DNA Cleavage Assay Research Articles

System for Rapid and Reproducible Identification of MLL-AF9 Translocations Induced by Dietary Compounds and Nutritional Supplements

AbstractAbstract 1275▪▪This icon denotes a clinically relevant abstractInfant acute leukemias account for ∼30% of all malignancy seen in childhood across the Western world. They are aggressive and characterized by rapid onset shortly after birth. The majority of these (∼80% ALL and ∼60% AML) have rearrangements involving the MLL gene. Although MLL fusion to more than 75 genes have been identified, AF9 is one of its most common translocation partners. MLL breakpoint sequences associated with infant acute leukemia are similar to those in secondary AML following exposure to the topoisomerase II (topoII) poison etoposide. This similarity led to the hypothesis that exposure during preganancy to biochemically similar compounds may promote infant acute leukemia. Some studies have shown an epidemiological link between bioflavonoid intake and increased incidence of MLL-rearranged infant leukemias. These bioflavonoids have also been shown to inhibit topoII in in vitro DNA cleavage assays and produce MLL rearrangements by inverse PCR in mice. Hundreds of unregulated nutritional supplements are widely available and perceived to prevent cardiovascular disease, inflammation, and cancer; however, their potential to promote leukemic translocations should be determined. Our goal was to create a model system that allows for rapid quantifiable screening of a large number of compounds to determine their potential to promote MLL-AF9 bcr translocations, and does not rely on inverse PCR that requires elimination of artifacts or less physiologically relevant internal deletions or intronic alterations from analysis. Thus, we developed reporter stem cell lines that contain two transgene constructs—(1) the MLL bcr fragment containing a genetically-engineered GFP 5’ exon, and (2) the AF9 bcr containing a genetically-engineered GFP 3’ exon. A translocation between the two bcrs reconstitutes the full-length GFP transcript resulting in quantifiable green fluorescence. Cells were treated with etoposide, quercetin, genestein, luteolin, dipyrone, or benzoquinone for 1 hour at 25mM – 200mM concentrations then allowed to repair and proliferate in culture. GFP+ fluorescent colonies as a result of MLL-AF9 translocations were readily scorable by 96 hours in a dose-dependent manner. [Display omitted] [Display omitted] [Display omitted] As previously demonstrated, totipotent stem cell viability was extremely impaired by multiple concentrations of etoposide, but the surviving fraction of hematopoietic stem cells that differentiated into multiple lineage sub-types exhibited a significant number of MLL-AF9 translocations (frequency of 1.5×10−4). Bioflavonoids genistein and quercetin (75mM) were also potent promoters of MLL-AF9 translocations in totipotent stem cells and hematopoietic stem cells at roughly similar frequencies as etoposide. By contrast, more differentiated hematopoietic progenitor cells had a significantly decreased (3-log) number of colonies with MLL-AF9 translocations following exposure to multiple compounds. This decrease is likely due to differences in DNA damage and repair responses or the potential of cells carrying translocations to proliferate in culture. The accuracy and significance of this model system is apparent from treatment of stem cells with benzoquinone that was not sufficient to produce MLL-AF9 translocations following exposure to concentrations up to 125mM (frequency < 0.1×10−6). Benzoquinone is a non-bioflavonoid thought to have a distinct mechanism of action and clinically associated with multiple leukemias. This system is a direct measure of the sensitivity of the MLL and AF9 bcrs to topoII poisons and bioflavonoids independent of their normal chromatin context and independent of formation of a leukemic fusion protein. In addition, the system allows for rapid and reproducible screening of hundreds of compounds that may have the potential to promote leukemogenic translocations in early stem cell and more differentiated cell subpopulations analogous to the events observed in infant acute leukemias. Disclosures:No relevant conflicts of interest to declare.

G-quadruplex DNA cleavage preference and identification of a perylene diimide G-quadruplex photocleavage agent using a rapid fluorescent assay

A rapid fluorescence assay for G-quadruplex DNA cleavage was used to investigate the preference of TMPyP4 photochemical and Mn·TMPyP4 oxidative cleavage. Both agents most efficiently cleave the c-Myc promoter G-quadruplex. Direct PAGE analysis of selected assay samples showed that for a given cleavage agent, different cleavage products are formed from different G-quadruplex structures. Cleavage assays carried out in the presence of excess competitor nucleic acid structures revealed the binding selectivity of cleavage agents, while comparisons with duplex cleavage efficiency employing a dual-labeled hairpin oligonucleotide revealed neither agent prefers G-quadruplex over duplex substrates. Finally, this assay was used to identify the perylene diimide Tel11 as a photocleavage agent for the c-Myc G-quadruplex.

Curcumin reduces the antimicrobial activity of ciprofloxacin against Salmonella Typhimurium and Salmonella Typhi

Typhoidal and non-typhoidal infection by Salmonella is a serious threat to human health. Ciprofloxacin is the last drug of choice to clear the infection. Ciprofloxacin, a gyrase inhibitor, kills bacteria by inducing chromosome fragmentation, SOS response and reactive oxygen species (ROS) in the bacterial cell. Curcumin, an active ingredient from turmeric, is a major dietary molecule among Asians and possesses medicinal properties. Our research aimed at investigating whether curcumin modulates the action of ciprofloxacin. We investigated the role of curcumin in interfering with the antibacterial action of ciprofloxacin in vitro and in vivo. RT-PCR, DNA fragmentation and confocal microscopy were used to investigate the modulation of ciprofloxacin-induced SOS response, DNA damage and subsequent filamentation by curcumin. Chemiluminescence and nitroblue tetrazolium reduction assays were performed to assess the interference of curcumin with ciprofloxacin-induced ROS. DNA binding and cleavage assays were done to understand the rescue of ciprofloxacin-mediated gyrase inhibition by curcumin. Curcumin interferes with the action of ciprofloxacin thereby increasing the proliferation of Salmonella Typhi and Salmonella Typhimurium in macrophages. In a murine model of typhoid fever, mice fed with curcumin had an increased bacterial burden in the reticuloendothelial system and succumbed to death faster. This was brought about by the inhibition of ciprofloxacin-mediated downstream signalling by curcumin. The antioxidant property of curcumin is crucial in protecting Salmonella against the oxidative burst induced by ciprofloxacin or interferon γ (IFNγ), a pro-inflammatory cytokine. However, curcumin is unable to rescue ciprofloxacin-induced gyrase inhibition. Curcumin's ability to hinder the bactericidal action of ciprofloxacin and IFNγ might significantly augment Salmonella pathogenesis.

Enhanced DPPH radical scavenging activity and DNA protection effect of litchi pericarp extract by Aspergillus awamoribioconversion

BackgroundLitchi (Litchi chinensis Sonn.) pericarp is a major byproduct which contains a significant amount of polyphenol. This study was designed to biotransformation litchi pericarp extract (LPE) by Aspergillus awamori to produce more bioactive compounds with stronger antioxidant activities.ResultsThe study exhibited that the 2,2-diphenyl-1-picrylhydrazyl radical scavenging activities significantly (p < 0.05) increased from 15.53% to 18.23% in the water-extracted fraction and from 25.41% to 36.82% in the ethyl acetate-extracted fraction. Application of DNA cleavage assay further demonstrated the enhanced protection effect of the fermented phenolics on DNA damage. It is also noted that the water-extracted fraction of the fermented LPE possessed a much stronger capacity than the ethyl acetate-extracted fraction to prevent from damage of supercoiled DNA. Interestingly, it was found that some new compounds such as catechin and quercetin appeared after of A. awamori fermentation of LPE, which could account for the enhanced antioxidant activity.ConclusionThe DPPH radical scavenging activity and DNA protection effect of LPE were increased by Aspergillus awamori bioconversion while some compounds responsible for the enhanced antioxidant activity were identified. This study provided an effective way of utilizing fruit pericarp as a readily accessible source of the natural antioxidants in food industry and, thus, extended the application area such as fruit by-products.

Expanding LAGLIDADG endonuclease scaffold diversity by rapidly surveying evolutionary sequence space.

LAGLIDADG homing endonucleases (LHEs) are a family of highly specific DNA endonucleases capable of recognizing target sequences ∼20 bp in length, thus drawing intense interest for their potential academic, biotechnological and clinical applications. Methods for rational design of LHEs to cleave desired target sites are presently limited by a small number of high-quality native LHEs to serve as scaffolds for protein engineering—many are unsatisfactory for gene targeting applications. One strategy to address such limitations is to identify close homologs of existing LHEs possessing superior biophysical or catalytic properties. To test this concept, we searched public sequence databases to identify putative LHE open reading frames homologous to the LHE I-AniI and used a DNA binding and cleavage assay using yeast surface display to rapidly survey a subset of the predicted proteins. These proteins exhibited a range of capacities for surface expression and also displayed locally altered binding and cleavage specificities with a range of in vivo cleavage activities. Of these enzymes, I-HjeMI demonstrated the greatest activity in vivo and was readily crystallizable, allowing a comparative structural analysis. Taken together, our results suggest that even highly homologous LHEs offer a readily accessible resource of related scaffolds that display diverse biochemical properties for biotechnological applications.

Rapid and highly efficient construction of TALE-based transcriptional regulators and nucleases for genome modification

Transcription activator-like effectors (TALEs) can be used as DNA-targeting modules by engineering their repeat domains to dictate user-selected sequence specificity. TALEs have been shown to function as site-specific transcriptional activators in a variety of cell types and organisms. TALE nucleases (TALENs), generated by fusing the FokI cleavage domain to TALE, have been used to create genomic double-strand breaks. The identity of the TALE repeat variable di-residues, their number, and their order dictate the DNA sequence specificity. Because TALE repeats are nearly identical, their assembly by cloning or even by synthesis is challenging and time consuming. Here, we report the development and use of a rapid and straightforward approach for the construction of designer TALE (dTALE) activators and nucleases with user-selected DNA target specificity. Using our plasmid set of 100 repeat modules, researchers can assemble repeat domains for any 14-nucleotide target sequence in one sequential restriction-ligation cloning step and in only 24 h. We generated several custom dTALEs and dTALENs with new target sequence specificities and validated their function by transient expression in tobacco leaves and in vitro DNA cleavage assays, respectively. Moreover, we developed a web tool, called idTALE, to facilitate the design of dTALENs and the identification of their genomic targets and potential off-targets in the genomes of several model species. Our dTALE repeat assembly approach along with the web tool idTALE will expedite genome-engineering applications in a variety of cell types and organisms including plants.

Characterization of a reductively-activated elimination pathway relevant to the biological chemistry of the kinamycins and lomaiviticins

The lomaiviticins (1 and 2) and kinamycins (3–5) are bacterial metabolites with potent antimicrobial and antiproliferative activities. Herein we establish that 1–5 are capable of generating electrophilic acylfulvene intermediates (6) under mildly reducing conditions. These acylfulvenes 6 are formed by a multistep process comprising two-electron reduction and loss of dinitrogen to form an ortho-quinone methide, followed by elimination. Based on these studies, the structure of the product formed from 1 in DNA-cleavage assays is proposed (26). We also show that the bis(hydroxynaphthoquinone) substructures of the lomaiviticins activate the metabolites toward reduction. Finally, based on COMPARE and time-dependent cell response profiling analyses, we show that kinamycin C (4) and the monomeric lomaiviticin aglycon (24) operate by a mechanism of action that is distinct from simple diazofluorenes, such as 23.

Label-free fluorescent assay for real-time monitoring site-specific DNA cleavage by EcoRI endonuclease

DNA cleavage reaction catalyzed by nucleases is essential in many important biological processes and medicinal chemistry. Therefore, it is important to develop reliable and facile methods to assay nuclease activity. With this goal in mind, we report a fluorescent assay for label-free, facile, and real-time monitoring of DNA cleavage by EcoRI endonuclease using SYBR Green I (SGI) as a signal probe. The fluorescence of SGI dramatically increased when the free SGI was mixed with double-stranded DNA (dsDNA) substrate. Upon interacting with EcoRI, which cleaves the dsDNA into small fragments, the weakened interaction between SGI and the shortened DNA fragments caused a decrease in fluorescence of SGI. EcoRI-DNA interaction was real-time studied by monitoring fluorescence change with the prolonging of interaction time. The important kinetic parameters, including Michaelis-Menten constant (K(M)) and maximum initial velocity (V(max)), were accurately calculated, which is consistent with previously reported studies. Site-specific DNA cleavage by EcoRI endonuclease has also been verified by gel electrophoresis analysis, which indicated that this method is a simple and effective approach to assay DNA cleavage reaction. Specificity investigation demonstrated that EcoRI-DNA interactions can be studied with high selectivity. Compared with previously reported methods, this approach is selective, simple, convenient and cost-efficient without any labeling of the probe or of the target.

DNA binding, nuclease activity and cytotoxicity studies of Cu(ii) complexes of tridentate ligands

Cu(II) complexes of three tridentate ligands, L(1), L(2) and L(3), [L(1), N-((1H-imidazole-2-yl)methyl)-2-(pyridine-2-yl)ethanamine; L(2), N-((1-methyl-1H-imidazole-2-yl)methyl)-2-(pyridine-2-yl)ethanamine; L(3), 2-(pyridine-2-yl)-N-((pyridine-2-yl)methyl)ethanamine] respectively, were synthesized and characterized. The single crystal X-ray structure of complex 1 reveals the pseudo octahedral coordination geometry around the copper center. Absorption and fluorescence experimental evidence show good DNA binding propensity (in the order of 10(5) M(-1)) of the complexes. Thermal denaturation and circular dichroism (CD) analyses reveal minor structural changes of calf thymus (CT) DNA in presence of complexes and groove and/or surface binding of the complexes to CT-DNA. Kinetic DNA cleavage assay shows pseudo-first-order kinetic reaction between the complex and supercoiled (SC) DNA. In addition, mechanistic SC DNA cleavage results show higher DNA cleavage activity in presence of reducing agent, due to the presence of hydroxyl radicals. In vitro cytotoxicity assay of the complexes demonstrate that the complexes have low toxicity for different cancer cell lines and IC(50) values were between 37 and 156 μM.

Amino Acid Substitutions at Position 95 in GyrA Can Add Fluoroquinolone Resistance to Mycobacterium leprae

Amino acid substitutions at position 89 or 91 in GyrA of fluoroquinolone-resistant Mycobacterium leprae clinical isolates have been reported. In contrast, those at position 94 in M. tuberculosis, equivalent to position 95 in M. leprae, have been identified most frequently. To verify the possible contribution of amino acid substitutions at position 95 in M. leprae to fluoroquinolone resistance, we conducted an in vitro assay using wild-type and mutant recombinant DNA gyrases. Fluoroquinolone-mediated supercoiling activity inhibition assay and DNA cleavage assay revealed the potent contribution of an amino acid substitution of Asp to Gly or Asn at position 95 to fluoroquinolone resistance. These results suggested the possible future emergence of quinolone-resistant M. leprae isolates with these amino acid substitutions and the usefulness of detecting these mutations for the rapid identification of fluoroquinolone resistance in leprosy.

A Biological Delivery Platform for Zinc Finger Nucleases Using Transferrin-Mediated Endocytosis

Abstract 1071Zinc finger nucleases (ZFNs) are custom-designed DNA binding proteins that produce DNA double-strand breaks (DSBs) at predetermined genomic sites, stimulating homology-directed repair in the presence of donor template by many orders of magnitude over the spontaneous rate. The ability to target specific genes with ZFN technology opens therapeutic opportunities for gene correction and selective gene silencing. Sickle cell anemia (SCA) is an ideal disease target because correction of the single gene β-globin mutation in patient-derived, autologous hematopoietic stem progenitor cells (HSPCs) promises to be curative. One of the barriers to ZFN-based gene correction is the lack of a nonviral delivery system that achieves bulk transport of the nucleases to hard-to-transfect target cells, such as embryonic and HSPCs. To address this challenge, we set out to develop a delivery platform that is (i) gentle to the cell, (ii) provides tunable delivery rates, and (iii) achieves improved spatio-temporal control of the nucleases. To reconcile these goals, we have explored a method for direct delivery of ZFNs as proteins by receptor-mediated endocytosis. We selected the transferrin receptor pathway as our lead candidate on the rationale that all nucleated cells, including HSPCs, must import elemental iron to remain viable under ex vivo culture conditions. To test the feasibility of this strategy, this initial work used a ZFN pair targeted against a model GFP transgene. We optimized expression by pilot scale fermentation in an Escherichia coli host-vector system and purification to homogeneity by serial chromatography. We conjugated the purified ZFNs to the iron carrier protein, transferrin (tf), using SPDP, an amine and sulfhydryl reactive heterobifunctional crosslinker. The resulting disulfide linkage is designed to undergo scission (“self-immolation”) upon entry into the intracellular reducing environment. In vitro DNA cleavage assays and surface plasmon resonance binding assays demonstrated that ZFNs remained competent for target sequence cleavage following conjugation, with only mild to quantitative impairment of activity. To analyze delivery in biological systems, we measured time- and dose-dependence of tf-mediated ZFN uptake in human osteosarcoma (U2OS 2–6–3) cells. ZFNs in DAPI stained cell nuclei were detected by indirect immunofluorescence and signal intensity was measured in projections of deconvolved depth coded z-stacks. Nuclear uptake of tf-ZFN protein occurred in >95% of cells, was dose-dependent and linear with time in the lower dose ranges, and reached saturation as early as 60 min. Importantly, maximal nuclear uptake was indistinguishable from ZFN plasmid treated cells. These results indicate that endocytic delivery of ZFNs readily traverses the cellular membrane, overcomes the potential hurdle of endosomal trapping, and targets the nucleus with high efficiency. To demonstrate gene targeting activity, we used the U2OS 2–6–3 cell assay which bears a tandem transgene array at a single locus that is cleavable by our GFP ZFNs. Cells were transfected with lacI-ECFP to mark the target locus, incubated with tf-ZFNs, fixed, and stained for 53BP1, a signaling protein that marks DSBs. Recruitment of 53BP1 to the target locus was observed in 13% (18/135) of tf-ZFN treated cells, whereas no recruitment (0/152) was observed in untreated cells. These findings demonstrate that tf-conjugated ZFNs retain cleavage activity after nuclear uptake in a significant percentage of cells. To determine whether the tf-ZFNs are capable of stimulating gene correction, we transfected primary mouse adult fibroblasts carrying a mutant GFP transgene with donor template, incubated with tf-ZFNs, and evaluated cells at 72 h for gene correction as evidenced by GFP expression. Flow cytometry revealed a gene correction rate of 1–2%, identical to ZFN plasmid transfected cells, demonstrating that the technology of shuttling ZFN proteins to the cell interior via the tf-receptor pathway can deliver bioactive ZFNs to the nuclear compartment, target specific gene sequences, and induce homology-directed repair in the presence of donor DNA. We are currently testing these methods in hematopoietic stem cells, with the ultimate goal of correcting the sickle globin allele. Toward this end, we plan to adapt these approaches for high-throughput transfer of ZFN proteins directly to the hematopoietic stem progenitor cell. Disclosures:No relevant conflicts of interest to declare.

Development of quantitative DNA cleavage assay for XPG endonuclease activity using endogenous nuclear proteins in human cell lines

XPG, a structure-specific DNA endonuclease responsible for the 3' incision of DNA lesions during nucleotide excision repair (NER), is associated with high risk of skin cancer as well as skeletal, neurological and developmental abnormalities when functionally defective. These observations have led to the model wherein the endonuclease activity of XPG is important for NER. Herein, we first demonstrate a sensitive assay of XPG cleavage activity using direct nuclear extracts as an XPG source. This method provided quantitative evaluation of the activity of endogenous XPG endonuclease derived from cells with high reproducibility. Our new assay takes advantage of 3'-end oligolabeling of the bubble-shaped substrate. Our results demonstrate efficient cleavage of the model substrate in two XPG wild-type cell lines (human fibroblasts and RKO colon cancer cells) in a time- and dose-dependent manner. In addition, XPG-deficient cells manifested lower cleavage activity relative to normal XPG cells, indicating that the incision activity of XPG was intrinsic in our methodology. It was also found that 7 mM MgCl2 and buffer pH 6.8 resulted in optimal endonucleolytic activity. Based on these results, our modified methodology has potential for quantitative monitoring of XPG cleavage activity in any cell type or tissue of interest.

Impact of the E540V Amino Acid Substitution in GyrB of Mycobacterium tuberculosis on Quinolone Resistance

Amino acid substitutions conferring resistance to quinolones in Mycobacterium tuberculosis have generally been found within the quinolone resistance-determining regions (QRDRs) in the A subunit of DNA gyrase (GyrA) rather than the B subunit of DNA gyrase (GyrB). To clarify the contribution of an amino acid substitution, E540V, in GyrB to quinolone resistance in M. tuberculosis, we expressed recombinant DNA gyrases in Escherichia coli and characterized them in vitro. Wild-type and GyrB-E540V DNA gyrases were reconstituted in vitro by mixing recombinant GyrA and GyrB. Correlation between the amino acid substitution and quinolone resistance was assessed by the ATP-dependent DNA supercoiling assay, quinolone-inhibited supercoiling assay, and DNA cleavage assay. The 50% inhibitory concentrations of eight quinolones against DNA gyrases bearing the E540V amino acid substitution in GyrB were 2.5- to 36-fold higher than those against the wild-type enzyme. Similarly, the 25% maximum DNA cleavage concentrations were 1.5- to 14-fold higher for the E540V gyrase than for the wild-type enzyme. We further demonstrated that the E540V amino acid substitution influenced the interaction between DNA gyrase and the substituent(s) at R-7, R-8, or both in quinolone structures. This is the first detailed study of the contribution of the E540V amino acid substitution in GyrB to quinolone resistance in M. tuberculosis.

팥(Phaseolus angularis) 열수 추출물의 산화적 DNA와 세포 손상 억제 효과

Abstract - In this study, we evaluated the protective effects of the hot water extract from red bean ( Phaseolus angularis ) against oxidative DNA and cell damage induced by hydroxyl radical. The antioxidant activities were evaluated by hydroxyl radical and hydrogen peroxide scavenging assay, and Fe 2+ -chelating assay. Although the extract with hot water didn’t scavenge the hydroxyl radical, it removed and chelated hydrogen peroxide and ferrous iron necessary for the induction of hydroxyl radical by 71% and 64% at 200 μg/ml, respectively. Its protective effect on oxidative DNA damage was carried using ψX-174 RF I plasmid DNA comparing the conversion level of supercoiled form of the plasmid DNA into open-circular form and linear form and the expres sion level of phospho-H2AX in NIH 3T3 cells. In ψX-174 RF I plasmid DNA cleavage assay, it inhibited oxidative DNA damage by 96% at 200 μg/ml. Also, it decreased the expression of phospho-H2AX by 50.1% at 200 μg/ml. Its protective effect against oxidative cell damage was measured by MTT assay and the expression level of p21 protein in NIH 3T3 cells. In MTT assay for the protective effect against the oxidative cell damage, it inhibited the oxidative cell death and the abnormal cell growth induced by hydroxyl radical. Also, it inhibited p21 protein expression by 98% at 200 μg/ml. In conclusion, the results of the present studies indicate that hot water extract from red bean exhibits antioxidant properties and inhibit oxidative DNA damage and the cell death caused by hydroxyl radical.

Deglycobleomycin A 6 analogues modified in the methylvalerate moiety

Previous studies have indicated that the methylvalerate subunit of bleomycin (BLM) plays an important role in facilitating DNA cleavage by BLM and deglycoBLM. Eleven methylvalerate analogues have been synthesized and incorporated into deglycoBLM congeners by the use of solid-phase synthesis. The effect of the valerate moiety in the deglycoBLM analogues has been studied by comparison with the parent deglycoBLM A 5 using supercoiled DNA relaxation and sequence-selective DNA cleavage assays. All of the deglycoBLM analogues were found to effect the relaxation of the plasmid DNA. Those analogues having aromatic C4-substituents exhibited cleavage efficiency comparable to that of deglycoBLM A 5. Some, but not all, of the deglycoBLM analogues were also capable of mediating sequence-selective DNA cleavage.

Synthesis and biological evaluation of novel 7-acyl homocamptothecins as Topoisomerase I inhibitors

A series of novel 7-acyl derivatives of homocamptothecin (hCPT) were designed and synthesized with the purpose to improve antitumor activity of hCPT, via Minisci free–radical reaction from 10-methoxyhomocamptothecin. All the compounds were evaluated for in vitro cytotoxicity against three cancer cell lines (A549, MDA-MB-435 and HCT116). For MDA-MB-435 cell line, compounds, 6a, 6b, 6k and all of 7-alkylcabonyl homocamptothecin derivatives showed higher in vitro inhibitory activities than topotecan (TPT). Furthermore, compounds 6d, 6e, and 6k showed highly potent inhibitory activities with the IC50 values from less than 1 nM to 2.2 nM. In Topoisomerase I (Topo I)-induced DNA cleavage assay, compounds 6a, 6d, and 6k, as compared to CPT, revealed higher Topo I inhibitory activity.

Glycosyloxyamine Neoglycosylation: A Model Study Using Calicheamicin

Glycosyloxyamine Neoglycosylation: A Model Study Using Calicheamicin

Synthesis, DNA Binding, and DNA Photocleavage of the Cobalt(III) Complexes [Co(bpy)2MDPPZ]3+, [Co(dmb)2MDPPZ]3+, and [Co(phen)2MDPPZ]3+and their Antimicrobial Activity

The ligand 7-methyl dipyrido[3,2-a:2′-3′-c]phenazine (MDPPZ) and its complexes [Co(bpy)2MDPPZ]3+(1) (bpy = 2,2′-bipyridine), [Co(dmb)2MDPPZ]3+ (2), (dmb = 4,4′-dimethyl-2,2′-bipyridine), and [Co(phen)2MDPPZ]3+(3) (phen = 1,10-phenanthroline) have been synthesized and characterized by UV-VIS, IR, EA, 1H, and 13C-NMR and mass spectra. The interactions of the three complexes with CT-DNA have been investigated by absorption and emission spectroscopy, DNA melting, viscosity measurements, and DNA cleavage assay. The experimental results suggested that these complexes bind to CT-DNA through an intercalative mode. Three complexes have also been found to promote the photo cleavage of plasmid pBR-322 DNA under irradiation. The inhibition studies suggest that the oxygen species are mainly responsible for the photocleavage by these complexes. Further, all the three Co(III) complexes synthesized were screened for their antimicrobial activity.

Antioxidant Activities and Inhibitory Effect on Oxidative DNA Damage of extracts from Abeliophylli distichi Folium

ABSTRACT Objective：In this study, we demonstrate the protective effect on oxidative DNA damage of leaf extracts from Abeliophylli distichi Folium via its antioxidant activity for the establishment of new value for the herbal medicine.Methods：Abeliophylli distichi Folium leaves were extracted with hot-wat er and ethylacetate (EtOAC). The 1,1-diphenyl-2- picrylhydrazyl (DPPH) radical and hydroxyl scav enging assay and Fe 2+ chelating assay were performed for antioxidative effect and φX-174 RF I DNA cleavage assay and intracellular DNA damage assay were used for inhibitory effect of intracellular DNA damage.Results：In DPPH, Hydroxyl radical scavenging activity and Fe 2+ chelating activity of EtOAC extracts were 94.72%, 62.88%, 41.13%, and hot-water extracts were 88.86%, 56. 7%, 37.4% at 200 ㎍/㎖, respectively. Also, those extracts showed protective effect of DNA damage against t he oxidative stress.Conclusion：These results indicated that the leaf extracts of Abeliophylli distichi Folium can be used as a natural antioxidants, which effectively inhibits the oxidative DNA damage.Key words：Abeliophylli distichi Folium, DPPH, Hydroxyl radical, Fe

Evolution of I-SceI Homing Endonucleases with Increased DNA Recognition Site Specificity

Elucidating how homing endonucleases undergo changes in recognition site specificity will facilitate efforts to engineer proteins for gene therapy applications. I-SceI is a monomeric homing endonuclease that recognizes and cleaves within an 18-bp target. It tolerates limited degeneracy in its target sequence, including substitution of a C:G+4 base pair for the wild-type A:T+4 base pair. Libraries encoding randomized amino acids at I-SceI residue positions that contact or are proximal to A:T+4 were used in conjunction with a bacterial one-hybrid system to select I-SceI derivatives that bind to recognition sites containing either the A:T+4 or the C:G+4 base pairs. As expected, isolates encoding wild-type residues at the randomized positions were selected using either target sequence. All I-SceI proteins isolated using the C:G+4 recognition site included small side-chain substitutions at G100 and either contained (K86R/G100T, K86R/G100S and K86R/G100C) or lacked (G100A, G100T) a K86R substitution. Interestingly, the binding affinities of the selected variants for the wild-type A:T+4 target are 4- to 11-fold lower than that of wild-type I-SceI, whereas those for the C:G+4 target are similar. The increased specificity of the mutant proteins is also evident in binding experiments in vivo. These differences in binding affinities account for the observed ∼36-fold difference in target preference between the K86R/G100T and wild-type proteins in DNA cleavage assays. An X-ray crystal structure of the K86R/G100T mutant protein bound to a DNA duplex containing the C:G+4 substitution suggests how sequence specificity of a homing enzyme can increase. This biochemical and structural analysis defines one pathway by which site specificity is augmented for a homing endonuclease.