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1.
Cellular DNA is not a uniform target for DNA-reactive drugs. At the nucleotide level, drugs recognize and bind short motifs of a few base pairs. The location of drug adducts at the genomic level depends on how these short motifs are distributed in larger domains. This aspect, referred to as region specificity, may be critical for the biological outcome of drug action. Recent studies demonstrated that certain minor groove binding (MGB) drugs, such as bizelesin, produce region-specific lesions in cellular DNA. Bizelesin binds mainly T(A/T)(4)A sites, which are on average scarce, but occasionally cluster in distinct minisatellite regions (200-1000 bp of approximately 85-100% AT), herein referred to as AT islands. Bizelesin-targeted AT islands are likely to function as strong matrix attachment regions (MARs), domains that organize DNA loops on the nuclear matrix. Distortion of MAR-like AT islands may be a basis for the observed inhibition of new replicon initiation and the extreme lethality of bizelesin adducts (<10 adducts/cell for cell growth inhibition). Hence, long AT-islands represent a novel class of critical targets for anticancer drugs. The AT island paradigm illustrates the potential of the concept of regional targeting as an essential component of the rational design of new sequence-specific DNA-reactive drugs.  相似文献   

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The adsorption of lysozyme on mixed phosphatidyl choline-cardiolipin vesicles was studied at pH 4.0 and 6.0. The binding constants at both pH were determined at 0 and 22 degrees C. The presence of maximum on the adsorption isotherm at pH 6.0 was interpreted as an indication of the formation of two types of the protein-lipid complexes. This interpretation was confirmed by electron-microscopic observations. On the other hand, at pH 4.0 only one type of the protein-lipid complex was formed. The lysozyme conformation in solution and in the protein-lipid complexes was studied by circular dichroism. It was found that at acidic pH the lysozyme molecule contains a higher per cent of alpha-helix segments than at neutral pH. As follows from the measurements of lysozyme distribution in two phase systems the increase in alpha-helicity results in the formation of hydrophobic patches on the surface of the protein molecule. The results of the present work and of the previous studies of the interaction of red- and oxy- form of cytochrome C with phospholipid allow the conclusion that for peripheral proteins the nature of protein-lipid interactions is determined by the protein alpha-helix content and by hydrophobic pattern of the protein molecule surface.  相似文献   

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The mechanism by which radiation-induced genomic instability is initiated, propagated and effected is currently under intense scrutiny. We have investigated the potential role of altered genomic methylation patterns in the cellular response to irradiation and have found evidence for widespread dysregulation of CpG methylation persisting up to 20 population doublings post-irradiation. Similar effects are seen with cells treated with medium from irradiated cells (the 'bystander effect') rather than subjected to direct irradiation. Using an arbitrarily primed methylation sensitive PCR screening method we have demonstrated that irradiation causes reproducible alterations in the methylation profile of a human keratinocyte cell line, HPV-G, and have further characterised one of these sequences as being a member of a retrotransposon element derived sequence family on chromosome 7; MLT1A. Multiple changes were also detected in the screen, which indicate that although the response of cells is predominantly hypermethylation, specific hypomethylation occurs as well. Sequence specific changes are also reported in the methylation of the pericentromeric SAT2 satellite sequence. This is the first demonstration that irradiation results in the induction of heritable methylation changes in mammalian cells, and provides a link between the various non-radiological instigators of genomic instability, the perpetuation of the unstable state and several of its manifestations.  相似文献   

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BACKGROUND: Base excision repair initiated by human thymine-DNA glycosylase (TDG) results in the generation of abasic sites (AP sites) in DNA. TDG remains bound to this unstable repair intermediate, indicating that its transmission to the downstream-acting AP endonuclease is a coordinated process. Previously, we established that posttranslational modification of TDG with Small Ubiquitin-like MOdifiers (SUMOs) facilitates the dissociation of the DNA glycosylase from the product AP site, but the underlying molecular mechanism remained unclear. RESULTS: We now show that upon DNA interaction, TDG undergoes a dramatic conformational change, which involves its flexible N-terminal domain and accounts for the nonspecific DNA binding ability of the enzyme. This function is required for efficient processing of the G.T mismatch but then cooperates with the specific DNA contacts established in the active site pocket of TDG to prevent its dissociation from the product AP site after base release. SUMO1 conjugation to the C-terminal K330 of TDG modulates the DNA binding function of the N terminus to induce dissociation of the glycosylase from the AP site while it leaves the catalytic properties of base release in the active site pocket of the enzyme unaffected. CONCLUSION: Our data provide insight into the molecular mechanism of SUMO modification mediated modulation of enzymatic properties of TDG. A conformational change, involving the N-terminal domain of TDG, provides unspecific DNA interactions that facilitate processing of a wider spectrum of substrates at the expense of enzymatic turnover. SUMOylation then reverses this structural change in the product bound TDG.  相似文献   

6.
A review of the literature on intracellular accumulation and distribution of anticancer drugs in sensitive and resistant tumor cells with the classic mechanism of multidrug resistance development, as well as on the role of ABC-transporters in the processes is presented. According to the data discussed the authors prove that clinical analysis of human tumor multidrug resistance phenotype always needs separate estimation of the functional activity of ABC-transporters, which control accumulation of MDR drugs in the cytoplasm and nucleus. The authors suggest a new term--"severity" of tumor phenotype of multidrug resistance.  相似文献   

7.
The influence of the human tumor suppressor PTEN on sensitivity of tumor cells to cytostatic drugs was studied. Rat ras-transformed (N-ras Asp12 ) fibroblasts were stably transfected with a full-size PTEN gene. Transfected clone was characterized by an enhanced expression of PTEN and a more normal phenotype in comparison with the parental cells. The effect of transient transfection with PTEN on the sensitivity of several malignant cell lines to the cytostatic drugs colchicine and adriablastine was studied. These drugs differ from each other in action mechanisms and intracellular targets. The tumor cell lines tested in this study included parental cell lines and stable sublines possessing drug resistance due to overexpression of P-glycoprotein. In all cell lines, introduction of exogenous PTEN caused a decrease in proliferation rates. This indicated that transgene was active. The chemosensitivity of some drug-resistant sublines was changed after PTEN transfection, but the drug sensitivity of parental cell lines remained unaffected. The effect of PTEN overexpression on chemosensitivity of malignant cells to cytostatic drugs was found to depend both on their mechanisms of action and on the origin of transfected cells. Our data suggest that PTEN is involved into the molecular mechanisms of drug resistance in cells studied.  相似文献   

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Endometriosis is characterized by infertility and pelvic pain in 10-15% of women of reproductive age. The genetic events involved in endometriotic cell expansion remain in large part unknown. To identify genomic changes involved in development of this disease, we examined a panel of 18 selected endometriotic tissues by comparative genomic hybridization (CGH), a molecular cytogenetic method that allows screening of the entire genome for chromosomal gains and/or losses. The study was performed on native, nonamplified DNA extracted from manually dissected endometriotic lesions. Recurrent copy number losses on several chromosomes were detected in 15 of 18 cases. Loss of chromosome 1p and 22q were detected in 50% of the cases. Additional common losses occurred on chromosomes 5p (33%), 6q (27%), 7p(22%), 9q (22%), 16 (22%) as well as on 17q in one case. Gain of DNA sequences were seen at 6q, 7q and 17q in three cases. To validate the CGH data, selective dual-color FISH was performed using probes for the deleted regions on chromosomes 1, 7 and 22 in parallel with the corresponding centromeric probes. Cases showing deletion by CGH all had two signals at 1p36, 7p22.1 and 22q12 in less than 30% of the nuclei in comparison to the double centromeric labels found in more than 85% of the cells. These findings indicate that genes localized to previously undescribed chromosomal regions play a role in development and progression of endometriosis.  相似文献   

12.
DNA methylation reprogramming, the erasure of DNA methylation patterns shortly after fertilization and their reestablishment during subsequent early development, is essential for proper mammalian embryogenesis. In contrast, the importance of this process in the development of non-mammalian vertebrates such as fish is less clear. Indeed, whether or not any widespread changes in DNA methylation occur at all during cleavage and blastula stages of fish in a fashion similar to that shown in mammals has remained controversial. Here we have addressed this issue by applying the techniques of Southwestern immunoblotting and immunohistochemistry with an anti-5-methylcytosine antibody to the examination of DNA methylation in early zebrafish embryos. These techniques have recently been utilized to demonstrate that development-specific changes in genomic DNA methylation also occur in Drosophila melanogaster and Dictyostelium discoideum, both organisms for which DNA methylation was previously not thought to occur. Our data demonstrate that genome-wide changes in DNA methylation occur during early zebrafish development. Although zebrafish sperm DNA is strongly methylated, the zebrafish genome is not detectably methylated through cleavage and early blastula stages but is heavily remethylated in blastula and early gastrula stages.  相似文献   

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DNA labeled for 15 minutes during UV induced repair synthesis is two-fold more sensitive to micrococcal nuclease than the bulk nuclear DNA. As the length of the labeling period increases from 15 minutes to 4 hours the nuclease sensitivity of repair labeled DNA approaches that of bulk chromatin. Pulse-chase experiments indicate that the nuclease sensitivity of the repaired DNA labeled during a brief pulse decreases with a half-life of about 15 minutes. In contrast to previous interpretations, we consider these results to mean that immediately after synthesis, chromatin labeled during repair has a conformation which renders it more susceptible to nuclease digestion than the bulk chromatin. With time these repaired regions are assembled into a nucleosome structure with normal nuclease sensitivity.  相似文献   

15.
S. Sato  H. Yano 《Protoplasma》1994,179(3-4):172-180
Summary The three-dimensional architecture of a filamentous nucleolar structure, called the “nucleolonema”, was investigated in onion root-tip cells by applying a silver impregnation technique to air-dried cells and serial ultrathin sections. The entire configuration of the nucleolonema was revealed when silver staining was applied to air-dried cells. The nucleolonema was knobbly or segmented along its entire length and showed great variation in thickness. Three categories of nucleolonema were discriminated depending on thickness; each had an average value of 0.5, 1.0, and 1.3 μm, respectively. Some root tips were embedded in Lowicryl K4M resin and cut into serial ultrathin sections about 100 nm thick. When these sections were subjected to silver impregnation, segments of nucleolonema were visualized. Most of them were found to contain achromatic holes. These holes apparently corresponded to the fibrillar centres seen with the electron microscope. According to the profiles of the holes, nucleolonema structures were classified into three types: (1) nucleolonema with no distinct holes, (2) those with beaded holes, and (3) those with cylindrical holes. The thicknesses were 0.7–0.8, 0.9–1.2, or 1.2–1.4 μm for nucleolonemata with no holes, beaded holes, or cylindrical holes, respectively. The argyrophilic wall of nucleolonemata with holes was about 0.4 μm thick, roughly compatible with the thinnest nucleolonema seen in air-dried specimens. The crescent-shaped segments were sometimes observed when the nucleolonema was sectioned transversely, suggesting that the achromatic holes are exposed to the nucleoplasm, in other words, the nucleolonema is partially degraded. Thus, the nucleolonema was not always structurally stable during interphase. The results suggest that the nucleolonemata gradually become knobbly and increase their thickness, with concomitant expansion of the fibrillar centres sometimes degrading into approximately 0.5 μm thick strands.  相似文献   

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Two new complexes [(Etdpa)MnCl2] and [(Adpa)Mn(Cl)(H2O)] (Etdpa = ethyl bis(2-pyridylmethyl)amino-2-propionate; Adpa = bis(2-pyridylmethyl)amino-2-propionic acid) were synthesized and characterized by spectral methods. The crystal structure of [(Etdpa)MnCl2] shows that the Mn(II) atom is coordinated by three N atoms (N1, N2, N3), one oxygen atom (O1) of the ligand (Etdpa) and two chloride atoms (Cl1, Cl2), forming a distorted octahedral geometry. The binding interaction between ct-DNA and the synthesized complexes was relatively weak, but they can inhibit the induced swelling of Ca2+-loaded mitochondria in a dose-dependent manner. The [(Adpa)Mn(Cl)(H2O)] can cause the obvious decrease of mitochondria membrane potential. The MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenpyltetra-zolium bromide) assay shows that the two Mn(II) complexes are more active against cancer cells. Especially [(Adpa)Mn(Cl)(H2O)] can inhibit the proliferation of glioma cells with IC50 9.5 μM. Experimental results indicate that the [(Adpa)Mn(Cl)(H2O)] could be a new potential antitumor complex to target the mitochondria.  相似文献   

18.
Binding of lamda-Cro protein and mutant CroV55C disulfide bonded dimer to synthetic olygonucleotide duplexes were studied using a competition with distamycin A. The equilibrium binding constants for lamda operator OR3 and duplexes contained symmetry left or right halves of OR3 with one base pair deletion or insertion in center of duplex were calculated. The higher binding constant for Cro was detected with 17 bp symmetry duplex consist two left halves of OR3, for the mutant CroV55C higher binding constant was detected with 16 bp derivate of this duplex with the central GC base pair deletion.  相似文献   

19.
Adenovirus virus-associated RNA (VAI) provides protection against the host antiviral response in part by inhibiting the interferon-induced double stranded RNA-activated protein kinase (PKR). VAI consists of three base-paired regions; the apical stem responsible for the interaction with double-stranded RNA binding motifs (dsRBMs) of PKR, the central stem required for inhibition, and the terminal stem. The solution conformation of VAI and VAI lacking the terminal stem were determined using SAXS that suggested extended conformations that are in agreement with their secondary structures. Solution conformations of VAI lacking the terminal stem in complex with the dsRBMs of PKR indicated that the apical stem interacts with both dsRNA-binding motifs whereas the central stem does not. Hydrodynamic properties calculated from ab initio models were compared to experimentally determined parameters for model validation. Furthermore, SAXS envelopes were used as a constraint for the in silico modeling of tertiary structure for RNA and RNA–protein complex. Finally, full-length PKR was also studied, but concentration-dependent changes in hydrodynamic parameters prevented ab initio shape determination. Taken together, results provide an improved structural framework that further our understanding of the role VAI plays in evading host innate immune responses.  相似文献   

20.
The adaptor protein Grb2 is a key element of mitogenetically important signaling pathways. With its SH2 domain it binds to upstream targets while its SH3 domains bind to downstream proteins thereby relaying signals from the cell membranes to the nucleus. The Grb2 SH2 domain binds to its targets by recognizing a phosphotyrosine (pY) in a pYxNx peptide motif, requiring an Asn at the +2 position C‐terminal to the pY with the residue either side of this Asn being hydrophobic. Structural analysis of the Grb2 SH2 domain in complex with its cognate peptide has shown that the peptide adopts a unique β‐turn conformation, unlike the extended conformation that phosphopeptides adopt when bound to other SH2 domains. TrpEF1 (W121) is believed to force the peptide into this unusual conformation conferring this unique specificity to the Grb2 SH2 domain. Using X‐ray crystallography, electron paramagnetic resonance (EPR) spectroscopy, and isothermal titration calorimetry (ITC), we describe here a series of experiments that explore the role of TrpEF1 in determining the specificity of the Grb2 SH2 domain. Our results demonstrate that the ligand does not adopt a pre‐organized structure before binding to the SH2 domain, rather it is the interaction between the two that imposes the hairpin loop to the peptide. Furthermore, we find that the peptide adopts a similar structure when bound to both the wild‐type Grb2 SH2 domain and a TrpEF1Gly mutant. This suggests that TrpEF1 is not the determining factor for the conformation of the phosphopeptide.  相似文献   

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