The unwinding free energy of 128 DNA octamers was correlated with the sum of interaction energies among DNA bases and their solvation energies. The former energies were determined by using the recently developed density functional theory procedure augmented by London dispersion energy (RI-DFT-D) that provides accurate hydrogen-bonding and stacking energies highly comparable with CCSD(T)/complete basis set limit benchmark data. Efficient tight-binding DFT covering dispersion energy was also used and yielded satisfactory results. The latter method can be used for extended systems. The solvation energy was determined by using a C-PCM continuum solvent at HF level calculations. Various models were adopted to correlate theoretical energies with experimental unwinding free energies. Unless all energy components (hydrogen-bonding, intra- and interstrand-stacking, and solvation energies) were included and weighted individually, no satisfactory correlation resulted. The most advanced model yielded very close correlation (RMSE=0.32 kcal mol(-1)) fully comparable with the entirely empirical correlation introduced in the original paper. Analysis of the theoretical results shows the importance of inter- and intramolecular stacking energies, and especially the latter term plays a key role in determining DNA-duplex stabilization.
Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
Nestr.
Projekt je zaměřen na studium bázových excizních oprav (BER) na buněčné i molekulární úrovni ve vztahu k strukturní a funkční organizaci aktivních replikonů a na cílené poškození BER s cílem nalézt nové možnosti léčby rakoviny. Očekáváme, že interakce enzymů zapojených v BER s modelovými duplexy DNA modifikovanými v kritických místech strukturně odlišnými nukleotidovými jednotkami (nukleosidové analogy a/nebo fosfonátové typy internukleotidových vazeb) umožní získání poznatků nutných pro cílené poškození BER systému. Projekt je navržen s cílem pokrýt celou dráhu od navržení a syntézy nukleosidových, nukleotidových a oligonukleotidových analogů, přes analýzu nukleosidových a nukleotidových koncentrací a mikroskopické analýzy DNA replikace a replikačních aparátů až po molekulární analýzu izolovaných DNA/proteinových komplexů.; The project is focused on the study of base excision repair (BER) at both cellular and molecular levels with respect to the structural and functional organization of active replicons and the targeted impairment of BER with the aim to find new possibilities of cancer treatment. We anticipate that interactions of BER enzymes with the model DNA duplexes modified in critical sites with the structurally diverse unnatural nucleotide units (nucleoside analogs and/or phosphonate types of the internucleotide linkages) enable the achievement of fundamental knowledge on the targeted impairment of base excision repair. The project is designed with the aim of covering the whole pathway from the design and synthesis of nucleoside, nucleotide, and oligonucleotide analogues, through the analysis of nucleoside and nucleotide pools, microscopy analysis of DNA replication and repair machineries up to the molecular analysis of isolated protein-DNA complexes.
- MeSH
- DNA Glycosylases MeSH
- Mass Spectrometry MeSH
- Microscopy MeSH
- Neoplasms therapy MeSH
- Nucleosides MeSH
- DNA Repair genetics MeSH
- Organophosphonates MeSH
- DNA Repair-Deficiency Disorders genetics MeSH
- DNA Replication MeSH
- Replicon MeSH
- Conspectus
- Patologie. Klinická medicína
- NML Fields
- onkologie
- genetika, lékařská genetika
- NML Publication type
- závěrečné zprávy o řešení grantu AZV MZ ČR
Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA cleavage and ATP-dependent DNA translocation activities located on motor subunit HsdR. Functional coupling of DNA cleavage and translocation is a hallmark of the Type I restriction systems that is consistent with their proposed role in horizontal gene transfer. DNA cleavage occurs at nonspecific sites distant from the cognate recognition sequence, apparently triggered by stalled translocation. The X-ray crystal structure of the complete HsdR subunit from E. coli plasmid R124 suggested that the triggering mechanism involves interdomain contacts mediated by ATP. In the present work, in vivo and in vitro activity assays and crystal structures of three mutants of EcoR124I HsdR designed to probe this mechanism are reported. The results indicate that interdomain engagement via ATP is indeed responsible for signal transmission between the endonuclease and helicase domains of the motor subunit. A previously identified sequence motif that is shared by the RecB nucleases and some Type I endonucleases is implicated in signaling.
- MeSH
- Adenosine Triphosphate chemistry metabolism MeSH
- DNA, Bacterial MeSH
- Escherichia coli genetics metabolism MeSH
- Exodeoxyribonuclease V chemistry genetics metabolism MeSH
- Gene Expression MeSH
- Nucleic Acid Conformation MeSH
- Crystallography, X-Ray MeSH
- Models, Molecular MeSH
- Mutation MeSH
- Plasmids chemistry metabolism MeSH
- Protein Subunits chemistry genetics metabolism MeSH
- Protein Sorting Signals MeSH
- Escherichia coli Proteins chemistry genetics metabolism MeSH
- Deoxyribonucleases, Type I Site-Specific chemistry genetics metabolism MeSH
- Signal Transduction MeSH
- DNA Cleavage MeSH
- Protein Structure, Tertiary MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Our 200ns MD simulations show that even fully modified oligonucleotides bearing the 3'-O-P-CH2-O-5' (but not 3'-O-CH2-P-O-5') phosphonate linkages can be successfully attached to the surface of Human RNase H. It enables to explain that oligonucleotides consisting of the alternating 3'-O-P-CH2-O-5' phosphonate and phosphodiester linkages are capable to elicit the RNase H activity (while the 3'-O-CH2-P-O-5' phosphonates are completely inactive). Stability of the binuclear active site of Human RNase H was achieved using the one-atom model for Mg(2+) in conjunction with a polarized phosphate group of the scissile bond, which is wedged between both magnesium ions. The reference MD simulation (lasting for 1000ns), which was produced using a well-established seven-point (with dummy atoms) model for Mg(2+) led to essentially the same results. The MD run (lasting for 500ns) produced for the Thermus thermophilus Argonaute enzyme shows the transferability of our approach for the stabilization of a binuclear active site. Glu512 was bound in the T. thermophilus Argonaute active site to the 2'-OH of the nucleotide adjacent to the scissile phosphate and one of the two active-site divalent metal ions in exactly the same way as Glu186 in Human RNase H. Glu512 thus completes the catalytic tetrad of Argonaute.
- MeSH
- DNA chemistry MeSH
- Catalytic Domain MeSH
- Nucleic Acid Conformation MeSH
- Protein Conformation MeSH
- Humans MeSH
- Ligands MeSH
- Organophosphonates chemistry MeSH
- Ribonuclease H chemistry MeSH
- RNA chemistry MeSH
- Molecular Dynamics Simulation * MeSH
- Protein Binding MeSH
- Binding Sites MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Mutations can be induced by environmental factors but also arise spontaneously during DNA replication or due to deamination of methylated cytosines at CpG dinucleotides. Sites where mutations occur with higher frequency than would be expected by chance are termed hotspots while sites that contain mutations rarely are termed coldspots. Mutations are permanently scanned and repaired by repair systems. Among them, the mismatch repair targets base pair mismatches, which are discriminated from canonical base pairs by probing altered elasticity of DNA. Using biased molecular dynamics simulations, we investigated the elasticity of coldspots and hotspots motifs detected in human genes associated with inherited disorders, and also of motifs with Czech population hotspots and de novo mutations. Main attention was paid to mutations leading to G/T and A+/C pairs. We observed that hotspots without CpG/CpHpG sequences are less flexible than coldspots, which indicates that flexible sequences are more effectively repaired. In contrary, hotspots with CpG/CpHpG sequences exhibited increased flexibility as coldspots. Their mutability is more likely related to spontaneous deamination of methylated cytosines leading to C > T mutations, which are primarily targeted by base excision repair. We corroborated conclusions based on computer simulations by measuring melting curves of hotspots and coldspots containing G/T mismatch.
- MeSH
- CpG Islands MeSH
- DNA chemistry genetics MeSH
- Humans MeSH
- Mutation * MeSH
- Nucleotide Motifs * MeSH
- Molecular Dynamics Simulation * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Oligonucleotides conduct electric charge via various mechanisms and their characterization and understanding is a very important and complicated task. In this work, experimental (temperature dependent steady state fluorescence spectroscopy, time-resolved fluorescence spectroscopy) and theoretical (Density Functional Theory) approaches were combined to study charge transfer processes in short DNA/DNA and RNA/DNA duplexes with virtually equivalent sequences. The experimental results were consistent with the theoretical model - the delocalized nature of HOMO orbitals and holes, base stacking, electronic coupling and conformational flexibility formed the conditions for more effective short distance charge transfer processes in RNA/DNA hybrids. RNA/DNA and DNA/DNA charge transfer properties were strongly connected with temperature affected structural changes of molecular systems - charge transfer could be used as a probe of even tiny changes of molecular structures and settings.
