The role of the Zn(II) binding domain in the mechanism of E. coli DNA topoisomerase I

Background  

Escherichia coli DNA topoisomerase I binds three Zn(II) with three tetracysteine motifs which, together with the 14 kDa C-terminal region, form a 30 kDa DNA binding domain (ZD domain). The 67 kDa N-terminal domain (Top67) has the active site tyrosine for DNA cleavage but cannot relax negatively supercoiled DNA. We analyzed the role of the ZD domain in the enzyme mechanism.     

The DNA relaxation activity and covalent complex accumulation of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> topoisomerase I can be assayed in <Emphasis Type="Italic">Escherichia coli</Emphasis>: application for identification of potential FRET-dye labeling sites

Background

Mycobacterium tuberculosis topoisomerase I (MtTOP1) and Escherichia coli topoisomerase I have highly homologous transesterification domains, but the two enzymes have distinctly different C-terminal domains. To investigate the structure-function of MtTOP1 and to target its activity for development of new TB therapy, it is desirable to have a rapid genetic assay for its catalytic activity, and potential bactericidal consequence from accumulation of its covalent complex.

Results

We show that plasmid-encoded recombinant MtTOP1 can complement the temperature sensitive topA function of E. coli strain AS17. Moreover, expression of MtTOP1-G116 S enzyme with the TOPRIM mutation that inhibits DNA religation results in SOS induction and loss of viability in E. coli. The absence of cysteine residues in the MtTOP1 enzyme makes it an attractive system for introduction of potentially informative chemical or spectroscopic probes at specific positions via cysteine mutagenesis. Such probes could be useful for development of high throughput screening (HTS) assays. We employed the AS17 complementation system to screen for sites in MtTOP1 that can tolerate cysteine substitution without loss of complementation function. These cysteine substitution mutants were confirmed to have retained the relaxation activity. One such mutant of MtTOP1 was utilized for fluorescence probe incorporation and fluorescence resonance energy transfer measurement with fluorophore-labeled oligonucleotide substrate.

Conclusions

The DNA relaxation and cleavage complex accumulation of M. tuberculosis topoisomerase I can be measured with genetic assays in E. coli, facilitating rapid analysis of its activities, and discovery of new TB therapy targeting this essential enzyme.

     

Molecular characterization of <Emphasis Type="Italic">tlyA</Emphasis> gene product,Rv1694 of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>: A non-conventional hemolysin and a ribosomal RNA methyl transferase

Background  

Mycobacterium tuberculosis is a virulent bacillus causing tuberculosis, a disease responsible for million deaths each year worldwide. In order to understand its mechanism of pathogenesis in humans and to help control tuberculosis, functions of numerous Mycobacterium tuberculosis genes are being characterized. In this study we report the dual functionality of tlyA gene product of Mycobacterium tuberculosis annotated as Rv1694, a 268 amino acid long basic protein.     

Dictyostelium cells bind a secreted autocrine factor that represses cell proliferation

Background  

Mycobacterium tuberculosis, an intracellular pathogen encounters redox stress throughout its life inside the host. In order to protect itself from the redox onslaughts of host immune system, M. tuberculosis appears to have developed accessory thioredoxin-like proteins which are represented by ORFs encoding WhiB-like proteins. We have earlier reported that WhiB1/Rv3219 is a thioredoxin like protein of M. tuberculosis and functions as a protein disulfide reductase. Generally thioredoxins have many substrate proteins. The current study aims to identify the substrate protein(s) of M. tuberculosis WhiB1.     

Synthesis, structure elucidation, DNA interaction, biological evaluation, and molecular docking of an isatin-derived tyramine bidentate Schiff base and its metal complexes

Abstract  

Three new transition metal complexes [CuL₂Cl₂], [NiL₂Cl₂], and [ZnL₂Cl₂], where L = Schiff base derived from isatin (1H-indole-2,3-dione) and tyramine (4-(2-aminoethyl)phenol), have been synthesized and characterized spectroscopically. The binding properties of these complexes with calf thymus DNA have been investigated by use of electronic absorption spectroscopy, viscosity measurement, cyclic voltammetry, and molecular docking analysis. Moreover, these complexes have been found to promote the cleavage of pUC19 DNA from the supercoiled form I to the open circular form II in the presence of hydrogen peroxide. The newly synthesized compounds were screened for antibacterial activity and subjected to molecular docking studies for inhibition of the enzyme Staphylococcus aureus sortase-A, which is a novel target for antibacterials.     

DNA biosensors for the detection of aflatoxin producing Aspergillus flavus and A. parasiticus

Abstract  

In this work, we report on the development of a DNA-based piezoelectric biosensor specific for the detection of an amplicon of the aflD gene of Aspergillus flavus and A. parasiticus. Expression of this gene is consistently correlated with a strain’s ability to produce aflatoxins that are considered very potent liver carcinogens in various animal species and humans. The DNA biosensor has been characterized with synthetic oligonucleotides and amplicons. Moreover, it has been applied to the analysis of real samples consisting of amplicons of DNA extracted from flours and feed contaminated with A. flavus and A. parasiticus.     

Insights into the role of Val45 and Gln182 of Escherichia coli MutY in DNA substrate binding and specificity

Background  

Escherichia coli MutY (EcMutY) reduces mutagenesis by removing adenines paired with guanines or 7,8-dihydro-8-oxo-guanines (8-oxoG). V45 and Q182 of EcMutY are considered to be the key determinants of adenine specificity. Both residues are spatially close to each other in the active site and are conserved in MutY family proteins but not in Methanobacterium thermoautotrophicum Mig.MthI T/G mismatch DNA glycosylase (A50 and L187 at the corresponding respective positions).     

Comparison of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> isocitrate dehydrogenases (ICD-1 and ICD-2) reveals differences in coenzyme affinity,oligomeric state,pH tolerance and phylogenetic affiliation

Background  

M.tb icd-1 and M.tb icd-2, have been identified in the Mycobacterium tuberculosis genome as probable isocitrate dehydrogenase (ICD) genes. Earlier we demonstrated that the two isoforms can elicit B cell response in TB patients and significantly differentiate TB infected population from healthy, BCG-vaccinated controls. Even though immunoassays suggest that these proteins are closely related in terms of antigenic determinants, we now show that M.tb icd-1 and M.tb icd-2 code for functional energy cycle enzymes and document the differences in their biochemical properties, oligomeric assembly and phylogenetic affiliation.     

Theoretical studies of 1,4-dihydropyridine-3,5-dicarboxamides as possible inhibitors of Mycobacterium tuberculosis enoyl reductase

Abstract  

The binding affinity of some novel 1,4-dihydropyridine-3,5-dicarboxamides to enoyl-ACP reductase (InhA) from M. tuberculosis was studied by the docking method. A molecular dynamics simulation lasting 230 ns in total was performed for 19 ligand–enzyme complexes to calculate the binding free energies of these ligands to the enzyme. All of the studied compounds stayed in the active site. An energetics analysis revealed that the van der Waals share is more important than electrostatic in binding of all ligands to the active site.     

The DNA polymerase activity of Pol ε holoenzyme is required for rapid and efficient chromosomal DNA replication in Xenopus egg extracts

Background  

DNA polymerase ε (Pol ε) is involved in DNA replication, repair, and cell-cycle checkpoint control in eukaryotic cells. Although the roles of replicative Pol α and Pol δ in chromosomal DNA replication are relatively well understood and well documented, the precise role of Pol ε in chromosomal DNA replication is not well understood.     

Biochemical characterization of the maltokinase from <Emphasis Type="Italic">Mycobacterium bovis</Emphasis> BCG

Background  

Maltose-1-phosphate was detected in Mycobacterium bovis BCG extracts in the 1960's but a maltose-1-phosphate synthetase (maltokinase, Mak) was only much later purified from Actinoplanes missouriensis, allowing the identification of the mak gene. Recently, this metabolite was proposed to be the intermediate in a pathway linking trehalose with the synthesis of glycogen in M. smegmatis. Although the M. tuberculosis H37Rv mak gene (Rv0127) was considered essential for growth, no mycobacterial Mak has, to date, been characterized.     

Kinetic characterisation of arylamine <Emphasis Type="Italic">N</Emphasis>-acetyltransferase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Background  

Arylamine N-acetyltransferases (NATs) are important drug- and carcinogen-metabolising enzymes that catalyse the transfer of an acetyl group from a donor, such as acetyl coenzyme A, to an aromatic or heterocyclic amine, hydrazine, hydrazide or N-hydroxylamine acceptor substrate. NATs are found in eukaryotes and prokaryotes, and they may also have an endogenous function in addition to drug metabolism. For example, NAT from Mycobacterium tuberculosis has been proposed to have a role in cell wall lipid biosynthesis, and is therefore of interest as a potential drug target. To date there have been no studies investigating the kinetic mechanism of a bacterial NAT enzyme.     

Specificity of DNA triple helix formation analyzed by a FRET assay

Background  

A third DNA strand can bind into the major groove of a homopurine duplex DNA to form a DNA triple helix. Sequence specific triplex formation can be applied for gene targeting, gene silencing and mutagenesis.     

DNA loops and semicatenated DNA junctions

Background  

Alternative DNA conformations are of particular interest as potential signals to mark important sites on the genome. The structural variability of CA microsatellites is particularly pronounced; these are repetitive poly(CA) · poly(TG) DNA sequences spread in all eukaryotic genomes as tracts of up to 60 base pairs long. Many in vitro studies have shown that the structure of poly(CA) · poly(TG) can vary markedly from the classical right handed DNA double helix and adopt diverse alternative conformations. Here we have studied the mechanism of formation and the structure of an alternative DNA structure, named Form X, which was observed previously by polyacrylamide gel electrophoresis of DNA fragments containing a tract of the CA microsatellite poly(CA) · poly(TG) but had not yet been characterized.     

Interaction and photodynamic activity of cationic porphyrin derivatives bearing different patterns of charge distribution with GMP and DNA

The interaction of amphiphilic cationic porphyrins, containing different patterns of meso-substitution by 4-(3-N,N,N-trimethylammoniumpropoxy)phenyl (A) and 4-(trifluoromethyl)phenyl (B) groups, with guanosine 5′-monophosphate (GMP) and calf thymus DNA have been studied by optical methods in phosphate buffer solution. The properties of these synthetic porphyrins were compared with those of representative standard of anionic 5,10,15,20-tetra(4-sulphonatophenyl)porphyrin (TPPS₄⁴⁻) and cationic 5,10,15,20-tetra(4-N,N,N-trimethylammonium phenyl)porphyrin (TMAP⁴⁺). Stable complexes with GMP were found for cationic porphyrins, except for monocationic AB₃⁺. The binding constant (K_GMP  10⁴ M⁻¹) follows the order: A₃B³⁺  ABAB²⁺ > A₄⁴⁺  TMAP⁴⁺. Also, interaction with DNA was observed for all evaluated cationic porphyrins. For these related cationic porphyrins, the binding constant (K_DNA  10⁵ M⁻¹) increases with the number of cationic charges. On the other hand, the photodynamic activity of porphyrins was analyzed in solution of GMP and DNA. Monocationic AB₃⁺ is a less effective sensitizer to oxidize GMP in comparison with the other cationic porphyrins, in agreement with the lack of detected interaction with this nucleotide. The electrophoretic analysis of DNA indicates that photocleavage takes place when the samples are exposed to photoexcited tricationic and tetracationic porphyrins. In the presence of sodium azide the DNA decomposition was diminished. Also, reduction in the DNA photocleavage was observed under anoxic condition, indicating that oxygen is essential for DNA photocleavage sensitized by these cationic porphyrins. In addition, an increase in DNA degradation was not observed in deuteriated water. Therefore, an important contribution of type I photoreaction processes could be occurring in the DNA photodamage sensitized by these cationic porphyrins. These results provide a better understanding of the characteristics needed for sensitizers to produce efficient DNA photocleavage.     

Effects of the ancillary ligands of polypyridyl cobalt(II) complexes on pH-induced spectral properties and DNA binding properties

Abstract  

Two new Co(II) complexes [Co(ipH)₂(bdipH)]²⁺ and [Co(8-HQ)₂(bdipH)] (ipH = imidazo[4,5-f][1,10]phenanthroline, bdipH = 2-(benzo[d][1,3]dioxol-4-yl)-1H-imidazo[4,5-f][1,10]phenanthroline, 8-HQ = 8-hydroxyquinoline) were synthesized and characterized in detail by elemental analysis, IR, and UV–Vis spectroscopic techniques. The effects of pH on the UV–Vis absorption and emission spectra of the complex were studied. The interaction of the two complexes with calf thymus DNA was explored by using viscosity measurements, electronic absorption titration, competitive binding experiments, and cyclic voltammetry. The experimental results show that complex [Co(ipH)₂(bdipH)]²⁺ exhibits pH-sensitive emission, the two complexes can bind to DNA in an intercalation mode, and the DNA binding affinity of complex [Co(ipH)₂(bdipH)]²⁺ (K _b = 2.11 × 10⁵ M⁻¹) is greater than that of complex [Co(8-HQ)₂(bdipH)] (K _b = 1.76 × 10⁵ M⁻¹). The results show that the size and shape of the ancillary ligand have significant effects on the binding affinity of DNA and complexes.     

DNA binding and oxidative cleavage activity of ternary (l-proline)copper(II) complexes of heterocyclic bases

Ternary copper(II) complexes [Cu(l-pro)(B)(H₂O)](NO₃) (1, 2) where l-pro = l-proline, B is a N,N-donor heterocyclic base, viz. 2,2′-bipyridine (bpy, 1), 1,10-phenanthroline (phen, 2), are synthesized, characterized, and their DNA binding and cleavage activity studied. The bpy complex (1) is structurally characterized by single-crystal X-ray crystallography. The complexes show the presence of a distorted square-pyramidal (4 + 1) CuN₃O₂ coordination geometry. Complex [Cu(l-pro)(bpy)(H₂O)](NO₃) (1) crystallizes in the triclinic space group P1 with unit cell parameters: a = 7.082(3) Å, b = 10.483(5) Å, c = 11.581(5) Å, α = 89.700(7)°, β = 83.488(8)°, γ = 84.109(8)° and V = 849.7(7) Å³. The one-electron paramagnetic complexes display a d–d band near 600 nm in water and show a cyclic voltammetric response due to Cu(II)/Cu(I) couple near 0.1 V (versus SCE) in Tris–HCl buffer–0.1 M KCl. Binding interactions of the complexes with calf thymus (CT) DNA have been investigated by emission, absorption, viscosity and DNA thermal denaturation studies. The phen complex displays significant binding propensity to the CT DNA giving an order: 2 (phen)  1 (bpy). The bpy complex does not show any apparent binding to the DNA and hence poor cleavage efficiency. Complex 2 shows efficient oxidative cleavage of SC-DNA in the presence of 3-mercaptopropionic acid (MPA) involving hydroxyl radical species as evidenced from the control data showing inhibition of DNA cleavage in the presence of DMSO and catalase.     

Chemical cleavage reactions of DNA on solid support: application in mutation detection

Background  

The conventional solution-phase Chemical Cleavage of Mismatch (CCM) method is time-consuming, as the protocol requires purification of DNA after each reaction step. This paper describes a new version of CCM to overcome this problem by immobilizing DNA on silica solid supports.     

Catalytic inhibition of topoisomerase II by a novel rationally designed ATP-competitive purine analogue

Background

Topoisomerase II poisons are in clinical use as anti-cancer therapy for decades and work by stabilizing the enzyme-induced DNA breaks. In contrast, catalytic inhibitors block the enzyme before DNA scission. Although several catalytic inhibitors of topoisomerase II have been described, preclinical concepts for exploiting their anti-proliferative activity based on molecular characteristics of the tumor cell have only recently started to emerge. Topoisomerase II is an ATPase and uses the energy derived from ATP hydrolysis to orchestrate the movement of the DNA double strands along the enzyme. Thus, interfering with ATPase function with low molecular weight inhibitors that target the nucleotide binding pocket should profoundly affect cells that are committed to undergo mitosis.

Results

Here we describe the discovery and characterization of a novel purine diamine analogue as a potent ATP-competitive catalytic inhibitor of topoisomerase II. Quinoline aminopurine compound 1 (QAP 1) inhibited topoisomerase II ATPase activity and decatenation reaction at sub-micromolar concentrations, targeted both topoisomerase II alpha and beta in cell free assays and, using a quantitative cell-based assay and a chromosome segregation assay, displayed catalytic enzyme inhibition in cells. In agreement with recent hypothesis, we show that BRCA1 mutant breast cancer cells have increased sensitivity to QAP 1.

Conclusion

The results obtained with QAP 1 demonstrate that potent and selective catalytic inhibition of human topoisomerase II function with an ATP-competitive inhibitor is feasible. Our data suggest that further drug discovery efforts on ATP-competitive catalytic inhibitors are warranted and that such drugs could potentially be developed as anti-cancer therapy for tumors that bear the appropriate combination of molecular alterations.     

Iron(II) Supramolecular Helicates Condense Plasmid DNA and Inhibit Vital DNA‐Related Enzymatic Activities

The dinuclear iron(II) supramolecular helicates [Fe₂L₃]Cl₄ (L=C₂₅H₂₀N₄) bind to DNA through noncovalent (i.e., hydrogen‐bonding, electrostatic) interactions and exhibit antimicrobial and anticancer effects. In this study, we show that the helicates condense plasmid DNA with a much higher potency than conventional DNA‐condensing agents. Notably, molecules of DNA in the presence of the M enantiomer of [Fe₂L₃]Cl₄ do not form intermolecular aggregates typically formed by other condensing agents, such as spermidine or spermine. The helicates inhibit the activity of several DNA‐processing enzymes, such as RNA polymerase, DNA topoisomerase I, deoxyribonuclease I, and site‐specific restriction endonucleases. However, the results also indicate that the DNA condensation induced by the helicates does not play a crucial role in these inhibition reactions. The mechanisms for the inhibitory effects of [Fe₂L₃]Cl₄ helicates on DNA‐related enzymatic activities have been proposed.