate inhibitors with respect to drug designing. The theme for this review is how secondary metabolites or natural products inactivate the action of DNA topoisomerases and open new avenues towards isolation and characterization of compounds for the development of novel drugs with anticancer potential.

Inhibitory Activities of Helichrysum. Taxa on Mammalian Type I DNA Topoisomerase

DNA topoisomerases are essential enzymes that regulate the conformational changes in DNA topology by catalyzing the concerted breakage and rejoining of DNA strands during normal cellular growth. During the past few years, there has been considerable pharmacological interest in these enzymes because the inhibitors of DNA topoisomerases represent a major class of anticancer drugs. In this study, we investigated the effects of the extracts, prepared from a number of Helichrysum. taxa, on mammalian DNA topoisomerase I via in vitro. supercoil relaxation assays using plasmid substrate, pBR322. Cytotoxic alkaloid, camptothecin, an inhibitor of eukaryotic topoisomerase I, was used as reference compound throughout the assays. Quantitative comparisons of the supercoiled and relaxed DNA band intensities on agarose gels suggested that the extracts from H. pallasii. (Sprengel) Ledeb., H. armenium DC subsp.. araxinum. (Kirp.) Takht, and H. plicatum DC subsp.. plicatum. manifested a considerable inhibition on the enzyme in a dose-dependent manner. The inhibition of mammalian DNA topoisomerase I by Helichrysum. extracts is a significant result because they can be potential sources of anticancer drugs.

Structure and function of the shufflon in plasmid r64

Conservative site-specific recombination plays key roles in creating biological diversity in prokaryotes. Most site-specific inversion systems consist of two recombination sites and a recombinase gene. In contrast, the shufflon multiple inversion system of plasmid R64 consists of seven sfx recombination sites, which separate four invertible DNA segments, and the rci gene encoding a site-specific recombinase of the integrase family. The rci product mediates recombination between any two inverted sfx sites, resulting in the inversion of four DNA segments independently or in groups. Random shufflon inversions construct seven pilV genes encoding constant N-terminal segment with different C-terminal segments. The pilV products are tip-located adhesins of the type IV pilus, called the thin pilus, of R64 and recognize lipopolysaccharides of recipient bacterial cells during R64 liquid matings. Thus, the shufflon determines the recipient specificity of liquid matings. Rci protein of R64 was overexpressed, purified, and used for in vitro recombination reactions. The cleavage and rejoining of DNA strands in shufflon recombinations were found to take place in the form of a 5' protruding 7-hp staggered cut within sfx sequences. Thus, the sfx sequence is asymmetric: only the 7-bp spacer sequence and the right arm sequence are conserved among various R64 sfxs, whereas the sfx left arm sequences are not conserved. Rci protein was shown to bind to entire sfx sequences, suggesting that it binds to the right arms of the sfx sequences in a sequence-specific manner and to their left arms in a non-sequence-specific manner. The sfx left arm sequences greatly affected the shufflon inversion frequency. The artificial symmetric sfx sequence, in which the sfx left arm was changed to the inverted repeat sequence of the right arm, exhibited the highest inversion frequency. Rci-dependent deletion of a DNA segment flanked by two symmetric sfx sequences in direct orientation was observed, suggesting that the asymmetry of sfx sequences may prevent recombination between sfx sequences in direct orientation in the R64 shufflon. The Rci C-terminal domain was not required for recombination using the symmetric sfx sequence. A model, where the C-terminal domain of Rci protein plays a key role in the sequence-specific and non-specific binding of Rci to asymmetric sfx sites, was proposed. Site-specific recombination in the temperate phage Mx8 of M. xanthus was also described. The Mx8 attP site is located within the coding sequence of the Mx8 intP gene. Therefore, the integration of Mx8 into the M. xanthus chromosome results in the conversion of the intP gene into a new gene, intP. As a result of this conversion, the 112-amino-acid C-terminal sequence of the intP product is replaced with a 13-amino acid sequence of the intR product. The C-terminal domain of Mx8 IntP recombinase is only required for integration and not for excision.

DNA topoisomerases as targets for anticancer drugs.

DNA topoisomerases are essential enzymes that regulate the conformational changes in DNA topology by catalysing the concerted breakage and rejoining of DNA strands during normal cellular growth. Over the past few years there has been considerable pharmacological interest in these enzymes because inhibitors of DNA topoisomerases represent a major class of anticancer drugs. This review highlights topoisomerase-targeting drugs that have shown promising anticancer activities. The mechanisms by which those drugs interfere with the catalytic cycles of type I and type II DNA topoisomerases and the factors involved in the development of resistance to these drugs are discussed.

Poly(ADP-ribose) polymerase in DNA damage-response pathway: implications for radiation oncology.

Poly(ADP-ribose) polymerase (PARP) catalyzes the transfer of successive units of ADP-ribose moiety from NAD(+) covalently to itself and other nuclear acceptor proteins. PARP is a zinc finger-containing protein, allowing the enzyme to bind to either double- or single-strand DNA breaks without any apparent sequence preference. The catalytic activity of PARP is strictly dependent on the presence of strand breaks in DNA and is modulated by the level of automodification. Data from many studies show that PARP is involved in numerous biological functions, all of which are associated with the breaking and rejoining of DNA strands, and plays a pivotal role in DNA damage repair. Recent advances in apoptosis research identified PARP as one of the intracellular "death substrates" and demonstrated the involvement of polymerase in the execution of programmed cell death. This review summarizes the biological effects of PARP function that may have a potential for targeted sensitization of tumor cells to genotoxic agents and radiotherapy. Int. J. Cancer (Radiat. Oncol. Invest.) 90, 59-67 (2000).

Identification of Active Site Residues in the “GyrA” Half of Yeast DNA Topoisomerase II

Site-directed mutagenesis was carried out at 10 highly conserved polar residues within the C-terminal half of yeast DNA topoisomerase II, which corresponds to the A subunit of bacterial DNA gyrase, to identify amino acid side chains that augment the active site tyrosine Tyr-782 in the breakage and rejoining of DNA strands. Complementation tests show that alanine substitution at Arg-690, Asp-697, Lys-700, Arg-704, or Arg-781, but not at His-735, His-736, Glu-738, Gln-750, or Asn-828, inactivates the enzyme in vivo. Measurements of DNA relaxation and cleavage by purified mutant enzymes show that these activities are abolished in the R690A mutant and are much reduced in the mutants D697A, K700A, R704A, and R781A. When a Y782F polypeptide with a phenylalanine substituting for the active site tyrosine was expressed in cells that also express the R690A polypeptide, the resulting heterodimeric yeast DNA topoisomerase II was found to nick plasmid DNA. Thus in a dimeric wild-type enzyme, Tyr-782 in one protomer and Arg-690 in the other cooperate in trans in the catalysis of DNA cleavage. For the residues D697A, K700A, R704A, and R781A, their locations in the crystal structures of type II DNA topoisomerase fragments suggest that Arg-781 and Lys-700 might be involved in anchoring the 5' and 3' sides of the broken DNA, respectively, and the roles of Asp-697 and Arg-704 are probably less direct.

Recognition of core-type DNA sites by λ integrase

Escherichia coli phage λ integrase (Int) is a 40 kilodalton, 356 amino acid residue protein, which belongs to the λ Int family of site-specific recombinases. The amino-terminal domain (residues 1 to 64) of Int binds to “arm-type” DNA sites, distant from the sites of DNA cleavage. The carboxy-terminal fragment, termed C65 (residues 65 to 356), binds “core-type” DNA sites and catalyzes cleavage and ligation at these sites. It has been further divided into two smaller domains, encompassing residues 65 to 169 and 170 to 356, respectively. The latter has been characterized and its crystal structure has been determined. Although this domain catalyzes the cleavage and rejoining of DNA strands it, unexpectedly, does not form electrophorectically stable complexes with core-type DNA. Here we have investigated the critical features of λ Int binding to core-type DNA sites; especially, the role of the central 65 to 169 domain. To eliminate the complexities arising from λ Int’s heterobivalency we studied Int C65, which was shown to be as competent as Int, in binding to, and cleaving, core-type sites. Zero-length UV crosslinking was used to show that Ala125 and Ala126 make close contact with bases in the core-type DNA. Modification by pyridoxal 5′-phosphate was used to identify Lys103 at the protein-DNA interface. Since both of the identified loci are in the central domain, it was cloned and purified and found to bind to core-type DNA autonomously and specifically. The synergistic roles of the catalytic and the central, or core-binding (CB), domains in the interaction with core-type DNA are discussed for (Int and related DNA recombinases.

A Catalytic Domain of Eukaryotic DNA Topoisomerase I

Eukaryotic type IB topoisomerases catalyze the cleavage and rejoining of DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate. The 314-amino acid vaccinia topoisomerase is the smallest member of this family and is distinguished from its cellular counterparts by its specificity for cleavage at the target sequence 5'-CCCTT downward arrow. Here we show that Topo-(81-314), a truncated derivative that lacks the N-terminal domain, performs the same repertoire of reactions as the full-sized topoisomerase: relaxation of supercoiled DNA, site-specific DNA transesterification, and DNA strand transfer. Elimination of the N-terminal domain slows the rate of single-turnover DNA cleavage by 10(-3.6), but has little effect on the rate of single-turnover DNA religation. DNA relaxation and strand cleavage by Topo-(81-314) are inhibited by salt and magnesium; these effects are indicative of reduced affinity in noncovalent DNA binding. We report that identical properties are displayed by a full-length mutant protein, Topo(Y70A/Y72A), which lacks two tyrosine side chains within the N-terminal domain that contact the DNA target site in the major groove. We speculate that Topo-(81-314) is fully competent for transesterification chemistry, but is compromised with respect to a rate-limiting precleavage conformational step that is contingent on DNA contacts made by Tyr-70 and Tyr-72.

Mutational analysis of vaccinia virus topoisomerase identifies residues involved in DNA binding.

Vaccinia DNA topoisomerase catalyzes the cleavage and re-joining of DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate formed at a specific target sequence, 5'-(C/T)CCTT downward arrow. The 314 aa protein consists of three protease-resistant structural domains demarcated by protease-sensitive interdomain segments referred to as the bridge and the hinge. The bridge is defined by trypsin-accessible sites at Arg80, Lys83 and Arg84. Photocrosslinking and proteolytic footprinting experiments suggest that residues near the interdomain bridge interact with DNA. To assess the contributions of specific amino acids to DNA binding and transesterification chemistry, we introduced alanine substitutions at 16 positions within a 24 aa segment from residues 63 to 86(DSKGRRQYFYGKMHVQNRNAKRDR). Assays of the rates of DNA relaxation under conditions optimal for the wild-type topoisomerase revealed significant mutational effects at six positions; Arg67, Tyr70, Tyr72, Arg80, Arg84 and Asp85. The mutated proteins displayed normal or near-normal rates of single-turnover transesterification to DNA. The effects of amino acid substitutions on DNA binding were evinced by inhibition of covalent adduct formation in the presence of salt and magnesium. The mutant enzymes also displayed diminished affinity for a subset of cleavage sites in pUC19 DNA. Tyr70 and Tyr72 were subjected to further analysis by replacement with Phe, His, Gln and Arg. At both positions, the aromatic moiety was important for DNA binding.

Mutational analysis of 26 residues of vaccinia DNA topoisomerase identifies Ser-204 as important for DNA binding and cleavage.

Vaccinia DNA topoisomerase, a 314 amino acid type I enzyme, catalyzes the cleavage and rejoining of DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate formed at a specific target sequence, 5'-(C/T)CCTT downward arrow. To identify amino acids that participate in the DNA binding and transesterification steps, we introduced alanine substitutions at 18 positions within a centrally located 27 amino acid segment (181-RLYKPLLKLTDDSSPEEFLFNKLSERK-207) and at 8 positions near the N-terminus (1-MRALFYKDGK-10). All mutant proteins except two displayed wild-type activity in relaxing supercoiled DNA. F200A and S204A exhibited reduced rates of relaxation and were subjected to a kinetic analysis of the strand cleavage reaction under single-turnover and equilibrium conditions. The F200A and S204A mutations reduced the rate of single-turnover DNA cleavage by factors of 5 and 70, respectively. Both mutations shifted the cleavage-religation equilibrium in favor of the noncovalently bound state. The S204A mutation reduced the affinity of topoisomerase for CCCTT-containing DNA, but did not alter the site-specificity of DNA cleavage. Vaccinia residue Ser-204, which is conserved in all poxvirus topoisomerases, but not in the cellular homologues, may contribute to the unique cleavage site specificity of the poxvirus enzymes. Phe-200 is conserved in all members of the type IB topoisomerase family.

Mutational Analysis of 39 Residues of Vaccinia DNA Topoisomerase Identifies Lys-220, Arg-223, and Asn-228 as Important for Covalent Catalysis

Vaccinia DNA topoisomerase, a 314-amino acid type I enzyme, catalyzes the cleavage and rejoining of DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate. To identify amino acids that participate in the transesterification reaction, we introduced alanine substitutions at 39 positions within a conserved 57amino acid segment upstream of the active-site tyrosine. Purified wild type and mutant proteins were compared with respect to their activities in relaxing supercoiled DNA. The majority of mutant proteins displayed wild type topoisomerase activity. Mutant enzymes that relaxed DNA at reduced rates were subjected to kinetic analysis of the strand cleavage and religation steps under single-turnover and equilibrium conditions. For the wild type topoisomerase, the observed single-turnover cleavage rate constant (kcl) was 0.29 s-1 and the cleavage-religation equilibrium constant (Kcl) was 0.22. The most dramatic mutational effects were seen with R223A; removal of the basic side chain reduced the rates of cleavage and religation by factors of 10(-4.3) and 10(-5.0), respectively, and shifted the cleavage-religation equilibrium in favor of the covalently bound state (Kcl = 1). Introduction of lysine at position 223 restored the rate of cleavage to 1/10 that of the wild type enzyme. We conclude that a basic residue is essential for covalent catalysis and suggest that Arg-223 is a constituent of the active site. Modest mutational effects were observed at two other positions (Lys-220 and Asn-228), at which alanine substitutions slowed the rates of strand cleavage by 1 order of magnitude and shifted the equilibrium toward the noncovalently bound state. Arg-223 and Lys-220 are conserved in all members of the eukaryotic type I topoisomerase family; Asn-228 is conserved among the poxvirus enzymes.

DNA ligase activity on chromatin and its analogs. Rejoining of DNA strands in polylysine-DNA complexes and in reconstituted chromatins

A highly purified DNA ligase from rat liver nuclei has been tested on DNA containing single-strand breaks ("nicks"); the DNA was present in several types of complexes which were chosen to serve as models for chromatin. These model systems included complexes of polylysine or histones with DNA as well as reconstituted chromatin preparations. In all these cases, the limit of ligase sealing was measured as a function of the ratio of polypeptide or protein to DNA. With an excess of either polylysine or histones, the ligase is totally prevented from sealing nicks in the DNA. However, at ratios of histones to DNA similar to those occurring in chromatin, about half of the nicks are accessible to the ligase. In the reconstitution of chromatin, the proteins are dissociated from the DNA by exposure to high ionic strength either with or without urea. If such procedures are carried out in the presence of labeled nicked DNA, the proteins will redistribute over this ligase substrate as well. When the chromatin is reconstituted at protein/DNA ratios similar to those occurring in chromatin, once more only about half of the nicks are accessible to the ligase. Similar results were obtained with preparations reconstituted with either rat liver or duck reticulocyte chromatin. The rate of ligase action has been measured on a variety of the complexes. While the rate falls as the DNA is increasingly covered with polylysine or histones, this is largely or entirely due to the decrease in concentration of sealable sites. At saturating concentrations of these DNA complexes, the original rate on uncovered DNA is approached.

Sensitivity of the newly synthesized and template DNA of lymphoma cells to damage by methyl methanesulphonate, and the nature of associated “repair” processes

Using alkaline sucrose gradient centrifugation, single-strand breaks can be shown to occur in [ 14C]thymidine labelled P388F lymphoma cells after treatment with methyl methanesulphonate at levels which allow for a high level of survival of the cells. The pattern of fragmentation of newly synthesized and template labelled DNA differs and these differences are discussed. The “repair” of this damage within 2-h after treatment has been studied and no evidence for rejoining of DNA strands during this time period was obtained. The increase in molecular weight following incubation of treated, newly labelled cells is now considered to be due to normal DNA synthesis on prelabelled fragments. The labelled template DNA shows two new features. A higher molecular weight component which appears to be broken internally by methyl methanesulphonate, but requires further incubation to release the fragments, and a characteristic distribution of labelled fragments which show no evidence of single-strand rejoining within 2-h post-treatment.