After the final set of protein-DNA complexes was selected, we used a sequence of processing steps to generate a uniform set of PDB files that can be readily used in computational analysis. First, we processed all dsDNA molecules to conform to a standardized format in which the two strands of DNA are treated as separate chains, the chains are ordered in a 5′ to 3′ orientation, all overhangs are removed, and the basepairs aligned so that they are physically matched. Since many structures in the PDB do not conform to this standard, we devel- oped scripts to reformat all PDB files in the database accordingly. Second, we extracted protein chains with multiple HTH domains and single HTH domains that span multiple chains, and formatted these protein chains so that each individual HTH domain is spanned by a single chain in an individual PDB file, along with its cognate DNA molecule. Finally, we processed the final set of PDB files with the PDB2PQR [33, 34] utility to carry out the protonation and dewatering steps. PDB2PQR is run with default settings using the AMBER molecular mechanics force field .
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Binding. The recognition pattern of the probe α helix has been well characterized; each finger binds adjacent 3 bp subsites on the DNA using amino acids at positions -1, 2, 3 and 6 relative to the start of the α helix, -1 being the residue position preceding the helix [2,24,76]. Although exceptions to this rule have been observed in specific examples [29,77], experiments have shown that by alter- ing the amino-acid types at the key positions, different subsite sequences are recognized, suggesting that these residue positions are usually sufficient for specific binding [30,78]. By varying the number of fingers used in a protein chain, this relatively simple motif allows recogni- tion of a wide range of binding sites with different degrees of specificity. For example, a protein with five fingers is expected to bind a site very selectively, whereas a protein with only a single finger would bind a wide range of sites containing the required 3 bp sequence. However, the structure of the human glioblastoma protein suggests that binding is not always straightforward; of the five fingers in the structure, one does not contact the DNA at all and only two appear to make specific contacts with bases . As described earlier, the protein subunits in this study have been split into distinct domains, each containing a single zinc-finger motif. The pairwise sequence identities of the aligned domains are all high, ranging from 73% (for example, human zinc-finger protein, 1udbA1, and Drosophila tramtrack protein, 2drpA1) to 100% (for example, mouse Zif268 protein, 1aayA1, and artificial protein, 1mey). All domains are structurally very similar, returning SSAP scores of over 90.
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and incubated for 24 hours prior to transfection. The media was then replaced with fresh growth medium in each well. pDNA was diluted with phosphate-buffered saline (pH 7.4) to 0.5 mg/ mL, and then added to an appropriate amount of the copolymer in the same buffer with an equal volume to obtain the desired N/P ratios. The complexes were mixed using a vortex mixer for 30 seconds and then allowed to stand for 30 minutes at room temperature. Complexes containing 2 µ g EGFP plasmid were subsequently added to the wells and incubated with the cells initially for 6 hours. The medium was then replaced with 1 mL of fresh complete medium, and the cells were incubated for an additional 24–72 hours at 37 ° C. As a positive control, transfection with Lipofectamine 2000/DNA complexes was performed according to the manufacturer’s protocol. Briefly, Lipofectamine 2000/DNA complexes were incubated with cells in 1 mL of serum-free medium for 6 hours, and further incubated with fresh complete medium for 24–72 hours at 37 ° C. Naked DNA was used on the cell cultures and examined as described above. All transfection experiments were per- formed in triplicate. The cells were transferred to an inverted fluorescence microscope (Leica DMI 4000B) for imaging.
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to internalize plasmid DNA into cells was performed using pEGFP-N1 labeled with YOYO-1 on MCF-7 and MCF-7/ADR cells (Figure 9A and B). The cellular uptake of YOYO-1-labeled naked DNA was negligible, and complexing with PEI 800 achieved no obvious improvement in internalization. However, when DNA was complexed with SP, cellular uptake increased significantly in both cell lines, as indicated by the mean fluorescence intensity and percentage of cellular uptake. Although the number of green cells treated with PEI 25,000-DNA complexes was similar to that treated with SP-DNA complexes, the mean fluorescence intensity of the PEI 25,000-DNA complexes was much lower than that of the SP-DNA complexes. These results showed that SP-DNA complexes could internalize plasmid DNA into cells more effectively than PEI 25,000-DNA complexes. For vectors with a positive surface charge, uptake into cells may occur predominantly via adsorptive endocytosis. 37 In addition, the
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Image Processing—Particle picking was carried out automat- ically using Boxer (EMAN suite (19)). Analysis of the contrast transfer function and correction was completed using CTFIT (EMAN suite (19)). Image analysis was performed with IMAGIC-5 (20). Images were normalized to the same S.D. and bandpass-filtered. The low-resolution cutoff was ⬃ 100 Å to remove uneven background in particle images, and the high- resolution cutoff was ⬃ 7 Å. Images were subjected to an align- ment procedure, followed by statistical analysis. Alignment and classification of images were performed as described previously (20) and yielded classes representing characteristic views of the molecule. Primary structural analysis of hMCM and hMCM plus DNA complexes was performed using an ab initio approach in which the orientations of the best 10 –15 image classes were determined by angular reconstitution using C1 startup. Three-dimensional maps were calculated using the exact filtered backprojection algorithm (20). Structural analysis was performed using several starting models with several dif- ferent sets of image classes for ab initio reconstructions. The first reconstructions were used for the following rounds of alignment and classification of images. The structures of the complexes were refined by an iterative procedure with the number of classes gradually increased. The final reconstruction for hMCM alone was calculated from the best 100 classes con- taining ⬃ 11 images each. For hMCM plus DNA, the final reconstruction was calculated from the best 155 classes con- taining ⬃ 10 images each. Resolution of the map was assessed using the 0.5 threshold of Fourier shell correlation (21), which corresponds to 23 Å. Domain fitting into the three-dimensional map of hMCM and hMCM plus DNA complexes was per- formed manually with UCSF Chimera (22). Illustrations were generated using UCSF Chimera. Surface representations (unless stated otherwise) are displayed at a threshold level of 3 (S.D. of densities within EM maps), which corresponds to ⬃ 100% of the expected mass at a specific protein density of 0.84 kDa/Å 3 .
Progress in the treatment of hepatocellular carcinoma (HCC), a common tumor worldwide, has been disappoint- ing. Inhibitors of topoisomerases are being widely studied as potential inducers of tumor cell apoptosis. Our aims were to determine whether topoisomerase-directed drugs would in- duce apoptosis in a human HCC cell line (Hep 3B) and, if so, to investigate the mechanism. The topoisomerase I poi- son camptothecin (CPT) induced apoptosis of Hep 3B cells in a time- and concentration-dependent manner. In con- trast, the topoisomerase II poison etoposide failed to induce apoptosis despite the apparent stabilization of topoiso- merase II–DNA complexes. Unexpectedly, CPT-induced apoptosis in this cell type occurred without any detectable cleavage of poly(ADP-ribose) polymerase or lamin B, poly- peptides that are commonly cleaved in other cell types un- dergoing apoptosis. Likewise, Hep 3B cell apoptosis occurred without a detectable increase in interleukin-1 b –converting enzyme (ICE)-like or cysteine protease P32 (CPP32)-like protease activity. In contrast, trypsin-like protease activity (cleavage of Boc-Val-Leu-Lys-chloromethylaminocoumarin in situ) increased threefold in cells treated with CPT but not etoposide. Tosyl-lysyl chloromethyl ketone inhibited the trypsin-like protease activity and diminished CPT-induced apoptosis. These data demonstrate that ( a) apoptosis is in- duced in Hep 3B cells after stabilization of topoisomerase I-DNA complexes but not after stabilization of topoisome- rase II-DNA complexes as measured by alkaline filter elution; (b) Hep 3B cell apoptosis occurs without activation of ICE- like and CPP32-like protease activity; and (c) a trypsin-like protease activity appears to contribute to apoptosis in this cell type. (J. Clin. Invest. 1996. 98:2588–2596.) Key words: camptothecin • CPP32 • etoposide • interleukin-1 b –convert-
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The melting points were measured on an electro thermal melting point apparatus and were not corrected. Fourier-transform infrared spectra were recorded using the KBr disc technique on a JASCO 410 FTIR spectrophotometer. Elemental (CHN) analysis was performed using an Exeter CE-440 elemental analyzer. UV- visible absorption spectra were measured in DMF (≈10 -5 mole -1 ) using a Pye–Unicam 8800a UV-visible automatic scanning spectrophotometer. Molar Conductivity was measured on a systronic conductivity bridge with a dip-type cell, using 1×10 -3 M solution of complexes in DMF. 1 HNMR spectra of the ligands and their complexes were recorded on a Varian Gemini-200 spectrometer (200 MHZ) and (300MHZ) using DMSO-d 6 as
Electronic absorption spectroscopy is widely employed to determine the DNA binding affinity of metal complexes. The present copper(II) complexes do not exhibit any intense d–d or charge transfer band to monitor their interaction with DNA. So the intense ligand based π–π* absorption band is used to monitor the interaction of the complexes with calf thymus DNA. Complexes bound to DNA through intercalation, which involves a strong stacking interaction of the planar aromatic rings of the coordinated ligand with the base pairs of DNA, usually result in hypochromism and red shift of ligand-band or charge transfer bands . All the present complexes exhibit significant hypochromism(15– 27%) on the incremental addition of DNA with varying red shifts (Fig. 3, Table 1). Further, as the extent of hypochromism is commonly consistent with the strength of intercalative interaction, it is evident that all the complexes exhibit almost the same DNA binding affinities . All these observations reveal that the present complexes intercalate less strongly. Moreover, the uncoordinated substituted group of the ligand chain may be involved in secondary interactions like hydrogen bonding with DNA possessing several hydrogen bonding sites accessible both in the minor and major grooves . Similar hydrogen bonding interactions have been proposed for [Co(NH 3 ) 6 ] 3+ bound
We then investigated the e ﬀ ect of biologically relevant electron donors on the formation of azidyl radicals from the photoactivation of 1. The strongest electron donor in DNA is the base guanine, but surprisingly, the presence of 2 molar equiv of 5 ′ -guanosine monophosphate (GMP) had little e ﬀ ect on the production of azidyl radicals from 1 induced by blue light (see the SI). No precipitate formed in the presence of GMP, in contrast to its absence, consistent with electron transfer from the azido ligands to Pt IV to give azidyl radicals
single amino acid; ideally, this product would localize to the nucleus for mismatch binding. The focus of this work was to synthesize rhodium complexes bearing single amino acids that would serve as the proteolytic cleavage product depicted in Figure 6.2. The goal was to synthesize an effective rhodium amino acid conjugate that 1) still demonstrated mismatch specificity, and 2) maximized nuclear and minimized mitochondrial uptake. Prior work in our group investigated appending peptides to rhodium and ruthenium complexes in an effort to improve cellular uptake and nuclear localization. A D-octaarginine appendage conjugated to a rhodium ancillary ligand (Figure 6.3) did bestow fast nuclear uptake in HeLa cells, but the complex no longer exhibited specific binding to mismatches. 8 A shorter peptide appendage, the RrRK nuclear targeting signal, was tethered to a ruthenium complex (Figure 6.3) and did impart an enhanced cellular uptake compared to free complex, but it was found that a higher concentration was required to accumulate in the nucleus compared to an octaarginine conjugate. 9 The identity of the conjugated peptide also greatly altered the nuclear uptake. For example, an SrSr sequence appended to the ruthenium complex exhibited a much lower nuclear accumulation compared to
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We performed Western Blot experiment to systematically quantify the different oligonucleotides’ gene silencing activity. After gene expression, HeLa cells were washed three times with phosphate saline buffer (PBS) and then scraped into 0.1 mL of RIPA lysis buffer containing the protease inhibitor. After incubation for 10 min on ice, the lysate was transferred to new tubes, followed by centrifuging at 14,000 g for 15 min at 4°C. After analyzing lysates by SDS- PAGE gel, the analysis verified the downregulation of EGFP expression in cells treated with our antisense DNAs. Moreover, our Se-oligonucleotide has about 20% more gene silencing activity than the native one. Finally, the relative eGFP expression was calculated as the ratio of the fluorescence intensity on the control of beta-actin expression. Our quantitative result proved that DNA 4 and 5 with CH 3 Se-modifications, silenced the eGFP gene expression more effectively. The
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interact with π-donors stronger. Higher affinity to the DNA base pairs may additionally be supported by slightly higher hydrophobicity of the nickel complex in comparison to the copper derivative, which may be particularly important for groove binding. At the same time, in the mixed sample (DNA, 2Ni and 2Cu; Fig. 7, grey curve), 2Ni influences visibly stronger on the metal complex absorbance region of the LD spectrum than analogous copper derivative. The DNA LD signals intensity is however only slightly reduced. Since both complexes intercalate and both are most likely able to undergo groove binding, it seems like either the groove binding occurs within different sites of the DNA or the nickel complex intercalation
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126.96.36.199 Viscosity measurements: Spectroscopic methods provide important but insufficient evidence about the nature of binding of metal complexes to DNA. However, hydrodynamic measurements are least ambiguous in understanding the mode of binding. One such method is measuring the viscosity of DNA solution in buffer with the increasing concentration of the complex solution. An increase in the DNA viscosity is indicative of intercalation of the complex with DNA, as intercalation leads to an increase in the length of DNA helix with the insertion of complex between the DNA base pairs. Whereas a decrease or no change in the viscosity of DNA suggests either electrostatic interaction or groove binding of the complex to DNA, which is a result of bend or kink in the structure of DNA 29 . Figure 7 displays the changes
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In this report, coordination chemistry of a Schiff base ligand, obtained from the reaction of 1,2-Di(2- thienyl)-1,2-ethanedione (Thenil), hydrazine hydrate and 2-Hydroxy-5-methylbenzaldehyde is described. Cu(II), Ni(II),and Zn(II) complexes have been synthesized using the Schiff base ligand and characterized by using spectral, physicochemical and elemental analyses. The interaction of the synthesized complexes with CT- DNA was investigated by electronic absorption, competitive fluorescence titration, viscosity measurements and circular dichroic analyses. The results suggest that complexes interact with CT- DNA by intercalative modes. Among the investigated complexes, the one containing copper as the central metal ion showed better binding affinity than the other two complexes containing Zinc and nickel ions as metal counterparts respectively.
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described herein, and which also lacks an azo group, has also been reported to be inactive . In general, the activity of compounds is not affected by the cisplatin resistance mechanisms, suggesting their modes of action differ. Their efficacy is in some cases better than that of cisplatin in resistant cells. The mononuclear compounds can all bind to the isolated model base 9-EtGua, but their DNA binding neither results in kinking like with cisplatin nor in the coiling as known for the dimetallo helicates . At this stage we cannot exclude the possibility that the target of these compounds is DNA, since they all bind to DNA in a non-cisplatin mode.
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stranded DNA substrates. Fen1 plays an essential role in key cellular processes, such as DNA replica- tion and repair, and mutations that compromise Fen1 expression levels or activity have severe health implications in humans. The nuclease activity of Fen1 and other FEN family members can be stimulated by processivity clamps such as proliferating cell nuclear antigen (PCNA); however, the exact mechanism of PCNA activation is currently unknown. Here, we have used a combination of ensemble and single-molecule Fo¨rster resonance energy transfer together with protein-induced fluor- escence enhancement to uncouple and investigate the substrate recognition and catalytic steps of Fen1 and Fen1/PCNA complexes. We propose a model in which upon Fen1 binding, a highly dynamic substrate is bent and locked into an open flap conformation where specific Fen1/DNA interactions can be estab- lished. PCNA enhances Fen1 recognition of the DNA substrate by further promoting the open flap con- formation in a step that may involve facilitated threading of the 5 0 ssDNA flap. Merging our data
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There is a strong tendency toward studying biologically active compounds such as metal complexes which are applied as antitumor drugs in biochemistry and medicine [1, 2].Regarding metal complexes, studying their interaction with nucleic acid is of utmost importance since they have an important role in developing new drugs which have therapeutic effects[3,4]. Generally, metal complexes can interact with DNA via covalent interaction which incorporates the coordination of the nitrogenous base or the phosphate moiety of the nucleic acid to the central metal ion or non-covalent interaction. This latter case includes intercalation, groove binding or external electrostatic binding . DNA repair can be hampered by metal complexes through their interference with enzymes or proteins involved in DNA replication or DNA repair. Imidazole is incorporated into many important biological molecules. The high therapeutic properties of the imidazole related drugs have encouraged the medicinal chemists to synthesize a large number of novel chemotherapeutic agents. Imidazole has become an important part of many pharmaceuticals. Synthetic imidazoles are present in many fungicides and antifungal, antiprotozoal, and antihypertensive medications [6-13]. Imidazole and its derivatives are reported to be physiologically and pharmacologically active and find applications in the treatment of several diseases. In view of all
10. Rayati S., Torabi N., Ghamei A., Mohebbi S., Wojtczak A., Kozakiewicz A., Vanadyl tetradentate Schiff base complexes as catalyst for C–H bond activation of olefins with tert-butylhydroperoxide: Synthesis, characterization and structure, Inorg. Chim. Acta., 361(5), 1239-1245 (2008) 11. Edmund K., Romanowski G., Nowicki W., Kwiatkowski M., Suwi ń ska K., Chiral dioxovanadium(V) complexes with single condensation products of 1,2- diaminocyclohexane and aromatic o-hydroxycarbonyl compounds: Synthesis, characterization, catalytic properties and structure, Polyhedron, 26(12), 2559-2568 (2007) 12. Ashok M., Prasad A.V.S.S., Reddy P.M. and Ravinder V.,
3.2. The Smc5/6 complex and stalled replication forks A second function of the Smc5/6 complex in DNA repair is the repair of collapsed replication forks . Smc6 localizes to collapsed replication forks in budding yeast . Inactivation of Smc5/6 caused accumulation of X-shaped HR intermediates that could be formed by the regression of stalled replication forks in rDNA. Furthermore, the SUMO ligase activity of Mms21 is required for preventing the accumulation of the X- shaped DNA molecules at damaged replication forks , although the relevant substrate of Mms21 in this process is unknown. Overexpression of BRC1 (a BRCT domain protein required for DNA repair during S phase) or the bacterial resolvase RuvA rescued the repli- cation-arresting defects of nse5, nse6, and smc6 mutants in S. pombe [76,85,86]. The structure-specific endonu- cleases Slx1/4 and Mus81/Eme1 are required for the BRC1-mediated suppression of Smc5/6 mutant pheno- types. Moreover, inactivation of the Mph1 helicase sup- pressed the accumulation of aberrant recombination intermediates in smc6 and mms21/nse2 mutants in S. cerevisiae . Smc5/6 has also been shown to facilitate the resolution of sister-chromatid linkages during mito- sis [88,89]. Finally, the Smc5/6 complex is required for loading RPA and Rad52 onto stalled replication forks to maintain them in recombination-competent configura- tions . Collectively, these investigations indicate that the Smc5/6 complex promotes HR-dependent rescue of stalled replication forks by stabilizing them in recombi- nation-component configurations and by facilitating the resolution or preventing the formation of certain recom- bination intermediates (Figure 3).
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Complexes with Schiff base Hbid, where Hbid=(E)-2-(2-hydroxybenzylideneamino) isoindoline-1,3-dione, displayed chemical nuclease activity by partial intercalation thus ability to inhibit the growth of both Gram-pos. and Gram-neg. bacteria  . Imidazole[4, 5-f][1, 10] phenanthroline derivatives available to inhibit c-myc gene expression in A549 cells via NF-kB pathway exhibit certain activities towards inhibiting tumor cells to some extend -. Although certain attention has been paid to the research on oxovanadium complexes as potential DNA intercalator, photocleavage and anticancer agents -, the oxovanadium compounds with imidazole[4, 5-f][1, 10] fluoro-phenanthroline derivatives has rarely been reported yet  .
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