A standard reference frame for the description of nucleic acid base-pair geometry.
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Base Pairing↗
Biomedical subjects
X J Lu
Publications and source records attributed to X J Lu.
Base sequence effects in bending induced by bulky carcinogen-DNA adducts: experimental and computational analysis.
The covalent binding of bulky mutagenic or carcinogenic compounds to DNA can lead to bending, which could significantly alter the interactions of DNA with critical replication and transcription proteins. The impact of adducts derived from the highly reactive bay region enantiomeric (+)- and (-)-anti-7,8-diol-9,10-epoxide derivatives of benzo[a]pyrene (BPDE) are of interest because the (+)-7R,8S,9S,10R-anti-BPDE enantiomer is highly tumorigenic in rodents, while the (-)-7S,8R,9R,10S-anti-BPDE enantiomer is not. Both (+)- and (-)-anti-BPDE bind covalently with DNA predominantly by trans addition at the exocyclic amino group of guanine to yield 10S (+)- and 10R (-)-trans-anti-[BP]-N(2)-dG adducts. We have synthesized a number of different oligonucleotides with single (+)- and (-)-trans-anti-[BP]-N(2)-dG adducts (G) in the base sequence context XG*Y, where X and Y are different DNA bases. The G* residues were positioned at or close to the center of 11 base pair ( approximately 1 helical turn) or 16 base pair ( approximately 1.5 turns) duplexes. All bases, except for X and Y and their partners, were identical. These sequences were self-ligated with T4 ligase to form multimers that yield a ladder of bands upon electrophoresis in native polyacrylamide gels. The extent of bending in each oligonucleotide was assessed by monitoring the decrease in gel mobilities of these linear, self-ligated oligomers, relative to unmodified oligonucleotides of the same base sequence. The extent of global bending was then estimated using a sequence-specific three-dimensional model from which the values of the base-pair step parameter roll adjacent to the lesion site could be extracted. We find that (+)-trans-anti-[BP]-N(2)-dG adducts are considerably more bent than the (-) isomers regardless of sequence and that A-T base pairs flanking the [BP]-N(2)-dG lesion site allow for local flexibility consistent with adduct conformational heterogeneity. Interestingly, the fit of computed versus observed gel mobilities using classical reptation treatments requires enhancement of unmodified DNA flexibility in gels, compared to aqueous salt solution. The differences in bending between the two stereoisomeric adduct duplexes and the observed base sequence context effects may play a significant role in the differential processing of these lesions by cellular replication, transcription, and repair enzymes.
Algorithms↗
A-form conformational motifs in ligand-bound DNA structures.
Recognition and biochemical processing of DNA requires that proteins and other ligands are able to distinguish their DNA binding sites from other parts of the molecule. In addition to the direct recognition elements embedded in the linear sequence of bases (i.e. hydrogen bonding sites), these molecular agents seemingly sense and/or induce an "indirect" conformational response in the DNA base-pairs that facilitates close intermolecular fitting. As part of an effort to decipher this sequence-dependent structural code, we have analyzed the extent of B-->A conformational conversion at individual base-pair steps in protein and drug-bound DNA crystal complexes. We take advantage of a novel structural parameter, the position of the phosphorus atom in the dimer reference frame, as well as other documented measures of local helical structure, e.g. torsion angles, base-pair step parameters. Our analysis pinpoints ligand-induced conformational changes that are difficult to detect from the global perspective used in other studies of DNA structure. The collective data provide new structural details on the conformational pathway connecting A and B-form DNA and illustrate how both proteins and drugs take advantage of the intrinsic conformational mechanics of the double helix. Significantly, the base-pair steps which exhibit pure A-DNA conformations in the crystal complexes follow the scale of A-forming tendencies exhibited by synthetic oligonucleotides in solution and the known polymorphism of synthetic DNA fibers. Moreover, most crystallographic examples of complete B-to-A deformations occur in complexes of DNA with enzymes that perform cutting or sealing operations at the (O3'-P) phosphodiester linkage. The B-->A transformation selectively exposes sugar-phosphate atoms, such as the 3'-oxygen atom, ordinarily buried within the chain backbone for enzymatic attack. The forced remodeling of DNA to the A-form also provides a mechanism for smoothly bending the double helix, for controlling the widths of the major and minor grooves, and for accessing the minor groove edges of individual base-pairs.
Animals↗
Protein-directed DNA structure. I. Raman spectroscopy of a high-mobility-group box with application to human sex reversal.
Protein-directed reorganization of DNA underlies mechanisms of transcription, replication, and recombination. A molecular model for DNA reorganization in the regulation of gene expression is provided by the sequence-specific high-mobility-group (HMG) box. Structures of HMG-box complexes with DNA are characterized by expansion of the minor groove, sharp bending toward the major groove, and local unwinding of the double helix. The Raman vibrational signature of such DNA reorganization has been identified in a study of the SRY HMG box, encoded by the human male-determining region of the Y chromosome. We observe in the human SRY-HMG:DNA complex extraordinarily large perturbations to Raman bands associated with vibrational modes of the DNA backbone and accompanying large increases in intensities of Raman bands attributable to base unstacking. In contrast, DNA major-groove binding, as occurs for the bZIP protein GCN4 [Benevides, J. M., Li, T., Lu, X.-J., Srinivasan, A. R., Olson, W. K., Weiss, M. A., and Thomas, G. J., Jr. (2000) Biochemistry 39, 548-556], perturbs the Raman signature of DNA only marginally. Raman markers of minor-groove recognition in the human SRY-HMG:DNA complex are due primarily to perturbation of specific vibrational modes of deoxyribose moieties and presumably reflect desolvation at the nonpolar interface of protein and DNA. These Raman markers may be diagnostic of protein-induced DNA bending and are proposed as a baseline for comparative analysis of mutations in SRY that cause human sex reversal.
Amino Acid Sequence↗
Protein-directed DNA structure II. Raman spectroscopy of a leucine zipper bZIP complex.
Mechanisms of transcription may involve protein-directed changes in DNA structure and DNA-directed changes in protein structure. We have employed Raman spectroscopy to characterize vibrational signatures associated with such induced molecular fitting for two classes of transcription factors-the basic leucine-zipper (bZIP) motif and the high-mobility-group (HMG) box-each with a DNA target site. Results for bZIP are described here; findings for the HMG-box are reported in the preceding paper in this issue [Benevides, J. M., Chan, G., Lu, X.-J., Olson, W. K., Weiss, M. A., and Thomas, G. J., Jr. (2000) Biochemistry 39, 537-547]. The yeast activator GCN4 provides a well-studied example of bZIP recognition, wherein B-DNA serves essentially as a template for protein folding. Analysis of Raman spectra of the 57-residue GCN4 bZIP domain, its AP-1 binding site, and their specific complex confirms a DNA-induced increase in alpha-helicity, attributable to folding of GCN4 basic arms with virtually no change in B-DNA structure, consistent with previous X-ray and NMR structure determinations. The absence of DNA perturbations in the bZIP model contrasts sharply with the HMG box, where DNA structure perturbations predominate. The bZIP and HMG-box models represent two opposing extremes in a range of induced fits identifiable by Raman spectroscopy. Previously characterized lambda repressor/operator complexes [Benevides, J. M., Weiss, M. A., and Thomas, G. J. (1994) J. Biol. Chem. 269, 10869-10878] occupy an intermediate position within this range. A comprehensive tabulation of Raman markers proposed as diagnostic of different protein/DNA recognition motifs is presented. The results are analyzed in terms of available DNA crystal structures (Nucleic Acid Database) to identify details of DNA conformation that correlate with specific Raman recognition markers.
Base Sequence↗
Resolving the discrepancies among nucleic acid conformational analyses.
Growing interest in understanding the relationship between the global folding of nucleic acids and the sequence-dependent structure of individual base-pair steps has stimulated the development of new mathematical methods to define the geometry of the constituent base-pairs. Several approaches, designed to meet guidelines set by the nucleic acid community, permit rigorous comparative analyses of different three-dimensional structures, as well as allow for reconstruction of chain molecules at the base-pair level. The different computer programs, however, yield inconsistent descriptions of chain conformation. Here we report our own implementation of seven algorithms used to determine base-pair and dimer step parameters. Aside from reproducing the results of individual programs, we uncover the reasons why the different algorithms come to conflicting structural interpretations. The choice of mathematics has only a limited effect on the computed parameters, even in highly deformed duplexes. The results are much more sensitive to the choice of reference frame. The disparate schemes yield very similar conformational descriptions if the calculations are based on a common reference frame. The current positioning of reference frames at the inner and outer edges of complementary bases exaggerates the rise at distorted dimer steps, and points to the need for a carefully defined conformational standard.
Algorithms↗
Overview of nucleic acid analysis programs.
We outline the mathematical distinctions among seven of the most popular computer programs currently used to analyze the spatial arrangements of bases and base pairs in nucleic acid helical structures. The schemes fall into three basic categories on the basis of their definitions of rotational parameters: matrix-based, projection-based, and combined matrix- and projection-based. The approaches also define and construct base and base-pair coordinate frames in a variety of ways. Despite these mathematical distinctions, the computed parameters from some programs are strongly correlated and directly comparable. By contrast, other programs which use identical methodologies sometimes yield very different results. The choice of reference frame rather than the mathematical formulation has the greater effect on calculated parameters. Any factor which influences the reference frame, such as fitting or not fitting standard bases to the experimentally derived coordinates, will have a noticeable effect on both complementary base pair and dimer step parameters.
Least-Squares Analysis↗
DNA sequence-dependent deformability deduced from protein-DNA crystal complexes.
The deformability of double helical DNA is critical for its packaging in the cell, recognition by other molecules, and transient opening during biochemically important processes. Here, a complete set of sequence-dependent empirical energy functions suitable for describing such behavior is extracted from the fluctuations and correlations of structural parameters in DNA-protein crystal complexes. These elastic functions provide useful stereochemical measures of the local base step movements operative in sequence-specific recognition and protein-induced deformations. In particular, the pyrimidine-purine dimers stand out as the most variable steps in the DNA-protein complexes, apparently acting as flexible "hinges" fitting the duplex to the protein surface. In addition to the angular parameters widely used to describe DNA deformations (i.e., the bend and twist angles), the translational parameters describing the displacements of base pairs along and across the helical axis are analyzed. The observed correlations of base pair bending and shearing motions are important for nonplanar folding of DNA in nucleosomes and other nucleoprotein complexes. The knowledge-based energies also offer realistic three-dimensional models for the study of long DNA polymers at the global level, incorporating structural features beyond the scope of conventional elastic rod treatments and adding a new dimension to literal analyses of genomic sequences.
DNA↗
Structure and conformation of helical nucleic acids: rebuilding program (SCHNArP).
We present a program, SCHNArP, for rebuilding double-helical nucleic acid structures from a set of helical parameters. The parameter sets are based on mathematically reversible schemes that allow direct comparison of data from experimental X-ray crystal structures analyzed using the analysis program, SCHNAaP (see accompanying paper), and structures built using the rebuilding program, SCHNArP. The program uses either local CEHS helical parameters or global helical parameters. A number of standard parameter sets from the literature are included that allow comparison of oligomer and polymer structures generated using different models for sequence-dependent DNA bending. Exact atomic models are provided for the bases. Schematic models that trace the path of the backbone and use rectangular blocks for the bases can be generated.
Base Composition↗
Structure and conformation of helical nucleic acids: analysis program (SCHNAaP).
We present a new versatile program, SCHNAaP, for the analysis of double-helical nucleic acid structures. The program uses mathematically rigorous and fully reversible procedures for calculating the structural parameters: the Cambridge University Engineering Department Helix computation Scheme (CEHS) is used to determine the local helical parameters and an analogous procedure is used to determine the global helical parameters. These parameters form a complete set that conforms to the "Cambridge Accord" on definitions and nomenclature of nucleic acid structure parameters. In addition to the two standard Watson-Crick base-pairs, the program handles mismatched base-pairs and chemically modified bases. An analysis of the sugar-phosphate backbone conformation is included. Standardized base-stacking diagrams of each dinucleotide step with reference to the mid-step triad are generated. Structures are classified as one of the four polymorphic families, A/B, Z, W or R, although W- and R-DNA (two types of hypothetical structure) have yet to be observed experimentally.
Base Composition↗
DNA base-stacking interactions: a comparison of theoretical calculations with oligonucleotide X-ray crystal structures.
Experimental data on the conformational properties of dinucleotides taken from high-resolution X-ray crystal structures of oligonucleotides have been compared with theoretical energy calculations on the base-stacking interactions. The conformational properties of the dinucleotides determined by calculation agree well with the experimental data, which shows that the method used for computing the stacking interactions is reliable. In addition, the calculations provide insight into the origins of the major trends that are observed in the experimental data. The values of the step parameters roll, tilt and rise, are determined entirely by the van der Waals interactions, and this reflects the strong requirement that the bases remain stacked in close contact. Slide, shift and twist do not affect the vertical separation of the bases and are therefore less tightly constrained. Electrostatic interactions play an important role in determining the values of shift and slide, but the base-stacking interaction energy is essentially independent of the value of twist. Thus the experimental value of twist is most likely fixed by the constraints of the backbone, which are missing in these calculations.
Base Composition↗
Construction of double-helical DNA structures based on dinucleotide building blocks.
We present a new method for building full 3-D structures of DNA sequences. A database of the conformational properties of dinucleotide steps has been compiled using X-ray crystal structures of oligonucleotides. The protocol uses these dinucleotides as building blocks to generate three dimensional structures of any required sequence in any required conformation.
Crystallography, X-Ray↗
Spontaneous differentiation of trophoblast cells along the spongiotrophoblast cell pathway: expression of members of the placental prolactin gene family and modulation by retinoic acid.
The purpose of this study was to examine part of the trophoblast cell multilineage pathway and its modulation by retinoic acid. A method for studying trophoblast cell differentiation along the spongiotrophoblast cell pathway in vitro was established and characterized. Cells were isolated from junctional zones of Day 13 rat chorioallantoic placentas via mechanical dissection, enzymatic digestion, and enrichment through a Percoll cushion. The cells were cultured up to 8 days and analyzed for their purity, morphology, and ability to express members of the placental prolactin (PRL) family. Cell preparations contained minimal mesenchymal contamination as estimated by immunocytochemical analysis for vimentin. The cells expressed PRL-like protein-A (PLP-A), PLP-B, PLP-C, and placental lactogen-I variant (PL-Iv) indicative of their differentiated spongiotrophoblast cell phenotype. Expression of members of the PRL family increased markedly during culture. Temporally the increase in PLP-A expression preceded the increased expression of PLP-B (0.9 kb), PLP-C, and PL-Iv. These in vitro observations paralleled the behavior of spongiotrophoblast cells developing in situ. Some differences were evident, including the immediate activation of PLP-B (1.2 kb) following enzymatic isolation of the cells. These cells were also susceptible to experimental manipulation. Exposure to retinoic acid influenced the morphology of the cells and the profile of members of the placental PRL family expressed by in vitro differentiated cells. In summary, a culture system has been devised to examine the control of spongiotrophoblast cell differentiation and the regulation of expression of members of the placental PRL gene family. Spongiotrophoblast cells spontaneously differentiate in vitro through discrete developmental phases that are susceptible to modulation by retinoic acid.
Animals↗
Recapitulation of the pathway for trophoblast giant cell differentiation in vitro: stage-specific expression of members of the prolactin gene family.
The trophoblast giant cell lineage is characterized by endoreduplication and expression of members of the PRL gene family. This report describes the functional consequences following in vitro manipulation of a rat trophoblast cell line, termed Rcho-1. Rcho-1 cells can be cultured under conditions that promote proliferation or differentiation. Proliferation is maintained by culturing the cells in the presence of fetal bovine serum under subconfluent conditions. Differentiation is induced by growing the cells to confluence and removing the mitogenic source. Differentiation is characterized by continued synthesis of DNA in the absence of proliferation (endoreduplication) and the sequential expression of members of the PRL gene family. Western and Northern blot analyses demonstrated that placental lactogen-I (PL-I) was first expressed, followed sequentially by PL-II, PRL-like protein-A, and PRL-like protein-C. The ontogeny of expression of members of the PRL gene family by the Rcho-1 cells recapitulated the pattern of in situ expression by trophoblast giant cells of the junctional zone of the chorioallantoic placenta. A notable difference between in vivo trophoblast giant cell differentiation and in vitro Rcho-1 cell differentiation is the termination of PL-I expression in normal trophoblast giant cells developing in vivo and the continued expression of PL-I in differentiated Rcho-1 cell cultures. The Rcho-1 cell line provides a unique in vitro model for investigating the initiation and maintenance of the trophoblast giant cell differentiation pathway.
Animals↗
[Scanning electron microscopy of adult Trichuris trichiura].
The paper reports the result of the observation on the ultrastructure of the body surface of adult T. trichiura by scanning electron microscopy. Unique features of T. trichiura are described including the presence of labial-shape structures at the periphery of mouth, eight cephalic pores and a pair of amphids around the mouth pore and vesicle-shaped cuticular protuberances on the abdomen of T. trichiura. Cuticular pores were found scattering on the surface of T. trichiura. Detailed descriptions of male copulatory sheath and spicule were presented.
Animals↗
[Scanning electron microscopic observations on the copulatory spicules of the male Necator americanus and Ancylostoma duodenale].
Scanning electron microscopic observations were made on the morphological structures of the two copulatory spicules of the male Necator americanus and Ancylostoma duodenale. In both species, one of the two copulatory spicules was in the shape of a concave groove and the other, in the shape of an oblate tube. Owing to the difference in the concavity of the groove shaped copulatory spicule between the two species as shown by the cross sections, Necator americanus usually appear to have only one copulatory spicule whereas Ancylostoma duodenale usually show two separate copulatory spicules in appearance (Figs 1-10).
Ancylostoma↗
Isopentenyl-diphosphate isomerase: irreversible inhibition by 3-methyl-3,4-epoxybutyl diphosphate.
Isopentenyl-diphosphate:dimethylallyl-diphosphate isomerase (EC 5.3.3.2) catalyzes the 1,3-allylic rearrangement of the homoallylic substrate isopentenyl diphosphate (IPP) to its allylic isomer, dimethylallyl diphosphate (DMAPP). Incubation of yeast IPP isomerase with 3-methyl-3,4-epoxybutyl diphosphate (EIPP) resulted in a time-dependent first-order loss of activity characteristic of an active-site-directed irreversible process, where k2 = 0.63 +/- 0.10 min-1 and KI = 0.37 +/- 0.11 microM. A 1:1 covalent E-I complex was formed upon incubation with [1-14C]EIPP. The inhibited enzyme was treated with trypsin to give two radioactive fragments, which were purified by reversed-phase HPLC on a C18 column. The modified amino acid in each fragment was identified as C139 by sequencing the radiolabeled peptides. Incubation of IPP isomerase with [2,4,5-13C3]EIPP gave a 13C-labeled E-I complex. A 1H-13C heteronuclear multiquantum correlation spectrum had strong cross-peaks at 1.2/28 and 2.9/48 ppm, which we assigned to the labeled methyl group and C(4) methylene, respectively, of the inhibitor. In addition, a weak signal at 2.17/42 ppm may be from the C(2) methylene. Comparison of these chemical shifts with those of a synthetic adduct isolated from treatment of EIPP with cysteine indicates C139 attacks C(4) of EIPP to generate a thioether linkage between the enzyme and the inhibitor.
Amino Acid Sequence↗
[Scanning electron microscopic observations on larvae and young adults of Angiostrongylus cantonensis].
Scanning electron microscopic observations on the structure of the body surface of various larval stages and young adults of Angiostrongylus cantonensis were made. The mouth opening of the first and second stage larvae closes in "Y" form until well developed young adult stage. There are two rows of 6 sensory papillae each around the mouth. With development of the worm, the papillae of the outer row gradually degenerated and could hardly be seen in adult worms. A pair of amphidial pores was present on the external side of lateral papillae of the inner row, being conspicuous in the fourth-stage larvae. There was one excretory pore on the ventral side of the anterior end. The copulatory bursa of the male worms began to develop in the third stage larvae and became well developed in the 25-day young adults. The processes of the development of copulatory bursa were described. The gonopore could be seen in the female worm as early as in the first-stage larvae but the anal pore appeared only in the fourth-stage larvae, both of them did not develop completely until the young adult stage of 11 day old (Figs. 1-18).
Angiostrongylus↗