DNA Cleavage

Described herein is the synthesis, structure, and monoesterase and diesterase activities of a new mononuclear (2,4-BDNPP). When covalently linked to 3-aminopropyl-functionalized silica, 1 undergoes disproportionation to form a dinuclear species (APS-1), whose catalytic efficiency is increased when compared to the homogeneous reaction due to second coordination sphere effects which increase the substrate to complex association constant. The anchored catalyst APS-1 can be recovered and reused for subsequent hydrolysis reactions (five times) with only a slight loss in activity. In the presence of DNA, we suggest that 1 is also converted into the dinuclear active species as observed with APS-1, and both were shown to be efficient in DNA cleavage.

Repetitive DNA sequences, such as are present in micro-and mini-satellites, telomeres, and trinucleotide repeats (linked to fragile X syndrome, Huntington disease, etc.), account for nearly 30% of the human genome. These domains exhibit enhanced susceptibility to oxidative attack to yield base modifications, strand breaks and abasic sites, have a propensity to adopt non-canonical DNA forms modulated by the position(s) of the lesion(s), and, when not properly processed, can contribute to genome instability that underlies aging and disease development. Knowledge of the repair efficiencies of DNA damage within such repetitive sequences is therefore crucial for understanding the impact of such domains on genomic integrity. In the present study, using strategically designed oligonucleotide substrates, we determined the ability of human apurinic/ apyrimidinic endonuclease 1 (APE1) to cleave at AP sites in a collection of tandem DNA repeat landscapes involving telomeric and CAG/CTG repeat sequences. Our studies reveal the differential influence of domain sequence, conformation, and AP site location/relative positioning on the efficiency of APE1 binding and strand incision. Intriguingly, our data demonstrate that APE1 endonuclease efficiency correlates with the thermodynamic stability of the DNA substrate. We discuss how these results have both predictive and mechanistic consequences for understanding the success and failure of repair protein activity associated with such oxidatively sensitive, conformationally plastic/dynamic repetitive DNA domains.

Repetitive DNA sequences, such as are present in micro-and mini-satellites, telomeres, and trinucleotide repeats (linked to fragile X syndrome, Huntington disease, etc.), account for nearly 30% of the human genome. These domains exhibit enhanced susceptibility to oxidative attack to yield base modifications, strand breaks and abasic sites, have a propensity to adopt non-canonical DNA forms modulated by the position(s) of the lesion(s), and, when not properly processed, can contribute to genome instability that underlies aging and disease development. Knowledge of the repair efficiencies of DNA damage within such repetitive sequences is therefore crucial for understanding the impact of such domains on genomic integrity. In the present study, using strategically designed oligonucleotide substrates, we determined the ability of human apurinic/ apyrimidinic endonuclease 1 (APE1) to cleave at AP sites in a collection of tandem DNA repeat landscapes involving telomeric and CAG/CTG repeat sequences. Our studies reveal the differential influence of domain sequence, conformation, and AP site location/relative positioning on the efficiency of APE1 binding and strand incision. Intriguingly, our data demonstrate that APE1 endonuclease efficiency correlates with the thermodynamic stability of the DNA substrate. We discuss how these results have both predictive and mechanistic consequences for understanding the success and failure of repair protein activity associated with such oxidatively sensitive, conformationally plastic/dynamic repetitive DNA domains.

The binding selectivity of the M(phen)(edda) (M = Cu, Co, Ni, Zn; phen = 1,10phenanthroline, edda = ethylenediaminediacetic acid) complexes towards ds(CG)6, ds(AT)6 and ds(CGCGAATTCGCG) B-form oligonucleotide duplexes were studied by CD spectroscopy and molecular modeling. The binding mode is intercalation and there is selectivity towards AT-sequence and stacking preference for A/A parallel or diagonal adjacent base steps in their intercalation. The nucleolytic properties of these complexes were investigated and the factors affecting the extent of cleavage were determined to be: concentration of complex, the nature of metal(II) ion, type of buffer, pH of buffer, incubation time, incubation temperature, and the presence of hydrogen peroxide or ascorbic acid as exogenous reagents. The fluorescence property of these complexes and its origin were also investigated. The crystal structure of the Zn(phen)(edda) complex is reported in which the zinc atom displays a distorted trans-N4O2 octahedral geometry; the crystal packing features double layers of complex molecules held together by extensive hydrogen bonding that interdigitate with adjacent double layers via π…π interactions between 1,10-phenanthroline residues. The structure is compared with that of the recently described copper(II) analogue and, with the latter, included in molecular modeling.

Crystal structure analysis of the zinc complex establishes it as a distorted octahedral complex, bis(3-methylpicolinato-κ2 N,O)2(1,10-phenanthroline-κ2 N,N)-zinc(II) pentahydrate, [Zn(3-Me-pic)2(phen)]•5H2O. The trans-configuration of carbonyl oxygen atoms of the carboxylate moieties and orientation of the two planar picolinate ligands above and before the phen ligand plane seems to confer DNA sequence recognition to the complex. It cannot cleave DNA under hydrolytic condition but can slightly be activated by hydrogen peroxide or sodium ascorbate. Circular Dichroism and Fluorescence spectroscopic analysis of its interaction with various duplex polynucleotides reveals its binding mode as mainly intercalation. It shows distinct DNA sequence binding selectivity and the order of decreasing selectivity is ATAT > AATT > CGCG. Docking studies lead to the same conclusion on this sequence selectivity. It binds strongly with G-quadruplex with human tolemeric sequence 5′-AG3(T2AG3)3-3′, can inhibit topoisomerase I efficiently and is cytotoxic against MCF-7 cell line.

The opposing activities of 53BP1 and BRCA1 influence pathway choice in DNA double-strand-break repair. How BRCA1 counteracts the inhibitory effect of 53BP1 on DNA resection and homologous recombination is unknown. Here we identify the site of BRCA1-BARD1 required for priming ubiquitin transfer from E2∼ubiquitin and demonstrate that BRCA1-BARD1's ubiquitin ligase activity is required for repositioning 53BP1 on damaged chromatin. We confirm H2A ubiquitination by BRCA1-BARD1 and show that an H2A-ubiquitin fusion protein promotes DNA resection and repair in BARD1-deficient cells. BRCA1-BARD1's function in homologous recombination requires the chromatin remodeler SMARCAD1. SMARCAD1 binding to H2A-ubiquitin and optimal localization to sites of damage and activity in DNA repair requires its ubiquitin-binding CUE domains. SMARCAD1 is required for 53BP1 repositioning, and the need for SMARCAD1 in olaparib or camptothecin resistance is alleviated by 53BP1 loss. Thus, BRCA1-BARD1 ligas...

Antimicrobial screening of several novel 4-thiazolidinones with benzothiazole moiety has been performed. These compounds were evaluated for antimicrobial activity against a panel of bacterial and fungal strains. The strains were treated with these benzothiazole derivatives at varying concentrations, and MIC's were calculated. Structures of these compounds have been determined by spectroscopic studies viz., FT-IR, 1H NMR, 13C NMR and elemental analysis. Significant antimicrobial activity was observed for some members of the series, and compounds viz. 3-(4-(benzo[d]thiazol-2-yl) phenyl-2-(4-methoxyphenyl)thiazolidin-4-one and 3-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(4-hydroxy phenyl)thiazolidin-4-one were found to be the most active against E.coli and C. albicans with MIC values in the range of 15.6-125 microg/ml. Preliminary study of the structure-activity relationship revealed that electron donating groups associated with thiazolidine bearing benzothiazole rings had a great effect ...

Pulsed gradient spin-echo nuclear magnetic resonance diffusion measurements have been used to show that platinum(II)-based intercalating agents self-stack in solution and form nanorods 0.45-3.9 nm in length (at 25 mM); their lengths are dependent on metal complex concentration, salt concentration and solution temperature.

Background DNA 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. Methods The 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 ...

The interpretation of in vitro cytotoxicity data of Cu(II)-1,10-phenanthroline (phen) complexes normally does not take into account the speciation that complexes undergo in cell incubation media and its implications in cellular uptake and mechanisms of action. We synthesize and test the activity of several distinct Cu(II)-phen compounds; up to 24 h of incubation, the cytotoxic activity differs for the Cu complexes and the corresponding free ligands, but for longer incubation times (e.g., 72 h), all compounds display similar activity. Combining the use of several spectroscopic, spectrometric, and electrochemical techniques, the speciation of Cuphen compounds in cell incubation media is evaluated, indicating that the originally added complex almost totally decomposed and that Cu(II) and phen are mainly bound to bovine serum albumin. Several methods are used to disclose relationships between structure, activity, speciation in incubation media, cellular uptake, distribution of Cu in cells, and cytotoxicity. Contrary to what is reported in most studies, we conclude that interaction with cell components and cell death involves the separate action of Cu ions and phen molecules, not [Cu(phen) n ] species. This conclusion should similarly apply to many other Cu-ligand systems reported to date.

The DNA cleavage activity of several β-diketonate vanadyl complexes is examined. Vanadyl acetylacetonate, V IV O(acac) 2 , 1, shows a remarkable activity in degrading plasmid DNA in the absence of any activating agents, air and photoirradiation. The cleaving activity of several related complexes V IV O(hd) 2 (2, Hhd=3,5-heptanedione), V IV O(acac-NH 2 ) 2 (3, Hacac-NH 2 =acetoacetamide) and V IV O(acac-NMe 2 ) 2 (4, Hacac-NMe 2 =N,N-dimethylacetoacetamide) is also evaluated. It is shown that 2 exhibits an activity similar to 1, while 3 and 4 are much less efficient cleaving agents. The different activity of the complexes is related to their stability towards hydrolysis in aqueous solution, which follows the order 1~2 >> 3~4. The nature of the pH buffer was also found to be determinant in the nuclease activity of 1 and 2. In a phospate buffered medium DNA cleavage by these agents is much more efficient than in tris, hepes, mes or mops buffers. The reaction seems to take place through a mixed mechanism, involving the formation of reactive oxygen species (ROS), namely OH radicals, and possibly also direct cleavage at phosphodiester linkages induced by the vanadium complexes.

Tridentate pyrazole-containing ligands of the Schiff base type, SalPz -HL 1 , Cl 2 SalPz -HL 2 and I 2 SalPz -HL 3 , were used to prepare a series of new Cu(II) complexes (CuSalPz -1, CuCl 2 SalPz -2 and CuI 2 SalPz -3). These new complexes have been studied by different analytical techniques (electrospray ionization mass spectrometry (ESI-MS), elemental analysis, FT-IR and EPR). The spectroscopic properties of 1-3 are consistent with the formation of Cu(II) complexes coordinated by monoanionic and tridentate (N,N,O)-chelators, behaving as monomeric species in aqueous solution, as shown by EPR studies. Crystals of 2 and 3, obtained by slow concentration of methanolic solutions of the compounds, were also analyzed by X-ray diffraction analysis. The X-ray structural study has shown that 2 crystallized as a dinuclear compound, [Cu 2 (μ-Cl) 2 (Cl 2 SalPz) 2 ], while the solid state structure determined for 3 is best described by monomeric units of [CuCl (I 2 SalPz)] displaying short Cu•••Cl intermolecular contacts. The in vitro evaluation of 1-3 comprised the study of their DNA-cleaving ability using plasmid DNA and the assessment of their cytotoxic activity against several human cancer cell lines (PC-3 prostate, MCF-7 breast and A2780 and A2780cisR-ovary). The studies with plasmid DNA have shown that 2 and 3 induce extensive DNA cleavage in the presence of different additives. The cytotoxic activity of 2 and 3 is comparable to the one presented by cisplatin, with the exception of the A2780 cell line where cisplatin is more active. It has been found that the introduction of halogen substituents in the phenolate rings of the chelators enhanced the cytotoxicity of the respective Cu(II) complexes.

Schiff base obtained by condensing salicylaldehyde with 2-dimethylamino-ethylamine in 1: 1 molar ratio readily forms a bis-complex with cupric perchlorate hexahydrate in methanol in the presence of KOH. The complex crystallizes from methanol in the monoclinic space group P2l/c with a = 11.221 (3), b = 19.462(2), c = 13.062(3), ~= 109.36(4)°, Z= 4, R = 0.050, R' = 0.045 and goodness-of-fit = 2.551.

Fluorescent double-stranded DNA (dsDNA) molecules labeled at both ends are commonly produced by annealing of complementary single-stranded DNA (ssDNA) molecules, labeled with fluorescent dyes at the same (3′ or 5′) end. Because the labeling efficiency of ssDNA is smaller than 100%, the resulting dsDNA have two, one or are without a dye. Existing methods are insufficient to measure the percentage of the doubly-labeled dsDNA component in the fluorescent DNA sample and it is even difficult to distinguish the doubly-labeled DNA component from the singly-labeled component. Accurate measurement of the percentage of such doubly labeled dsDNA component is a critical prerequisite for quantitative biochemical measurements, which has puzzled scientists for decades. We established a fluorescence correlation spectroscopy (FCS) system to measure the percentage of doubly labeled dsDNA (PDL) in the total fluorescent dsDNA pool. The method is based on comparative analysis of the given sample and a reference dsDNA sample prepared by adding certain amount of unlabeled ssDNA into the original ssDNA solution. From FCS autocorrelation functions, we obtain the number of fluorescent dsDNA molecules in the focal volume of the confocal microscope and PDL. We also calculate the labeling efficiency of ssDNA. The method requires minimal amount of material. The samples have the concentration of DNA in the nano-molar/L range and the volume of tens of microliters. We verify our method by using restriction enzyme Hind III to cleave the fluorescent dsDNA. The kinetics of the reaction depends strongly on PDL, a critical parameter for quantitative biochemical measurements.

The cancer chemotherapeutic potential of surfactant–cobalt(III) complexes, cis‐[Co(bpy)2(C14H29NH2)Cl](ClO4)2 · 3 H2O (1) and cis‐[Co(phen)2(C14H29NH2)Cl](ClO4)2 · 3 H2O (2) (bpy = 2,2′‐bipyridine, phen = 1,10‐phenanthroline) on MCF‐7 breast cancer cell was determined adopting MTT assay and specific staining techniques. The complexes affected the viability of the cells significantly and the cells succumbed to apoptosis as seen in the changes in the nuclear morphology and cytoplasmic features. Since the complex 2 appeared to be more potent, further assays were carried out on the complex 2. Single‐cell electrophoresis indicated DNA damage. The translocation of phosphatidyl serine and loss of mitochondrial potential was revealed by annexin V‐Cy3 staining and JC‐1 staining respectively. Western blot analysis revealed up‐regulation of pro‐apoptotic p53 and down‐regulation of anti‐apoptotic Bcl‐2 protein. Taken together, the surfactant–cobalt(III) complex 2 would be a potential candidate ...

Metal complexes of the type Mn(bpy) 2 (N 3 ) 2 (1), Co(bpy) 2 (N 3 ) 2 .3H 2 O (2) and Zn 2 (bpy) 2 (N 3 ) 4 (3) (Where bpy = 2,2-bipyridine) have been synthesized and characterized by elemental analysis and spectral (FT-IR, UV-vis) studies. The structure of complexes (1-3) have been determined by single crystal X-ray diffraction studies and the configuration of ligandcoordinated metal(II) ion was well described as distorted octahedral coordination geometry for Mn(II), Co(II) and distorted square pyramidal geometry for Zn(II) complexes. DNA binding interaction of these complexes (1-3) were investigated by UV-vis absorption, fluorescence circular dichroism spectral and molecular docking studies. The intrinsic binding constants K b of complexes 1, 2 and 3 with CT-DNA obtained from UV-vis absorption studies were 8.37x10 4 , 2.23x10 5 and 5.52x10 4 M -1 respectively. The results indicated that the three complexes are able to bind to DNA with different binding affinity, in the order 2 > 1 > 3. Complexes (1-3) exhibit a good binding propensity to bovine serum albumin (BSA) proteins having relatively high binding constant values. Gel electrophoresis assay demonstrated the ability of the complexes 1 -3 promote the cleavage ability of the pBR322 plasmid DNA in the presence of the reducing agent 3-mercaptopropionic acid (MPA) but with different cleavage mechanisms: the complex 3 cleaves DNA via hydrolytic pathway (T4 DNA ligase assay), while the DNA cleavage by complexes 1 and 2 follows oxidative pathway. The chemical nuclease activity follows the order: 2 > 1 > 3.

Accepted on 8 May, 2019 __________________________________________________________________________________________ ABSTRACT Thermal decomposition of ammonium per chlorate in the presence of lanthanide (Ln) nitrates, where Ln = La, Ce, Nd, Sm, and Eu is studied. The analytical techniques of mass spectrometry (MS), thermo gravimetric (TG), and differential Scanning Calorimetry (DSC) have been employed. In all the cases of Ammonium Per chlorate (AP)-Lanthanide nitrate mixtures, the major product evolved is H2O.Besides H2O, HCl, and NH3 are common products released. N2O, Cl2, and O2 have not been observed in the case of pure AP. In the case of AP-Samarium nitrate system NO is not observed, and O2is the other major product evolved. The cessation of decomposition of AP after an initial conversion of 30 percent is modified in the presence of nitrates of La, Nd, Sm, and Eu in the range between 35 % and 50%. In terms of heat release, Samarium nitrate gives better energy output (1207 J g).

ST1481 is the lead compound of a novel series of 7-modified camptothecins, the 7-oxyimino methyl derivatives, characterized by potent topoisomerase I inhibition and cytotoxic activity. Based on its therapeutic efficacy in a human non-small cell lung carcinoma model and its favorable pharmacological profile, the novel analogue was selected for further preclinical development. We investigated the growth-inhibitory effects of ST1481 and topotecan, used as a reference compound, in a panel of human tumor cell lines of various tumor types (ovarian carcinoma, glioblastoma, osteosarcoma, and melanoma), including sublines with acquired resistance to cisplatin. We explored the antitumor efficacy in a large panel of human tumor xenografts, with particular reference to intrinsically resistant tumor types, using oral administration and an intermittent treatment schedule. ST1481 showed a potent antiproliferative activity with comparable effects in all tested cell lines. Only U-87-MG glioma cells ...

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Despite the success of antiretroviral therapy (ART), HIV remains incurable due to the persistence of latent viral reservoirs that evade immune clearance and therapy. Current strategies targeting latency, such as "shock-and-kill" and "block-and-lock," have shown limited success and safety concerns. We propose a novel phased gene therapy approach using engineered HIV-derived lentiviral vectors to target both actively replicating and latent HIV. In the first phase, a Therapeutic Interfering Particle (TIP) or antiviral gene cassette is delivered to suppress active HIV replication. In the second phase, following suppression, a latency-reversing or cytotoxic payload is introduced via a second vector to reactivate and eliminate reservoir cells. This staged model leverages the natural tropism and replication biology of HIV to deliver therapy precisely and sequentially. If validated, this strategy could offer a path toward a functional or sterilizing cure for HIV infection.

Analogues of the tripyrrolic natural product prodigiosin bearing an additional methyl and a carbonyl group at the C-ring were synthesised and evaluated. In vitro anticancer activity screening (NCI) and the study of modes of action (copper-mediated cleavage of double-stranded DNA and transmembrane transport of chloride anions) showed that the presence of the methyl group is not detrimental to activity. Furthermore, although the presence of an ester conjugated to the prodigiosene C-ring seems to decrease both pK a and chloride transport efficiency compared to the natural product, these analogues still exhibit a high rate of chloride transport. All analogues exhibit good in vitro anticancer activity and reduced toxicity compared to the natural product: compare an acute systemic toxicity of 100 mg kg -1 in mice vs. 4 mg kg -1 for prodigiosin, pointing towards a larger therapeutic window than for the natural product.

Homing endonucleases, also known as meganucleases, are sequence-specific enzymes with large DNA recognition sites. These enzymes can be used to induce efficient homologous gene targeting in cells and plants, opening perspectives for genome engineering with applications in a wide series of fields, ranging from biotechnology to gene therapy. Here, we report the crystal structures at 2.0 and 2.1 Å resolution of the I-DmoI meganuclease in complex with its substrate DNA before and after cleavage, providing snapshots of the catalytic process. Our study suggests that I-DmoI requires only 2 cations instead of 3 for DNA cleavage. The structure sheds light onto the basis of DNA binding, indicating key residues responsible for nonpalindromic target DNA recognition. In silico and in vivo analysis of the I-DmoI DNA cleavage specificity suggests that despite the relatively few protein-base contacts, I-DmoI is highly specific when compared with other meganucleases. Our data open the door toward th...

DNA Binding Proteins.............................................................................................................................................14 Termination of replication in prokaryotes........................................................................................................14 Eukaryotic ribosomal barriers............................................................................................................................16

Sepantronium bromide (YM155) is a small molecule antitumor agent currently in phase II clinical trials. Although developed as survivin suppressor, YM155's primary mode of action has recently been found to be DNA damage. However, the mechanism of DNA damage by YM155 is still unknown. Knowing the mechanism of action of an anticancer drug is necessary to formulate a rational drug combination and select a cancer type for achieving maximum clinical efficacy. Using cell-based assays, we showed that YM155 causes extensive DNA cleavage and reactive oxygen species generation. DNA cleavage by YM155 was found to be inhibited by radical scavengers and desferal. The reducing agent DTT and the cellular reducing system xanthine/xanthine oxidase were found to reductively activate YM155 and cause DNA cleavage. Unlike quinones, DNA cleavage by YM155 occurs in the presence of catalase and under hypoxic conditions, indicating that hydrogen peroxide and oxygen are not necessary. Although YM155 is a ...

DNA binding properties of two previously synthesized copper complexes of vic-dioximes bearing thiosemicarbazone units (2E)-2-[4-(dimethylamino)benzylidene]-N-[(1Z,2E)-N-hydroxy-2-(hydroxyimino)ethanimidoyl]hydrazine carbothioamide (1) and (2E)-2-[4-(diethylamino)benzylidene]-N-[(1Z,2E)-N-hydroxy-2-(hydroxyimino)ethanimidoyl] hydrazine carbothioamide (2), were investigated using absorption spectroscopy, fluorescence spectroscopy, and agarose gel electrophoresis methods. Experimental studies suggested that the complexes bind to DNA through intercalation. Their intrinsic binding constants (Kb) were calculated as 1: 5.50 ± 0.25 × 104 M−1; 2: 2.10 ± 0.18 × 105 M−1. These complexes also promote the cleavage of plasmid pBR322, both in the absence and presence of hydrogen peroxide.

To date, DNA cleavage, caused by cleavage agents, has been monitored mainly by gel and capillary electrophoresis. However, these techniques are time-consuming, non-quantitative and require gel stains. In this work, a novel, simple and, importantly, a quantitative method for monitoring the DNA nuclease activity of potential anti-cancer drugs, at a DNA electrochemical sensor, is presented. The DNA sensors were prepared using thiol-modified oligonucleotides that self-assembled to create a DNA monolayer at gold electrode surfaces. The quantification of DNA double-strand breaks is based on calculating the DNA surface coverage, before and after exposure to a DNA cleavage agent. The nuclease properties of a model DNA cleavage agent, copper bis-phenanthroline ([Cu(phen)]), that can cleave DNA in a Fenton-type reaction, were quantified electrochemically. The DNA surface coverage decreased on average by 21% after subjecting the DNA sensor to a nuclease assay containing [Cu(phen)], a reductant...

Toward developing antileishmanial agents with mode of action targeted to DNA topoisomerases of Leishmania donovani, we have synthesized a large number of derivatives of betulin. The compound, a natural triterpene isolated from the cork layer of Betula spp. plants exhibits several pharmacological properties. Three compounds (disuccinyl betulin, diglutaryl dihydrobetulin, and disuccinyl dihydrobetulin) inhibit growth of the parasite as well as relaxation activity of the enzyme type IB topoisomerase [Leishmania donovani topoisomerase I (LdTOP1LS)] of the parasite. Mechanistic studies suggest that these compounds interact with the enzyme in a reversible manner. The stoichiometry of these compounds binding to LdTOP1LS is 1:1 (mole/mole) with a dis-sociation constant on the order of ϳ10 Ϫ6 M. Unlike CPT, these compounds do not stabilize the cleavage complex; rather, they abrogate the covalent complex formation. In processive mode of relaxation assay condition, these compounds slow down the strand rotation event, which ultimately affects the relaxation of supercoiled DNA. It is noteworthy that these compounds reduce the intracellular parasite burden in macrophages infected with wild-type L. donovani as well as with sodium antimony gluconate resistant parasite (GE1). Taken together, our data suggest that these betulin derivatives can be exploited as potential drug candidates against threatening drug resistant leishmaniasis.

This thesis discusses the construction and DNA-binding properties of homo-and heterometallic, oligonuclear complexes containing the [Ru(tpm)(dppz)] 2+ moiety. Specifically it explores how the nature of the tether affects the binding properties of these systems. Towards these goals, four new connecting ligands that possess potential DNA recognition sites in themselves have been prepared. The ligands have also been chosen to investigate the effects of changes in connectivity and linker rigidity on the binding properties of these metallo-intercalators. A series of mononuclear complexes incorporating the new Undertaking this PhD has been a truly life-changing experience for me and it would not have been possible to do without the support and guidance that I received from many people. I would like to express my special appreciation and thanks to my supervisor Professor Jim Thomas-you have been a tremendous mentor for me. I would like to thank you for encouraging my research and for allowing me to grow as a research scientist. Your advice on both my research and my career has been priceless. I am grateful for the support and guidance given to me by Dr. Niek Buurma for his help and patience in understanding ITC and also for allowing me to work in his research facility in Cardiff University. Also I would like to acknowledge the help of Dr. Andrea and Dr Jenny in the Staniland group for allowing me to do DNA photocleavage experiments in their lab'. A special thank you to all the technical staff at the University for their continued help and support. In the Chemistry department itself, my thanks go out to Sue and Dr Sandra van Meurs in NMR, Sharon and Simon in Mass spec., Jenny in Elemental, Harry in X-Ray and for the technical support of Keith and Stephen Atkin. Thank you to Dan in the glass workshop for constantly creating works of art to be used for scientific purposes in our lab. I would like to thank the gents in stores, Pete and Nick, as well as the ladies in accounts, Denise, Rachel and Louise. You all make the department a nice place to be around. -Caroline, Sasha, and Rachel, as well as the gents-Mike, Stuart, Tom, Sree, Sam, Shahryar, and Simon. I would like to thank them all for their friendship and the warmth they extended to me during my PhD, and for always being there and bearing with me during the ups and downs of life over the last 4 years. I could not have asked for more than I got from them. To Paul Jarman-from the day I began my PhD, you have become one of my best friends. We have seen and helped each other through both good times and bad. I will always remember the great conversations we had about premier league football, feeding birds in Western Park, and playing pool in the common room etc. I know that we will break bread together many more times. Jack and Claire's encouragement and love have made this journey bearable, and no amount of thanks will ever be enough to return that joy. V John Lennon said it best: "I get by with a little help from my friends" From my time in Sheffield, I want to thank my remarkable friends Ashley,

Schiff bases are one of the important class of organic ligands, which are obtained by the condensation of aldehydes/ ketones with primary amines. The metal complexes of Schiff bases are widely studied because they possess very good biological applications such as antimicrobial, larvicidal, antioxidant, antitubercular, antidiabetic, anti-inflammatory and antitumour properties. The present work deals with synthesis and characterization of a series of Schiff base transition metal complexes obtained from amino acid with general formula [ML 1 L 2 ](ClO 4) [where, M = Co 2+ , Cu 2+ , Ni 2+ and Zn 2+ ; L 1 = Schiff base ligand derived from L-methionine and cuminaldehyde; L 2 = N,N,N',N'-tetramethyl-1,3diaminopropane]. The synthesised complexes were characterized by physicochemical and spectral analyses such as UV-Vis., FTIR, GC-MS and ESR. They were also tested for in vitro biological activities. The FTIR spectra confirmed the bidentate coordination nature of the Schiff base via imine nitrogen and one of the oxygen atom of carboxylate group. The in vitro antibacterial studies were carried out against S. Aureus, E. Coli, P. aeruginosa, Bascillus as well as antifungal studies against A. flavus, Candida, Rhizhopus and A. Niger. Larvicidal activity was performed against Culex quinquefasciatus and antioxidant activity of the complexes was studied by H 2 O 2 and DPPH method. The results proved that the complexes have good biological activities.

Homing endonucleases, also known as meganucleases, are sequencespecific enzymes with large DNA recognition sites. These enzymes can be used to induce efficient homologous gene targeting in cells and plants, opening perspectives for genome engineering with applications in a wide series of fields, ranging from biotechnology to gene therapy. Here, we report the crystal structures at 2.0 and 2.1 Å resolution of the I-DmoI meganuclease in complex with its substrate DNA before and after cleavage, providing snapshots of the catalytic process. Our study suggests that I-DmoI requires only 2 cations instead of 3 for DNA cleavage. The structure sheds light onto the basis of DNA binding, indicating key residues responsible for nonpalindromic target DNA recognition. In silico and in vivo analysis of the I-DmoI DNA cleavage specificity suggests that despite the relatively few protein-base contacts, I-DmoI is highly specific when compared with other meganucleases. Our data open the door toward the generation of custom endonucleases for targeted genome engineering using the monomeric I-DmoI scaffold. gene targeting ͉ genetics ͉ protein-DNA interactions ͉ X-ray crystallography Author contributions: M.

A novel heterobinuclear mixed valence complex [Fe III Cu II (BPBPMP)(OAc) 2 ]ClO 4 , 1, with the unsymmetrical N 5 O 2 donor ligand 2-bis[{(2-pyridylmethyl)aminomethyl}-6-{(2hydroxybenzyl)(2-pyridylmethyl)}aminomethyl]-4-methylphenol (H 2 BPBPMP) has been synthesized and characterized. A combination of data from mass spectrometry, potentiometric titrations, X-ray absorption and electron paramagnetic resonance spectroscopy, as well as kinetics measurements indicates that in ethanol/water solutions an [Fe III -(µ)OH-Cu II OH 2 ]+ species is generated which is the likely catalyst for 2,4-bis(dinitrophenyl)phosphate and DNA hydrolysis. Insofar as the data are consistent with the presence of an Fe III -bound hydroxide acting as a nucleophile during catalysis, 1 presents a suitable mimic for the hydrolytic enzyme purple acid phosphatase. Notably, 1 is significantly more reactive than its isostructural homologues with different metal composition (Fe III M II , where M II is Zn II , Mn II , Ni II , or Fe II ). Of particular interest is the observation that cleavage of double-stranded plasmid DNA occurs even at very low concentrations of 1 (2.5 µM), under physiological conditions (optimum pH of 7.0), with a rate enhancement of 2.7×10 7 over the uncatalyzed reaction. Thus, 1 is one of the most effective model complexes to date, mimicking the function of nucleases.

Described herein is the synthesis, structure, and monoesterase and diesterase activities of a new mononuclear (2,4-BDNPP). When covalently linked to 3-aminopropyl-functionalized silica, 1 undergoes disproportionation to form a dinuclear species (APS-1), whose catalytic efficiency is increased when compared to the homogeneous reaction due to second coordination sphere effects which increase the substrate to complex association constant. The anchored catalyst APS-1 can be recovered and reused for subsequent hydrolysis reactions (five times) with only a slight loss in activity. In the presence of DNA, we suggest that 1 is also converted into the dinuclear active species as observed with APS-1, and both were shown to be efficient in DNA cleavage.

Background: The development of 3, 39-diindolyl methane (DIM) resistant parasite Leishmania donovani (LdDR50) by adaptation with increasing concentrations of the drug generates random mutations in the large and small subunits of heterodimeric DNA topoisomerase I of Leishmania (LdTOP1LS). Mutation of large subunit of LdTOP1LS at F270L is responsible for resistance to DIM up to 50 mM concentration. Methodology/Principal Findings: In search of compounds that inhibit the growth of the DIM resistant parasite and inhibit the catalytic activity of mutated topoisomerase I (F270L), we have prepared three derivatives of DIM namely DPDIM (2,29diphenyl 3,39-diindolyl methane), DMDIM (2,29-dimethyl 3,39-diindolyl methane) and DMODIM (5,59-dimethoxy 3,39diindolyl methane) from parent compound DIM. All the compounds inhibit the growth of DIM resistant parasites, induce DNA fragmentation and stabilize topo1-DNA cleavable complex with the wild type and mutant enzyme. The results suggest that the three derivatives of DIM can act as promising lead molecules for the generation of new anti-leishmanial agents.

The interactions and the photosensitizing activity of three antimalarial drugs quinine (Q), mefloquine (MQ) and quinacrine (QC) toward DNA was studied. Evidences obtained by absorption and emission spectroscopy and by linear dichroism measurements indicate that these derivatives bind the macromolecule with a high affinity (binding constants K a $ 10 5 M )1 ). The absorption characteristics of the drugs changed markedly by addition of DNA and their fluorescence was quenched with rate constants higher than that of diffusion. The geometry of binding involves predominantly the intercalation into the double helix. The DNA photocleavage properties of antimalarials was investigated using plasmid DNA as a model, at different [drug] ⁄ [DNA] ratios. The results indicate that mainly MQ and Q are able to induce significant photodamage to DNA. In particular the marked effect of the former drug is evidenced after treatment of photosensitized DNA by two base excision repair enzymes, formamydo-pyrimidine glycosilase (Fpg) and Endonuclease III (Endo III). From a mechanistic point of view, experiments carried out in different experimental conditions indicate that these drugs photoinduce DNA damage through singlet oxygen and ⁄ or radical cation production. These findings are further supported by the determination of two photoproducts of 2¢-deoxyguanosine, which are diagnostic for Type I and Type II pathways, namely 2,2-diamino(2-deoxy-b-D D-erythro-pentofu- ranosyl)-4-amino-5(2H)-oxazolone and (R,S)4-hydroxy-8-oxo-4,8-dihydro-2¢-deoxyguanosine (4-OH-8-oxo-dGuo). Laser flash photolysis experiments carried out in the presence of DNA indicates that the excitation produces mainly the triplet state for Q and the triplet and radical cation for QC. Moreover the singlet and triplet states and radical cations of the drugs are quenched by 2¢-deoxyguanosine monophosphate. The absorbances of these transients decrease with increasing DNA concentration.

DNA double-strand breaks (DSBs) activate a DNA damage response (DDR) that coordinates checkpoint pathways with DNA repair. ATM and ATR kinases are activated sequentially. Homologydirected repair (HDR) is initiated by resection of DSBs to generate 3 0 single-stranded DNA overhangs. How resection and HDR are activated during DDR is not known, nor are the roles of ATM and ATR in HDR. Here, we show that CtIP undergoes ATR-dependent hyperphosphorylation in response to DSBs. ATR phosphorylates an invariant threonine, T818 of Xenopus CtIP (T859 in human). Nonphosphorylatable CtIP (T818A) does not bind to chromatin or initiate resection. Our data support a model in which ATM activity is required for an early step in resection, leading to ATR activation, CtIP-T818 phosphorylation, and accumulation of CtIP on chromatin. Chromatin binding by modified CtIP precedes extensive resection and full checkpoint activation.

Three new transition metal (Co(II), Ni(II) and Cu(II)) complexes of Schiff's base ligand were prepared in situ by refluxing stoichiometric amounts of respective metal salts with 2-hydroxy-1-naphthaldehyde and 2-picolylamine. The structural feature of the synthesized complexes was derived from spectral (FT-IR, UV-Vis and ESImass) studies and elemental analysis. The single crystal X-ray diffraction analysis of complex 3 showed the structure as dimeric form [Cu(2-hnppa)Cl] 2 and each copper atom is penta-coordinated. The calculated τ value of [Cu(2-hnppa)Cl] 2 was 0.25 (where α = 157.84 and β = 172.18) indicating the square pyramidal geometry. Binding of these complexes (1-3) with calf thymus DNA was investigated by UV-Vis, fluorescence, viscosity measurements and computer aided molecular docking studies and the K b value of complexes 1-3 were 2.4 × 10 5 , 5.9 × 10 5 and 2.0 × 10 4 M -1 , respectively. The relative binding energies of the metal complexes 1-3 docked with DNA were -151.82, -151.86 and -151.80 eV, respectively. Complexes (1-3) exhibited a good binding capability with bovine serum albumin (BSA) with relatively high binding constant values. The complexes also act as a catalyst that enables efficient aerobic oxidation of a broad range of alcohols (benzyl alcohol, n-octanol) to the corresponding aldehydes at room temperature with ambient air as the oxidant.

The combination of yeast surface display and flow cytometric analyses and selections is being used with increasing frequency to alter specificity of macromolecular recognition, including both protein-protein and protein-nucleic acid interactions. Here we describe the use of yeast surface display and cleavage-dependent flow cytometric assays to increase the specificity of an engineered meganuclease. The re-engineered meganuclease displays a significantly tightened specificity profile, while binding its cognate target site with a slightly lower, but still sub-nanomolar affinity. When incorporated into otherwise identical megaTAL protein scaffolds, these two nucleases display significantly different activity and toxicity profiles in cellulo. The structural basis for reprogrammed DNA cleavage specificity was further examined via high-resolution X-ray crystal structures of both enzymes. This analysis illustrated the altered protein-DNA contacts produced by mutagenesis and selection, that...

A new ruthenium polypyridyl complex, [Ru(bpy) 2 (dpqp)] 2þ (bpy = 2,2 0 -bipyridne; dpqp = pyrazino[2 0 ,3 0 :5,6]pyrazino-[2,3-f][1,10]phenanthroline), shows strong luminescence in water at room temperature, a behavior that is strikingly different from that of the nonemissive "DNA light-switch" prototype [Ru(bpy) 2 (dppz)] 2þ (dppz = dipyrido[3,2-a:2 0 -3 0 -c]phenazine) under similar conditions. Variation of the absorption and emission spectra of [Ru(bpy) 2 (dpqp)] 2þ as a function of the pH is consistent with the occurrence of two ground-state protonation steps associated with the dpqp ligand and an apparent pK a * of 2.1. Electrochemistry and theoretical calculations indicate that the lowest unoccupied molecular orbital (LUMO) of [Ru(bpy) 2 (dpqp)] 2þ is localized on the distal portion of the dpqp ligand and lies at a lower energy than the dppz-based LUMO of [Ru(bpy) 2 (dppz)] 2þ . The combination of its strong DNA binding affinity and relatively long-lived triplet metal-to-ligand charge-transfer excited state in an aqueous solution results in more efficient DNA photocleavage by [Ru(bpy) 2 (dpqp)] 2þ than [Ru(bpy) 2 (dppz)] 2þ .

During V(D)J recombination, the RAG1/2 recombinase is thought to play an active role in transferring newly excised recombination ends from the RAG post-cleavage complex (PCC) to the non-homologous end joining (NHEJ) machinery to promote appropriate antigen receptor gene assembly. However, this transfer mechanism is poorly understood, partly because of the technical difficulty in revealing weak association of coding ends (CEs) with one of the PCCs, coding end complex (CEC). Using fluorescence resonance energy transfer (FRET) and anisotropy measurement, we present here real-time monitoring of the RAG1/2-catalyzed cleavage reaction, and provide unequivocal evidence that CEs are retained within the CEC in the presence of Mg 2+ . By examining the dynamic fluorescence changes during the cleavage reaction, we compared the stability of CEC assembled with core RAG1 paired with full-length RAG2, core RAG2 or a frameshift RAG2 mutant that was speculated to destabilize the PCC, leading to increased aberrant joining. While the latter two CECs exhibit similar stability, the full-length RAG2 renders a less stable CEC unless H3K4me3 peptides are added. Interestingly, the RAG2 mutant appears to modulate the structure of the RAG-12RSS pre-cleavage complex. Thus, the fluorescence-based detection offers a sensitive, quantitative and continuous assessment of pre-cleavage complex assembly and CEC stability.

The common mismatch repair system processed by MutS and MutL and their homologs was identified in Bacteria and Eukarya. However, no evidence of a functional MutS/L homolog has been reported for archaeal organisms, and it is not known whether the mismatch repair system is conserved in Archaea. Here, we describe an endonuclease that cleaves double-stranded DNA containing a mismatched base pair, from the hyperthermophilic archaeon Pyrococcus furiosus. The corresponding gene revealed that the activity originates from PF0012, and we named this enzyme Endonuclease MS (EndoMS) as the mismatch-specific Endonuclease. The sequence similarity suggested that EndoMS is the ortholog of NucS isolated from Pyrococcus abyssi, published previously. Biochemical characterizations of the EndoMS homolog from Thermococcus kodakarensis clearly showed that EndoMS specifically cleaves both strands of double-stranded DNA into 5protruding forms, with the mismatched base pair in the central position.

A variety of active chemicals found in medicinal plants can be used to develop new medications with few adverse effects. In vitro and in silico analyses were used to evaluate the anticancer properties of Juniperus procera fruit and leaf extracts. Here, we show that the methanolic extract from J procera fruit and leaf extracts inhibits 2 human ovarian cancer cell lines, A2780CP and SKOV-3. The leaf extract demonstrated strong cytotoxicity against A2780CP with an IC50 of 1.2 μg/mL, almost matching the IC50 of the anticancer medication doxorubicin (0.9 μg/mL). Higher antioxidant activity was observed in the fruit than leaf extract. The molecular docking results showed that the active component, podocarpusflavone A, was the best-docked chemical with the human topoisomerase II alpha enzyme. According to our knowledge, this is the first in vitro study to show the cytotoxicity of J procera extracts against the 2 previously described human ovarian cancer cell lines. The fact that the podocarpusflavone A molecule may have an inhibitory effect on the human topoisomerase II alpha enzyme was also revealed by this first in silico analysis. Our findings imply that the J procera fruit and leaf methanolic extract has anticancer characteristics that may guide future in vivo studies.

Novel bacterial type II topoisomerase inhibitors (NBTIs) stabilize single-strand DNA cleavage breaks by DNA gyrase but their exact mechanism of action has remained hypothetical until now. We have designed a small library of NBTIs with an improved DNA gyrase-binding moiety resulting in low nanomolar inhibition and very potent antibacterial activity. They stabilize single-stranded cleavage complexes and, importantly, we have obtained the crystal structure where an NBTI binds gyrase–DNA in a single conformation lacking apparent static disorder. This directly proves the previously postulated NBTI mechanism of action and shows that they stabilize single-strand cleavage through asymmetric intercalation with a shift of the scissile phosphate. This crystal stucture shows that the chlorine forms a halogen bond with the backbone carbonyls of the two symmetry-related Ala68 residues. To the best of our knowledge, such a so-called symmetrical bifurcated halogen bond has not been identified in a ...

Regioisomers of the functional group of the main ligand (L) on a series of [Ru(phen) 2 L] 2+ and [Ru(bpy) 2 L] 2+ complexes, where phen is 1,10 phenanthroline and bpy is 2,2′-bipyridine, were synthesised to investigate the interaction with deoxyribonucleic acid (DNA) as potential therapeutics. UV-Vis binding titrations, thermal denaturation and circular dichroism were used to evaluate their interaction with DNA. The conclusions indicated the significance of the auxiliary ligand; especially 1,10-phenanthroline has on the binding constants (K b ). The systematic variation of auxiliary ligand(phen or bpy), and polypyridyl ligand (4 , split in terms of functional group change were investigated for DNA interaction. The CPIP analogues in particular were investigated for the regioisomerism (ortho, meta, para) effect of the nitrile group on the ligand. It was found that both the DNA interaction could be tailored through the systematic variation of the electronic nature of the individual auxiliary ligand and to a lesser extent the functional group and regioisomeric change. Preliminary cell line studies have been carried out to determine the selectivity of the complexes against cell lines such as A375 (Skin Cancer), HeLa (Cervical Cancer), A549 (Lung Cancer), Beas2B (Lung Normal Cell) and . Complexes which had strong DNA interactions in the binding studies have proven to be the most efficacious against certain cell lines. Establishing well-defined structure property relationships when looking at trends in spectroscopic properties and DNA binding will aid in the intelligent design of potential therapeutic complexes.

The human DNA replication origin, located in the lamin B2 gene, interacts with the DNA topoisomerases I and II in a cell cycle-modulated manner. The topoisomerases interact in vivo and in vitro with precise bonds ahead of the start sites of bidirectional replication, within the prereplicative complex region; topoisomerase I is bound in M, early G1 and G1/S border and topoisomerase II in M and the middle of G1. The Orc2 protein competes for the same sites of the origin bound by either topoisomerase in different moments of the cell cycle; furthermore, it interacts on the DNA with topoisomerase II during the assembly of the pre-replicative complex and with DNA-bound topoisomerase I at the G1/S border. Inhibition of topoisomerase I activity abolishes origin firing. Thus, the two topoisomerases are closely associated with the replicative complexes, and DNA topology plays an essential functional role in origin activation.

Three Cu II complexes of bis-pyrazole based ligands have been synthesized and structurally characterized by X-ray crystallography. One of the ligand (L2) contains a methionine ester conjugated to a bis-pyrazole carboxylate through an amide linkage. The binding constant for complexes 1-3 with CT DNA are of the order of 10 4 M -1 . The crystal structure suggests that the axial Cu-O bonds (ca. 2.31(4) Å) are relatively labile and hence during the redox cycle with ascorbic acid and oxygen one or both the axial Cu-O bonds might open to promote copper oxygen reaction and generate ROS. The chemical nuclease activity of complexes 1-3 in dark, show complete relaxation of supercoiled DNA at 100 μM concentration in presence of ascorbic acid (H 2 A). The mechanistic investigation suggests that the complexes 1 and 2 show involvement of peroxo species whereas 3 shows involvement of both singlet oxygen and peroxo species in DNA cleavage. The singlet oxygen formation in dark is otherwise unfavourable but the presence of methionine as pendant arm in L2 might activate the generation of singlet oxygen from the metal generated peroxo species. The results of DNA cleavage studies suggest that methionine based copper(II) complexes can promote dual pathway for DNA cleavage. Probing the cytotoxic activity of these complexes on MCF-7, human breast cancer cell line shows that 3 is the most effective one with an IC 50 of 70(2) μM.

In the anticancer activity studies, Cu(II) complexes have been considered as the best alternative to cis-platin due to their biocompatibility and significant functions in biological systems. Synthesis of nanoparticles from the Schiff base transition metal complexes was one of the most suitable methods, because the products obtained were of good purity and with perfect structure. Based on this, herein, we have reported the synthesis of two CuO Nps from two Copper Schiff base complexes by the thermal decomposition method. The Schiff base complexes were characterized by FTIR, UVvis, 1 H NMR, PXRD, EPR spectra, and TGA studies. The CuO Nps synthesized were characterized by FTIR, XRD, SEM, and EDX analyses. All the studies confirm the formation of the CuO nanoparticles. The Cu(II) complexes as well as the CuO Nps were analyzed for the antibacterial and anticancer studies. Studies indicate the high biological activity of the Cu(II) complexes than the CuO Nps. The decrease in activity of the CuO Nps may be due to the diminishing size of the particles in complexes.