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The structure of HBV adr NC-1 DNA is analyzed and compared with another five strains of HBV DNAs. Some of the prokaryotic promoter-like sequences, palindrom sequences and ATAA are found. An enhancer core sequence and some other characteristics are also shown. In considering the frame and its regulatory sequence as a transcriptional unit some of the possible new frames are discussed.
Chemical ligation of oligonucleotides in double-stranded helices has been considered in its structural-kinetic aspect. A study was made of (i) two series of DNA duplexes with various arrangements of reacting groups in the ligation junction induced by mispairing or by alteration of furanose structure (the replacement of dT unit with rU, aU, IU, xU, dxT ones) and of (ii) eight synthetic water-soluble carbodiimides with different substituents at N1 and N3 atoms. We assumed that some information on the local structure of modified sites in the duplex can be obtained from kinetic parameters of oligonucleotide coupling reaction. The ratio of kinetic constants k3/(k2 + k3) for productive and nonproductive decomposition of the activated phosphomonoester derivative apparently reflects the reaction site structure: for a given duplex this parameter is virtually independent of the condensing agent composition. Based on the analysis of the chemical ligation kinetics a suggestion has been made about the conformation of some modified units in the double helix.
A realistic DNA target model has been developed and implemented in the biophysical simulation code PARTRAC. It describes five levels of the B-DNA structure (nucleotides, DNA helices, nucleosomes, chromatin fiber structure and chromatin fiber loops) on an atomic level for the whole genome inside a mammalian cell nucleus. The model is capable of describing regular solenoidal, crossed-linker or zigzag structures as well as repeating stochastic arrangements of nucleosomes in the chromatin fiber. Electron tracks resulting from monoenergetic electrons with energies up to 100 keV and from 220 kVp X rays, starting at random positions in the cell, were superimposed on four DNA target models with different chromatin fiber structures. The yields of SSBs, DSBs and short single- and double-stranded DNA fragments were determined from spatial coincidences with strand atoms. Two parameters of the model-the energy necessary to create an SSB and the distance between two breaks that would be scored as a DSB-were adapted to equate simulated and measured strand break yields after X irradiation of human fibroblast cells. The integral fractions of short single- and double-stranded fragments were rather similar for all condensed chromatin fiber structures; they agreed with experimental data for DNA fragments below 2 kbp. The simulated fragment size distributions in the range from 0.1 to 1.5 kbp reflected the fiber structure irrespective of strandedness or electron energy. The distributions using a stochastic arrangement of nucleosomes in the chromatin fiber were found to be in better accordance with experimental data than those obtained with regular fiber structures.
It is shown that osmium tetroxide, 2,2'-bipyridine can be applied as a probe of DNA structure in a bacterial cell. Using this probe we demonstrate (a) presence of structural distortions at the junctions between the right-handed B and left-handed Z DNA in a recombinant plasmid pRW751 and (b) unusual structure of the d(A-T)16 insert in pAT32 plasmid in E. coli cells and in in vitro.
The structure of a presumptive DNA intermediate in the integration of retroviral DNA was studied in a cell-free reaction with exogenously added target DNA. The product made by viral core particles of Moloney murine leukemia virus (Mo-MLV) containing linear viral DNA has a structure consistent with an integration mechanism similar to that observed during bacteriophage Mu transposition. In this intermediate, the 3' ends of the LTR sequences are joined to the target DNA, while the 5' ends of the viral DNA remain unjoined. The 5' ends of the LTR sequences in the intermediate are exactly the same as those found in the unintegrated linear double-stranded viral DNA. This result demonstrates that the linear form of Mo-MLV DNA can integrate directly without prior circularization.
We have determined the relative stabilities and melting behaviors of DNA hairpin structures as a function of the nonbonded residues in the loop. The specific family of hairpin structures we investigated in this work is formed by the 16-mer sequence d[CGAACG(X)4CGTTCG], where X is deoxyadenosine, deoxycytidine, deoxyguanosine, or deoxythymidine. As shown below, this 16-mer can fold back on itself to form a family of DNA hairpin structures that possess a common hexameric stem duplex and a nonbonded loop of 4 nucleotides. For the hairpin structures investigated in this work, we varied the loop composition from all purine residues to all pyrimidine residues. (Formula: see text). We thermodynamically characterized the relative stabilities and melting profiles of these hairpin structures by a combination of spectroscopic and calorimetric techniques. To establish a thermodynamic "baseline," we also conducted parallel studies on the isolated hexameric duplex d[CGAACG).(CG-TTCG)], which corresponds to the common stem duplex present in each hairpin structure. Our spectroscopic and calorimetric data reveal the following: (i) The hairpin structure with four dT residues in the loop exhibits the highest melting temperature, while the corresponding hairpin structure with four dA residues in the loop exhibits the lowest melting temperature. (ii) The free energy data at 25 degrees C reveal the following order of DNA hairpin stability for the four structures studied here: T loop greater than C loop greater than G loop greater than A loop. In other words, the pyrimidine-looped hairpins of four residues are more stable than the purine-looped hairpins. (iii) The loop-dependent order of hairpin stability is paralleled by a similar trend in the calorimetrically determined transition enthalpies for hairpin disruption. Thus, the enhanced stability of the pyrimidine-looped hairpin structures relative to purine-looped hairpin structures is enthalpic in origin. To develop insight into the molecular basis for the thermodynamic differences, proton NMR spectroscopy was used to probe for structural disparities between the most stable hairpin structure (T loop) and the least stable hairpin structure (A loop). Two-dimensional nuclear Overhauser enhancement spectroscopy revealed connectivities between the residues in the stem duplexes of both hairpin structures that are consistent with B-form DNA. In addition, the nonbonded residues in both the T and A loops exhibited the same connectivity patterns. However, on the 5' side of the stem-loop junction, the T-loop residue exhibited a connectivity with the adjacent base pair of the stem duplex that is not observed for the corresponding A-loop residue. This difference in connectivities at the stem-loop junction may provide a structural basis for our observation that the T-looped hairpin structure is more stable than the corresponding A-looped hairpin structure.
The inosine-containing sequence d(CCIGTACm(5)CGG) is shown to crystallize as a four-stranded DNA junction. This structure is nearly identical to the antiparallel junction formed by the parent d(CCGGTACm(5)()CGG) sequence [Vargason, J. M., and Ho, P. S. (2002) J. Biol. Chem. 277, 21041-21049] in terms of its conformational geometry, and inter- and intramolecular interactions within the DNA and between the DNA and solvent, even though the 2-amino group in the minor groove of the important G(3).m(5)C(8) base pair of the junction core trinucleotide (italicized) has been removed. In contrast, the analogous 2,6-diaminopurine sequence d(CCDGTACTGG) crystallizes as resolved duplex DNAs, just like its parent sequence d(CCAGTACTGG) [Hays, F. A., Vargason, J. M., and Ho, P. S. (2003) Biochemistry 42, 9586-9597]. These results demonstrate that it is not the presence or absence of the 2-amino group in the minor groove of the R(3).Y(8) base pair that specifies whether a sequence forms a junction, but the positions of the extracyclic amino and keto groups in the major groove. Finally, the study shows that the arms of the junction can accommodate perturbations to the B-DNA conformation of the stacked duplex arms associated with the loss of the 2-amino substituent, and that two hydrogen bonding interactions from the C(7) and Y(8) pyrimidine nucleotides to phosphate oxygens of the junction crossover specify the geometry of the Holliday junction.
BACKGROUND: Proteins can force DNA to adopt distorted helical structures that are rarely if ever observed in naked DNA. The ability to synthesize DNA that contains defined helical aberrations would offer a new avenue for exploring the structural and energetic plasticity of DNA. Here we report a strategy for the enforcement of non-canonical helical structures through disulfide cross-linking; this approach is exemplified by the design and synthesis of an oligonucleotide containing a pronounced bend. RESULTS: A localized bend was site-specifically introduced into DNA by the formation of a disulfide cross-link between the 5' adenines of a 5'-AATT-3' region in complementary strands of DNA. The DNA bend was characterized by high-resolution NMR structure determination of a cross-linked dodecamer and electrophoretic mobility assays on phased multimers, which together indicate that the cross-linked tetranucleotide induces a helical bend of approximately 30 degrees and a modest degree of unwinding. The enforced bend was found to stimulate dramatically the binding of an architecture-specific protein, HMG-D, to the DNA. DNase I foot-printing analysis revealed that the protein is recruited to the section of DNA that is bent. CONCLUSIONS: The present study reports a novel approach for the investigation of non-canonical DNA structures and their recognition by architecture-specific proteins. The mode of DNA bending induced by disulfide cross-linking resembles that observed in structures of protein-DNA complexes. The results reveal common elements in the DNA-binding mode employed by sequence-specific and architecture-specific HMG proteins.
We report here the crystal structure of the DNA hexamer duplex d(CGCGCA)*d(TGCGCG) at 1.71 A resolution. The crystals, in orthorhombic space group, were grown in the presence of cobalt hexammine, a known inducer of the left-handed Z form of DNA. The interaction of this ion with the DNA helix results in a change of the adenine base from the common amino tautomeric form to the imino tautomer. Consequently the A:T base pair is disrupted from the normal Watson-Crick base pairing to a 'wobble' like base pairing. This change is accommodated easily within the helix, and the helical parameters are those expected for Z-DNA. When the cobalt hexammine concentration is decreased slightly in the crystallization conditions, the duplex crystallizes in a different, hexagonal space group, with two hexamer duplexes in the asymmetric unit. One of these is situated on a crystallographic 6-fold screw axis, leading to disorder. The tautomeric shift is not observed in this space group. We show that the change in inter-helix interactions that lead to the two different space groups probably arise from the small decrease in ion concentration, and consequently disordered positions for the ion.
Protein HMGB1 has long been known as one of the most abundant non-histone proteins in the nucleus of mammalian cells, and has regained interest recently for its function as an extracellular cytokine. As a DNA-binding protein, HMGB1 facilitates DNA-protein interactions by increasing the flexibility of the double helix, and binds specifically to distorted DNA structures. We have previously observed that HMGB1 binds with extremely high affinity to a novel DNA structure, hemicatenated DNA loops (hcDNA), in which double-stranded DNA fragments containing a tract of poly(CA).poly(TG) form a loop maintained at its base by a hemicatenane. Here, we show that the single HMGB1 domains A and B, the HMG-box domain of sex determination factor SRY, as well as the prokaryotic HMGB1-like protein HU, specifically interact with hcDNA (Kd approximately 0.5 nM). However, the affinity of full-length HMGB1 for hcDNA is three orders of magnitude higher (Kd<0.5 pM) and requires the simultaneous presence of both HMG-box domains A and B plus the acidic C-terminal tail on the molecule. Interestingly, the high affinity of the full-length protein for hcDNA does not decrease in the presence of magnesium. Experiments including a comparison of HMGB1 binding to hcDNA and to minicircles containing the CA/TG sequence, binding studies with HMGB1 mutated at intercalating amino acid residues (involved in recognition of distorted DNA structures), and exonuclease III footprinting, strongly suggest that the hemicatenane, not the DNA loop, is the main determinant of the affinity of HMGB1 for hcDNA. Experiments with supercoiled CA/TG-minicircles did not reveal any involvement of left-handed Z-DNA in HMGB1 binding. Our results point to a tight structural fit between HMGB1 and DNA hemicatenanes under physiological conditions, and suggest that one of the nuclear functions of HMGB1 could be linked to the possible presence of hemicatenanes in the cell.
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Anti-DNA antibodies are a major contributor to the pathogenesis associated with the autoimmune disease systemic lupus erythematosus in mice and human. The accumulation of a large body of structural information on autoimmune anti-DNA antibodies over the past several years, particularly from mice, has provided considerable insight into the structure, function, and biology of this important class of autoantibodies. Even though the germline repertoire of light and heavy chain variable regions that may encode DNA-specific antibodies is very large in mice, there are individual light and heavy chain variable region genes that have been recurrent and preferentially expressed among anti-DNA hybridomas. This has been particularly true for hybridomas producing antibodies that bind duplex, B-form, mammalian DNA (dsDNA). Recurrent somatically derived variable region structures, particularly arginines in the third complementary-determining region of the heavy chain (VH-CDR3), have also been recurrent and preferentially expressed among monoclonal anti-DNA antibodies. In fact specificity for dsDNA can be correlated to the relative amino acid position at which arginines are expressed within VH-CDR3 of anti-DNA. Most important from the results of structural analyses of monoclonal anti-DNA autoantibodies has been the realization that autoimmunity to DNA results from a clonally selective, antigen-specific immune response to DNA. Autoimmune antibodies to DNA have all of the characteristics of secondary immune antibodies. In further support of this hypothesis, we have been able to induce anti-DNA antibodies in normal, nonautoimmune mice by immunization with immunogenic DNA-peptide complexes. The induced antibodies have all of the structural and functional characteristics of autoimmune anti-DNA including the pathogenetic potential to induce glomerulonephritis. This review summarizes the results of research from our laboratory that support the above conclusions.
The nucleotide sequences of the cDNAs of the genes for the structural proteins C, preM, M and E of the tick-borne encephalitis viral strain 205 have been determined. The complete nucleotide sequence of the 5'-end nonstructural region of the viral genome has been studied for the first time. The difference in the amino acids sequences of the structural proteins from different strains (205, Sofiin and Neidorf) of the virus is discussed.
DNA bases in the three-base-pair (3bp) region of duplexes with the two major lesions of cisplatin (cis-PtCl(2)(NH(3))(2)) with DNA, namely d(XGG) and d(XAG) ( = N7-platinated base), differ in their relative positions by as much as approximately 3.5 A in structures in the literature. Such large differences impede drug design and assessments of the effects of protein binding on DNA structure. One recent and several past structures based on NMR-restrained molecular dynamics (RMD) differ significantly from the reported X-ray structure of an HMG-bound XGG 16-mer DNA duplex (Ohndorf, U.-M.; Rould, M. A.; He, Q.; Pabo, C. O.; Lippard, S. J. Nature 1999, 399, 708). This 16-mer structure has several significant novel and unique features (e.g., a bp step with large positive shift and slide). Hypothesizing that novel structural features in the XGG or XAG region of duplexes elude discovery by NMR methods (especially because of the flexible nature of the 3bp region), we studied an oligomer with only G.C bp's in the XGGY site by NMR methods for the first time. This 9-mer gave a 5'-G N1H signal with a normal shift and intensity and showed clear NOE cross-peaks to C NHb and NHe. We assigned for the first time (13)C NMR signals of a duplex with a GG lesion. These data, by adding NMR-based criteria to those inherent in NOESY and COSY data, have more specifically defined the structural features that should be present in an acceptable model. In particular, our data indicated that the sugar of the X residue has an N pucker and that the GG cross-link should have a structure similar to the original X-ray structure of cis-Pt(NH(3))(2)(d(pGpG)) (Sherman S. E.; Gibson, D.; Wang, A. H.-J.; Lippard, S. J. J. Am. Chem. Soc. 1988, 110, 7368). With these restrictions added to NOE restraints, an acceptable model was obtained only when we started our modeling with the 16-mer structural features. The new X-ray/NMR-based model accounted for the NOESY data better than NOE-based models, was very similar in structure to the 16-mer, and differed from solely NOE-based models. We conclude that all XGG and XAG (X = C or T) duplexes undoubtedly have structures similar to those of the 16-mer and our model. Thus, protein binding does not change greatly the structure of the 3bp region. The structure of this region can now be used in understanding structure-activity relationships needed in the design of new carrier ligands for improving Pt anticancer drug activity.
Epigenetic regulatory mechanisms heritably alter patterns of gene expression without changes in DNA sequence. Epigenetic states are often correlated with developmentally imposed alterations in genomic DNA methylation and local chromatin structure. Pl-Blotched is a stable epigenetic allele of the maize anthocyanin regulatory gene, purple plant1(pl). Pl-Blotched plants display a variegated pattern of pigmentation that contrasts sharply with the uniformly dark purple pigmentation of plants carrying the dominant Pl-Rhoades allele. Previously, we showed that the lower level of pigmentation in Pl-Blotched is correlated with lower pl mRNA levels and increased DNA methylation at some sites. To explore how DNA methylation, chromatin structure, and developmental stage might contribute to the expression of Pl-Blotched, we used methylation-sensitive restriction enzymes and DNaseI sensitivity assays to compare the methylation status and chromatin structure of Pl-Blotched and Pl-Rhoades at different stages in development. Both alleles exhibit developmentally sensitive changes in methylation. In Pl-Blotched, methylation of two diagnostic HpaII/MspI sites increases progressively, coincident with the juvenile-to-adult transition in growth. In seedlings, the chromatin encompassing the coding region of the gene is less sensitive to DNaseI digestion in Pl-Blotched than in Pl-Rhoades. Developmental maturation from seedling to adult is accompanied by expansion of this closed chromatin domain to include the promoter and downstream flanking sequences. We provide evidence to show that chromatin structure, rather than DNA methylation, is the primary epigenetic determinant for the phenotypic differences between Pl-Blotched and Pl-Rhoades.
Eukaryotic DNA synthesis is thought to occur in multienzyme complexes present at numerous discrete sites throughout the nucleus. We demonstrate here that cellular DNA replication sites identified by bromodeoxyuridine labeling are relocated in cells infected with herpes simplex virus such that they correspond to viral prereplicative structures containing the HSV DNA replication protein, ICP8. Thus components of the cellular DNA replication apparatus are present at viral prereplicative sites. Mutant virus strains expressing defective ICP8 do not alter the pattern of host cell DNA replication sites, indicating that functional ICP8 is required for the redistribution of cellular DNA replication complexes. This demonstrates that a specific protein molecule can play a role in the organization of DNA replication proteins at discrete sites within the cell nucleus.
Transcriptional regulation in papillomaviruses depends on sequence-specific binding of the regulatory protein E2 to several sites in the viral genome. Crystal structures of bovine papillomavirus E2 DNA targets reveal a conformational variant of B-DNA characterized by a roll-induced writhe and helical repeat of 10.5 bp per turn. A comparison between the free and the protein-bound DNA demonstrates that the intrinsic structure of the DNA regions contacted directly by the protein and the deformability of the DNA region that is not contacted by the protein are critical for sequence-specific protein/DNA recognition and hence for gene-regulatory signals in the viral system. We show that the selection of dinucleotide or longer segments with appropriate conformational characteristics, when positioned at correct intervals along the DNA helix, can constitute a structural code for DNA recognition by regulatory proteins. This structural code facilitates the formation of a complementary protein-DNA interface that can be further specified by hydrogen bonds and nonpolar interactions between the protein amino acids and the DNA bases.
The DNA intermediates and final products formed by the Type I restriction endonuclease, EcoB, were further characterized. DNA cleaved on only one strand (hemi-restricted DNA) contains gaps of approximately 70-100 nucleotides, while the fully restricted products contain 3'-single-stranded tails averaging approximately 70-100 nucleotides for each strand cleaved. The gaps and tails are formed with the release of an equal number of nucleotides as small oligonucleotides that are soluble in acid. After purification, neither the hemi-restricted nor the fully restricted DNAs are cleaved again by EcoB. There is no apparent specificity for which strand of a duplex is initially cleaved by EcoB, nor is there specificity with respect to the composition of the 3'-terminal nucleotide formed on the DNA or the 3'- or 5'-terminal nucleotides of the acid-soluble oligonucleotides released during DNA cleavage. The structure formed at the 5' terminus of the DNA product which blocks phosphorylation by T4 polynucleotide kinase remains unknown, but its removal with phage lambda exonuclease allows at least some reutilization of recognition sites by EcoB as well as phosphorylation of the newly formed 5' termini. To explain the complex mechanism of this enzyme, it is suggested that the unidentified 5'-tails prevent wasteful rerestriction from occurring, whereas the 3'-single-stranded tails create DNA which, when nonhomologous to chromosomal DNA, cannot be rescued because such tails are not substrate for DNA polymerases. However, when homologous chromosomal DNA exists, the randomly cleaved large fragments with these tails can easily be assimilated by recA-mediated genetic recombination, thus stimulating DNA exchange between related organisms.