NMR solution structure of an oxaliplatin 1,2-d(GG) intrastrand cross-link in a DNA dodecamer duplex

We have determined, at high resolution, the NMR solution structure of an oxaliplatin-GG DNA dodecamer in the AGGC sequence context by 2D NMR studies. Homonuclear assignment strategies resulted in unambiguous assignment of 203 out of 249 protons, which corresponds to assignment of approximately 81% of the protons. Assignments of H5' and H5" protons were tentative due to resonance overlap. The structure of the oxaliplatin duplex was calculated using the program CNS with a simulated annealing protocol. A total of 510 experimental restraints were employed in the structure calculation. Of 20 calculated structures, the 15 with the lowest energy were accepted as a family. The RMSD of the 15 lowest energy structures was 0.68 A, indicating good structural convergence. The theoretical NOESY spectrum obtained by back-calculation from the final average structure showed excellent agreement with the experimental data, indicating that the final structure was in good agreement with the experimental NMR data. Significant conformational differences were observed between the oxaliplatin-GG 12-mer DNA we studied and all previous solution structures of cisplatin-GG DNA duplexes. For example, the oxaliplatin-GG adduct shows much less distortion at the AG base-pair step than the cisplatin-GG adducts. In addition, the oxaliplatin-GG structure also has a narrow minor groove and an overall axis bend of about 31 degrees, both of which are very different from the recent NMR structures for the cisplatin-GG adducts. These structural differences may explain some of the biological differences between oxaliplatin- and cisplatin-GG adducts.     

NMR structure of an intracellular loop peptide derived from prostaglandin EP3alpha receptor

We found that a peptide (EP3a: TIKALVSRCRAKAAV) corresponding to the N-terminal site of the intracellular third loop of human prostaglandin EP3α receptor could activate G protein α-subunit directly. The activity was almost same as Mastoparan-X, a G protein activating peptide from wasp venom. The three-dimensional molecular structure of the peptide in SDS-d₂₅ micelles was determined by 2D ¹H NMR spectroscopy. The structure of EP3a consists of a positive charge cluster on the C-terminal helical site. The cluster was also found in several corresponding receptor peptides. Therefore, the positive charge cluster on the helical structure might play a crucial role in activation of G protein.     

Metropolis Monte Carlo calculations of DNA structure using internal coordinates and NMR distance restraints: An alternative method for generating a high-resolution solution structure

Summary A new method, a restrained Monte Carlo (rMC) calculation, is demonstrated for generating high-resolution structures of DNA oligonucleotides in solution from interproton distance restraints and bounds derived from complete relaxation matrix analysis of two-dimensional nuclear Overhauser effect (NOE) spectral peak intensities. As in the case of restrained molecular dynamics (rMD) refinement of structures, the experimental distance restraints and bounds are incorporated as a pseudo-energy term (or penalty function) into the mathematical expression for the molecular energy. However, the use of generalized helical parameters, rather than Cartesian coordinates, to define DNA conformation increases efficiency by decreasing by an order of magnitude the number of parameters needed to describe a conformation and by simplifying the potential energy profile. The Metropolis Monte Carlo method is employed to simulate an annealing process. The rMC method was applied to experimental 2D NOE data from the octamer duplex d(GTA-TAATG)·d(CATTATAC). Using starting structures from different locations in conformational space (e.g. A-DNA and B-DNA), the rMC calculations readily converged, with a root-mean-square deviation (RMSD) of <0.3 Å between structures generated using different protocols and starting structures. Theoretical 2D NOE peak intensities were calculated for the rMC-generated structures using the complete relaxation matrix program CORMA, enabling a comparison with experimental intensities via residual indices. Simulation of the vicinal proton coupling constants was carried out for the structures generated, enabling a comparison with the experimental deoxyribose ring coupling constants, which were not utilized in the structure determination in the case of the rMC simulations. Agreement with experimental 2D NOE and scalar coupling data was good in all cases. The rMC structures are quite similar to that refined by a traditional restrained MD approach (RMSD<0.5 Å) despite the different force fields used and despite the fact that MD refinement was conducted with additional restraints imposed on the endocyclic torsion angles of deoxyriboses. The computational time required for the rMC and rMD calculations is about the same. A comparison of structural parameters is made and some limitations of both methods are discussed with regard to the average nature of the experimental restraints used in the refinement.Abbreviations MC Monte Carlo - rMC restrained Monte Carlo - MD molecular dynamics - rMD restrained molecular dynamics - DG distance geometry - EM energy minimization - 2D NOE two-dimensional nuclear Overhauser effect - DQF-COSY double-quantum-filtered correlation spectroscopy - RMSD root-mean-square deviationTo whom correspondence should be addressed.     

Spatial distribution of GC- and AT-rich DNA sequences within human chromosome territories

Previous topological analyses of DNA sequence organization in the interphase chromosome mainly focused on the spatial distribution of individual gene copies within chromosome territories. In order to achieve a more comprehensive view into the subchromosomal arrangement of DNA, we isolated the GC-richest/gene-richest fraction (H3 isochores) as well as AT-richest/gene-poorest fraction of human genomic DNA (L1+L2 isochores) and visualized the respective DNA within individual chromosome territories by means of dual-color FISH. Application of confocal laser scanning microscopy and dedicated 3D image analysis software, which differentiated territory subvolumes by peeling shells one voxel in width, revealed a significant difference in the intraterritorial distribution of these two DNA sequence classes. While the H3 isochores were found localized in all subvolumes of the territories at similar frequency, simultaneously detected L1+L2 isochores were observed more to the interior of the same chromosome territories. Thus the GC-rich sequences display a much higher variability in their intraterritorial localization than AT-rich DNA fragments.     

HMG-D complexed to a bulge DNA: an NMR model

An NMR model is presented for the structure of HMG-D, one of the DROSOPHILA: counterparts of mammalian HMG1/2 proteins, bound to a particular distorted DNA structure, a dA(2) DNA bulge. The complex is in fast to intermediate exchange on the NMR chemical shift time scale and suffers substantial linebroadening for the majority of interfacial resonances. This essentially precludes determination of a high-resolution structure for the interface based on NMR data alone. However, by introducing a small number of additional constraints based on chemical shift and linewidth footprinting combined with analogies to known structures, an ensemble of model structures was generated using a computational strategy equivalent to that for a conventional NMR structure determination. We find that the base pair adjacent to the dA(2) bulge is not formed and that the protein recognizes this feature in forming the complex; intermolecular NOE enhancements are observed from the sidechain of Thr 33 to all four nucleotides of the DNA sequence step adjacent to the bulge. Our results form the first experimental demonstration that when binding to deformed DNA, non-sequence-specific HMG proteins recognize the junction between duplex and nonduplex DNA. Similarities and differences of the present structural model relative to other HMG-DNA complex structures are discussed.     

DNA sequence,structure, and phylogenetic relationship of the small subunit rRNA coding region of mitochondrial DNA fromPodospora anserina

Summary DNA sequence analysis and the localization of the 5 and 3 termini by S1 mapping have shown that the mitochondrial (mt) small subunit rRNA coding region fromPodospora anserina is 1980 bp in length. The analogous coding region for mt rRNA is 1962 bp in maize, 1686 bp inSaccharomyces cerevisiae, and 956 bp in mammals, whereas its counterpart inEscherichia coli is 1542 bp. TheP. anserina mt 16S-like rRNA is 400 bases longer than that fromE. coli, but can be folded into a similar secondary structure. The additional bases appear to be clustered at specific locations, including extensions at the 5 and 3 termini. Comparison with secondary structure diagrams of 16S-like RNAs from several organisms allowed us to specify highly conserved and variable regions of this gene. Phylogenetic tree construction indicated that this gene is grouped with other mitochondrial genes, but most closely, as expected, with the fungal mitochondrial genes.     

Insertion sequence IS5 contains a sharply curved DNA structure at its terminus

Summary It was demonstrated that insertion sequence IS5 contains a sequence-directed bent (sharply curved) DNA structure at its terminus, close to one of its 16 bp terminal repeats. The minimal number of copies of IS5 related sequences and the locations of the latter on the Escherichia coli K12 W3110 chromosome were determined. Evidence is presented of the occurrence of IS5 mediated translocation and duplication of a large DNA segment on the E. coli chromosome.     

NMR structure of a 4 x 4 nucleotide RNA internal loop from an R2 retrotransposon: identification of a three purine-purine sheared pair motif and comparison to MC-SYM predictions

The NMR solution structure is reported of a duplex, 5'GUGAAGCCCGU/3'UCACAGGAGGC, containing a 4 × 4 nucleotide internal loop from an R2 retrotransposon RNA. The loop contains three sheared purine-purine pairs and reveals a structural element found in other RNAs, which we refer to as the 3RRs motif. Optical melting measurements of the thermodynamics of the duplex indicate that the internal loop is 1.6 kcal/mol more stable at 37°C than predicted. The results identify the 3RRs motif as a common structural element that can facilitate prediction of 3D structure. Known examples include internal loops having the pairings: 5'GAA/3'AGG, 5'GAG/3'AGG, 5'GAA/3'AAG, and 5'AAG/3'AGG. The structural information is compared with predictions made with the MC-Sym program.     

Molecular Dynamics Simulation in Solvent of the Estrogen Receptor Protein DNA Binding Domain in Complex with a Non-Consensus Estrogen Response Element DNA Sequence

Abstract

We investigated protein/DNA interactions, using molecular dynamics simulations computed between a 10 Angstom water layer model of the estrogen receptor (ER) protein DNA binding domain (DBD) amino acids and DNA of a non-consensus estrogen response element (ERE) consisting of 29 nucleotide base pairs. This ERE nucleotide sequence occurs naturally upstream of the Xenopus laevis Vitelligenin AI gene. The ER DBD is encoded by three exons. Namely, exons 2 and 3 which encode the two zinc binding motifs and a sequence of exon 4 which encodes a predicted alpha helix. We generated a computer model of the ER DBD using atomic coordinates derived from the average of 30 nuclear magnetic resonance (NMR) spectroscopy coordinate sets. Amino acids on the carboxyl end of the ER DBD were disordered in both X-ray crystallography and NMR determinations and no coordinates were reported. This disordered region includes 10 amino acids of a predicted alpha helix encoded in exon 4 at the exon 3/4 splice junction. These amino acids are known to be important in DNA binding and are also believed to function as a nuclear translocation signal sequence for the ER protein. We generated a computer model of the predicted alpha helix consisting of the 10 amino acids encoded in exon 4 and attached this helix to the carboxyl end of the ER DBD at the exon 3/4 splice junction site. We docked the ER DBD model within the DNA major groove halfsites of the 29 base pair non-consensus ERE and flanking nucleotides. We constructed a solvated model with the ER DBD/ERE complex surrounded by a ten Angstrom water layer and conducted molecular dynamics simulations. Hydrogen bonding interactions were monitored. In addition, van der Waals and electrostatic interaction energies were calculated. Amino acids of the ER DBD DNA recognition helix formed both direct and water mediated hydrogen bonds at cognate codon-anticodon nucleotide base and backbone sites within the ERE DNA right major groove halfsite. Amino acids of the ER DBD exon 4 encoded predicted alpha helix formed direct and water mediated H-bonds with base and backbone sites of their cognate codon-anticodon nucleotides within the minor grooves flanking the ERE DNA major groove halfsites. These interactions together induced bending of the DNA into the protein.     

NMR solution structure of the acylphosphatase from Escherichia coli

The solution structure of Escherichia coli acylphosphatase (E. coli AcP), a small enzyme catalyzing the hydrolysis of acylphosphates, was determined by (1)H and (15)N NMR and restrained modelling calculation. In analogy with the other members of AcP family, E. coli AcP shows an alpha/beta sandwich domain composed of four antiparallel and one parallel beta-strand, assembled in a five-stranded beta-sheet facing two antiparallel alpha-helices. The pairwise RMSD values calculated for the backbone atoms of E. coli and Sulfolobus solfataricus AcP, Bovine common type AcP and Horse muscle AcP are 2.18, 5.31 and 5.12 A, respectively. No significant differences are present in the active site region and the catalytic residue side chains are consistently positioned in the structures.     

NMR structure of a human homologous methuselah gene receptor peptide

Human APG1 gene is homologous to Drosophila methuselah gene associated with extended life span. A peptide (APG1: RNGKRSNRTLREE) corresponding to a predicted region of the intracellular third loop of G protein-coupled receptor coded in human APG1 gene could activate Gi protein alpha subunit directly. The three-dimensional molecular structure of the peptide in SDS-d25 micelles was determined by 2D 1H NMR spectroscopy. APG1 formed an alpha-helical structure at the C-terminal site and a positive charge cluster at the N-terminal site. The cluster was also found in several other Gi protein-coupled receptor peptides. Therefore, the positive charge cluster on the helical structure might be engaged in G protein activation.     

Presence of an SAR-like sequence in junction regions between an introduced transgene and genomic DNA of cultured tobacco cells: its effect on transformation frequency

A 12.5-kb DNA fragment with junction regions between the transgene and genomic DNA was cloned from a transgenic tobacco cell line obtained by microprojectile bombardment of plasmid pCaMVNEO. Nucleotide sequence analysis of the fragment (DDBJ accession no. D84238) showed that it carried a 7.7-kb core sequence (concatemer of a complete pCaMVNEO and a partial pCaMVNEO) and two identical 1.3-kb junction sequences that flanked both the 5' and 3' ends of the core sequence and had inverted orientations. These sequences had topoisomerase II (Topo II) cleavage sites and adenine and thimine-rich sequences known to be specific to nuclear scaffold-attachment regions (SARs). An in vitro binding assay showed that a 507-bp fragment (designated TJ1) from the 1.3-kb sequence had the ability to bind to nuclear scaffold preparations of cultured tobacco cells, confirmation that the 1.3-kb sequence is an SAR. Insertion of TJ1 at the 5' and 3' sides of the expression cassette for the npt II gene increased transformant yields 5- to 10-fold and the NPT II enzyme activity per copy of the gene 5-fold. TJ1 enhances the integration or expression of the transgene, or both. Clearly, TJ1 is very useful for producing transgenic plants. This is the first report on an SAR-like sequence that is located in the transgene locus and enhances transformation efficiency in eukaryotic cells. The possible role of TJ1-SAR in the molecular evolution of plant genome is discussed.     

DNA序列分析中的信息熵应用现状

信息熵理论是生物信息学研究的一个重要工具,它在DNA序列分析中有着广泛的应用。本文详细介绍了近年来诸多DNA序列分析问题中信息熵应用的研究进展,并分析了未来该问题的研究方向。     

NMR structure of the 101-nucleotide core encapsidation signal of the Moloney murine leukemia virus

The full length, positive-strand genome of the Moloney Murine Leukemia Virus contains a "core encapsidation signal" that is essential for efficient genome packaging during virus assembly. We have determined the structure of a 101-nucleotide RNA that contains this signal (called mPsi) using a novel isotope-edited NMR approach. The method is robust and should be generally applicable to larger RNAs. mPsi folds into three stem loops, two of which (SL-C and SL-D) co-stack to form an extended helix. The third stem loop (SL-B) is connected to SL-C by a flexible, four-nucleotide linker. The structure contains five mismatched base-pairs, an unusual C.CG base-triple platform, and a novel "A-minor K-turn," in which unpaired adenosine bases A340 and A341 of a GGAA bulge pack in the minor groove of a proximal stem, and a bulged distal uridine (U319) forms a hydrogen bond with the phosphodiester of A341. Phylogenetic analyses indicate that these essential structural elements are conserved among the murine C-type retroviruses.     

Stereoselective Nucleoside Deuteration for NMR Studies of DNA

A procedure has been elaborated for stereoselective deuterium substitution of one of the diastereotopic 5′-protons in 2′-deoxynucleotides. The synthetic scheme uses the reduction of the 5-oxosugar derivative with deuterated Alpine-Borane. The resulting deuterosugar is converted into pyrimidine nucleosides and incorporated into DNA using standard protocols. Comparison of two-dimensional NMR spectra of the fully protonated and partially deuterated duplexes allowed us to assign diastereotopic 5′ protons, increasing the number of experimental restraints used for structure determination.     

DNA sequence organization in finger millet (Eleusine coracana)

Approximately 39 to 49% of the genome of finger millet consists of repetitive DNA sequences which intersperse with 18% of single copy DNA sequences of 1900 nucleotide pairs. Agarose gel filtration and electrophoresis experiments have yielded the sizes of interspersed repeated sequences as 4000–4200 nucleotide pairs and 150–200 nucleotide pairs. Approximately 20% of the repeated DNA sequences (4000–4200 nucleotide pairs) are involved in long range interspersion pattern, while 60% of the repeated DNA sequences (150–200 nucleotide pairs) are involved in short period interspersion pattern. Based on the data available in literature and the results described here on DNA sequence organization in plants, it is proposed that plants with haploid DNA content of more than 2.5 pg exhibit mostly the short period interspersion pattern, while those with haploid DNA content of less than 2.5 pg show diverse patterns of genome organization. NCL Communication No.: 2708