95 research outputs found
Synthesis, base pairing properties and trans-lesion synthesis by reverse transcriptases of oligoribonucleotides containing the oxidatively damaged base 5-hydroxycytidine
The synthesis of a caged RNA phosphoramidite building block containing the oxidatively damaged base 5-hydroxycytidine (5-HOrC) has been accomplished. To determine the effect of this highly mutagenic lesion on complementary base recognition and coding properties, this building block was incorporated into a 12-mer oligoribonucleotide for Tm and CD measurements and a 31-mer template strand for primer extension experiments with HIV-, AMV- and MMLV-reverse transcriptase (RT). In UV-melting experiments, we find an unusual biphasic transition with two distinct Tm's when 5-HOrC is paired against a DNA or RNA complement with the base guanine in opposing position. The higher Tm closely matches that of a C-G base pair while the lower is close to that of a C-A mismatch. In single nucleotide extension reactions, we find substantial misincorporation of dAMP and to a lesser extent dTMP, with dAMP almost equaling that of the parent dGMP in the case of HIV-RT. A working hypothesis for the biphasic melting transition does not invoke tautomeric variability of 5-HOrC but rather local structural perturbations of the base pair at low temperature induced by interactions of the 5-HO group with the phosphate backbone. The properties of this RNA damage is discussed in the context of its putative biological function
A genotyping strategy based on incorporation and cleavage of chemically modified nucleotides
Structure of the uncomplexed DNA repair enzyme endonuclease VIII indicates significant interdomain flexibility
Escherichia coli endonuclease VIII (Nei) excises oxidized pyrimidines from DNA. It shares significant sequence homology and similar mechanism with Fpg, a bacterial 8-oxoguanine glycosylase. The structure of a covalent Nei–DNA complex has been recently determined, revealing critical amino acid residues which are important for DNA binding and catalysis. Several Fpg structures have also been reported; however, analysis of structural dynamics of Fpg/Nei family proteins has been hindered by the lack of structures of uncomplexed and DNA-bound enzymes from the same source. We report a 2.8 Å resolution structure of free wild-type Nei and two structures of its inactive mutants, Nei-E2A (2.3 Å) and Nei-R252A (2.05 Å). All three structures are virtually identical, demonstrating that the mutations did not affect the overall conformation of the protein in its free state. The structures show a significant conformational change compared with the Nei structure in its complex with DNA, reflecting a ∼50° rotation of the two main domains of the enzyme. Such interdomain flexibility has not been reported previously for any DNA glycosylase and may present the first evidence for a global DNA-induced conformational change in this class of enzymes. Several local but functionally relevant structural changes are also evident in other parts of the enzyme
Сравнительное исследование фармакокинетики и биораспределения карбазольного соединения CBL0100 в составе различных топических лекарственных форм на морских свинках
An experimental drug CBL0100 is developed as an antifungal agent for topical application. In order to assess the effect of composition of the formulation on transdermal absorption, a comparative absorption study of its pharmacokinetics and biodistribution after a single topical application on the guinea pigs' skin. Two different formulations of CBL0100 (prototypes of final dosage forms for topical applications) - cream emulsion and polyethylene glycol vehicles with a content of active substance 1, 0,2 and 0,02% mass. in each of these formulations were used in the study. CBL0100 concentration in blood plasma and homogenates of organs and tissues of animals was analyzed by HPLC - MS/MS. During the preliminary blood-to-plasma partitioning in vitro experiment it was determined that CBL0100 is found mostly in the cell fraction of the blood, which, based upon the pharmacokinetic curves shape affects elimination from plasma. It was revealed that after a single topical application active compound enters the systemic circulation and intensively distributed into the spleen, liver, kidneys and lungs of guinea pigs. At the same time, its content in the muscular tissue and the heart is negligible; CBL0100 moderately penetrates into the brain. In general, it was shown that the emulsion composition provides improved bioavailability of CBL0100 compared to the polyethylene glycol composition.Экспериментальный препарат CBL0100 разрабатывается в качестве противогрибкового средства для местного применения. Для оценки влияния состава лекарственной формы на трансдермальное всасывание было проведено сравнительное исследование его фармакокинетики и биораспределения после однократного нанесения на кожу морских свинок. В исследовании использовали две различные прописи CBL0100 (прототипы готовых лекарственных форм для топических аппликаций) - крема на эмульсионной (ЭМ) и полиэтиленгликолевой (ПЭГ) основах с содержанием активного вещества 1, 0,2 и 0,02% по массe. Концентрацию активного вещества в плазме крови и гомогенатах органов и тканей животных определяли методом ВЭЖХ-МС/ МС. В предварительном эксперименте по изучению распределения CBL0100 между плазмой и клеточными компонентами крови in vitro установлено, что исследуемое соединение преимущественно обнаруживается в клеточной фракции крови, что, судя по форме фармакокинетических кривых, влияет на скорость его элиминации из плазмы. Показано, что после однократного накожного нанесения крема CBL0100 проникает в системный кровоток и интенсивно распределяется в селезёнку, печень, почки и лёгкие морских свинок. В то же время его содержание в мышечной ткани и сердце незначительно; в мозг CBL0100 проникает умеренно. Установлено, что пропись на ЭМ основе обеспечивает повышенную по сравнению ПЭГ основой биодоступность CBL0100
Development of a ‘clickable’ non-natural nucleotide to visualize the replication of non-instructional DNA lesions
The misreplication of damaged DNA is an important biological process that produces numerous adverse effects on human health. This report describes the synthesis and characterization of a non-natural nucleotide, designated 3-ethynyl-5-nitroindolyl-2′-deoxyriboside triphosphate (3-Eth-5-NITP), as a novel chemical reagent that can probe and quantify the misreplication of damaged DNA. We demonstrate that this non-natural nucleotide is efficiently inserted opposite an abasic site, a commonly formed and potentially mutagenic non-instructional DNA lesion. The strategic placement of the ethynyl moiety allows the incorporated nucleoside triphosphate to be selectively tagged with an azide-containing fluorophore using ‘click’ chemistry. This reaction provides a facile way to quantify the extent of nucleotide incorporation opposite non-instructional DNA lesions. In addition, the incorporation of 3-Eth-5-NITP is highly selective for an abasic site, and occurs even in the presence of a 50-fold molar excess of natural nucleotides. The biological applications of using 3-Eth-5-NITP as a chemical probe to monitor and quantify the misreplication of non-instructional DNA lesions are discussed
Unexpected Role for Helicobacter pylori DNA Polymerase I As a Source of Genetic Variability
Helicobacter pylori, a human pathogen infecting about half of the world population, is characterised by its large intraspecies variability. Its genome plasticity has been invoked as the basis for its high adaptation capacity. Consistent with its small genome, H. pylori possesses only two bona fide DNA polymerases, Pol I and the replicative Pol III, lacking homologues of translesion synthesis DNA polymerases. Bacterial DNA polymerases I are implicated both in normal DNA replication and in DNA repair. We report that H. pylori DNA Pol I 5′- 3′ exonuclease domain is essential for viability, probably through its involvement in DNA replication. We show here that, despite the fact that it also plays crucial roles in DNA repair, Pol I contributes to genomic instability. Indeed, strains defective in the DNA polymerase activity of the protein, although sensitive to genotoxic agents, display reduced mutation frequencies. Conversely, overexpression of Pol I leads to a hypermutator phenotype. Although the purified protein displays an intrinsic fidelity during replication of undamaged DNA, it lacks a proofreading activity, allowing it to efficiently elongate mismatched primers and perform mutagenic translesion synthesis. In agreement with this finding, we show that the spontaneous mutator phenotype of a strain deficient in the removal of oxidised pyrimidines from the genome is in part dependent on the presence of an active DNA Pol I. This study provides evidence for an unexpected role of DNA polymerase I in generating genomic plasticity
Противоопухолевое действие кураксина CBL0137 на моделях аденокарциномы толстой кишки
Curaxins represent low molecular weight carbazole derivatives, which simultaneously activate p53-dependent apoptosis and inhibit NF-kBdependent signal transduction pathways. Antitumor activity of curaxin CBL0137 was demonstrated in vivo on the model of solid transplantable mouse colon adenocarcinoma Akatol. Significant tumor growth inhibition caused by CBL0137 treatment was observed. On the 37th day after tumor transplantation for CBL0137 oral doses 5, 10, 15 and 20 mg/kg the tumor growth inhibition indexes were 53, 50, 56 and 74 %, respectively. CBL0137 treatment extended life span of mice in all study groups receiving this test article. Maximum lifespan increase (81,7 %) was observed in the group of mice treated with 20 mg/kg dose of CBL0137. The cytotoxicity of CBL0137 was assessed on human colon cancer cells in vitro. We demonstrated that CBL0137 inhibits the expression COX2, which in its turn is known to exhibit antiapoptotic, pro-angiogenic and pro- metastatic properties. Thus, we demonstrated antitumor effect of CBL0137 against colon cancer in vitro and in vivo.Кураксины представляют собой низкомолекулярные карбазольные производные, обладающие афинностью к ДНК, способные одновременно активировать р53-зависимый апоптоз и ингибировать NF-kB-зависимый сигнальный путь. На модели перевиваемой аденокарциномы толстой кишки (Aкатол) на мышах линии BALB/c продемонстрировано противоопухолевое действие кураксина CBL0137. Наблюдалось значительное тормозящее действие CBL0137 на рост данной аллографтной опухоли: на 37-е сутки после перевивки опухоли при пероральном введении препарата в дозах 5, 10, 15 и 20 мг/кг в день торможение роста опухоли составило 53, 50, 56 и 74 % соответственно. Значимое увеличение продолжительности жизни наблюдалось во всех группах животных, получавших CBL0137. Максимальный эффект (81,7 %) был получен при введении препарата в дозе 20 мг/кг в день. В экспериментах in vitro при обработке клеток аденокарциномы толстой кишки человека HTC116 наблюдался значительный цитотоксический эффект. CBL0137 проявил способность ингибировать экспрессию гена COX2, обладающего антиапоптотическим действиеми способностью стимулировать неоангиогенез и метастазирование. Таким образом, в системах in vivo и in vitro продемонстрировано противоопухолевое действие CBL0137 в отношении аденокарциномы толстой кишки
Location of abasic sites in oligodeoxynucleotides by tandem mass spectrometry and by a chemical cleavage initiated by an unusual reaction of the ODN with MALDI matrix
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