Formaldehyde as a probe of DNA structure. 3. Equilibrium denaturation of DNA and synthetic polynucleotides.
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Adenosine Monophosphate↗
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T4 endonuclease V promotes the formation of multimeric DNA structures.
Electron microscopy of UV-irradiated circular DNA molecules which had been treated with T4 endonuclease V revealed the formation of multimeric DNA structures in addition to the expected conversion of the superhelical DNA molecules into nicked circular and linear forms. The multimeric DNA molecules could be distinguished in electron micrographs from catenated molecules which were present in the original DNA preparation by a combination of rotary and single angle heavy metal shadowing. The complexity and frequency of these structures increased with time of reaction with endonuclease V. Their formation, as well as the endonuclease activity of enzyme, was dependent on UV irradiation of the DNA, and the complexes could be disrupted by prior phenol extraction and ethanol precipitation. Preparations of endonuclease V estimated to be 98% pure by mass promoted the same complex formation between DNA molecules as did preparations estimated to be only 5-10% pure. In addition to these intermolecular structures, the formation of complexes between regions on the same DNA molecules was manifest as discrete double-stranded 'loops' 200-300 base pairs in length. DNA 'bubble structures' were also observed and may represent folding of the 'loops' onto adjacent segments of DNA. These results suggest that at least one active form of T4 endonuclease V may be a multimeric complex of enzyme molecules in association with DNA.
DNA, Bacterial↗
Transcriptional activation by protein-induced DNA bending: evidence for a DNA structural transmission model.
Integration host factor (IHF) is a DNA-bending protein that binds to an upstream activating sequence (UAS1) and, on a negatively supercoiled DNA template, activates transcription from the ilvPG promoter of the ilvG-MEDA operon of Escherichia coli. The transcriptional initiation site of the ilvGMEDA operon is located 92 bp downstream of UAS1. Activation is still observed when the orientation of the upstream IHF binding site is reversed. This manipulation places the IHF binding site on the opposite face of the DNA helix, directs the IHF-induced DNA bend in the opposite direction, and presents the opposite face of the nonsymmetrical, heterodimeric, IHF molecule to the downstream RNA polymerase. Lymphoid enhancer-binding factor, LEF-1, is a DNA-bending, lymphoid-specific, mammalian transcription factor that shares no amino acid sequence similarity with IHF. When the IHF site in UAS1 is replaced with a LEF-1 site, LEF-1 activates transcription from the downstream ilvPG promoter in E. coli as well as it is activated by its natural activator, IHF. These results suggest that specific interactions between IHF and RNA polymerase are not required for activation. The results of DNA structural studies show that IHF forms a protein-DNA complex in the UAS1 region that, in the absence of RNA polymerase, alters the structure of the DNA helix in the -10 hexanucleotide region of the downstream ilvPG promoter. The results of in vitro abortive transcription assays show that IIIF also increases the apparent rate of RNA polymerase isomerization from a closed to an open complex. We suggest, therefore, that IHF activates transcription by forming a higher-order protein-DNA complex in the UAS1 region that structurally alters the DNA helix in a way that facilitates open complex formation at the downstream ilvPG promoter site.
Bacterial Proteins↗
The site-specific cleavage of synthetic Holliday junction analogs and related branched DNA structures by bacteriophage T7 endonuclease I.
Various branched DNA structures were created from synthetic, partly complementary oligonucleotides combined under annealing conditions. Appropriate mixtures of oligonucleotides generated three specific branched duplex DNA molecules: (i) a Holliday junction analog having a fixed (immobile) crossover bounded by four duplex DNA branches, (ii) a similar Holliday junction analog which is capable of limited branch migration and, (iii) a Y-junction, with three duplex branches and fixed branch point. Each of these novel structures was specifically cleaved by bacteriophage T7 gene 3 product, endonuclease I. The cleavage reaction "resolved" the two Holliday structure analogs into pairs of duplex DNA products half the size of the original molecules. The point of cleavage in the fixed-junction molecules was predominantly one nucleotide removed to the 5' side of the expected crossover position. Multiple cleavage positions were mapped on the Holliday junction with the mobile, or variable, branch point, to sites consistent with the unrestricted movement of the phosphodiester crossover within the region of limited dyad symmetry which characterizes this molecule. Based on the cleavage pattern observed with this latter substrate, the enzyme displayed a modest degree of sequence specificity, preferring a pyrimidine on the 3' side of the cleavage site. Branched molecules that were partial duplexes (lower order complexes which possessed single-stranded as well as duplex DNA branches) were also substrates for the enzyme. In these molecules, the cleaved phosphodiester bonds were in duplex regions only and predominantly one nucleotide to the 5' side of the branch point. The phosphodiester positions 5' of the branch point in single-stranded arms were not cleaved. Under identical reaction conditions, individually treated oligonucleotides were completely refractory. Thus, cleavage by T7 endonuclease I displays great structural specificity with an efficiency that can vary slightly according to the DNA sequence.
Base Sequence↗
DNA structural variation affects complex formation and promoter melting in ribosomal RNA transcription.
Eukaryotic ribosomal RNA promoters exhibit an unusual conservation of non-canonical DNA structure (curvature, twist angle and duplex stability) despite a lack of primary sequence conservation. This raises the possibility that rRNA transcription factors might utilize structural anomalies in their sequence recognition process. We have analyzed in detail the interaction of the polymerase I transcription factor TIF-IB from Acanthmoeba castellanii with the CORE promoter. TIF-IB interacts primarily with the minor groove of the promoter. By correlating the effects on transcription and on DNA structure of promoter point mutations, we show that the TIF-IB interaction is strongly inhibited by increases in minor groove width. This suggests that a particular DNA structure is required for interaction with the transcription factor. In addition, TIF-IB induces a small bend in the promoter upon binding. Modeling of this bend reveals that it requires an additional narrowing of the minor groove, which would favor binding to mutants with narrower grooves. We also discuss how this narrowing would induce a small destabilization of the helix upstream of the transcription start site. Telestability predicts this would result in destabilization of the sequence that melts during initiation, suggesting that TIF-IB may have a role in stimulating melting.
Acanthamoeba↗
Reversible helix/coil transitions of left-handed Z-DNA structures. Comparison of the thermodynamic properties of poly(dG).poly(dC), poly[d(G-C)].poly[d(G-C)], and poly(dG-m5dC).poly(dG-m5dC).
In contrast to poly(dG).poly(dC), which remains in the B-DNA conformation under all experimental conditions the polynucleotides with the strictly alternating guanine/cytosine or guanine/5'-methylcytosine sequences can change from the classical right-handed B-DNA structure to the left-handed Z-DNA structure when certain experimental conditions such as ionic strength or solvent composition are fulfilled. Up to now the investigation of the helix/coil transition of left-handed DNA structures was not possible because the transition temperature exceeds 98 degrees C. By applying moderate external pressure to the surface of the aqueous polymer solution in the sample cell the boiling point of the solvent water is shifted up the temperature scale without shifting the transition temperature, so that we can measure the helix/coil transition of the polynucleotides at all experimental conditions applied. It can thus be shown that the Z-DNA/coil transition is cooperative and reversible. The Tm is 125 degrees C for poly(dG-m5dC).poly(dG-m5dC) in 2mM Mg2+, 50mM Na+, pH 7.2 and 115 degrees c for poly[d(G-C)].poly[d(G-C)] in 3.04M Na+. The transition enthalpy per base pair was determined by the help of an adiabatic scanning microcalorimeter.
DNA↗
Cleavage of insertion/deletion mismatches, flap and pseudo-Y DNA structures by deoxyinosine 3'-endonuclease from Escherichia coli.
Deoxyinosine 3'-endonuclease, an Escherichia coli repair enzyme that recognizes and cleaves DNA containing deoxyinosine and base mismatches, can cleave heteroduplexes containing a hairpin or unpaired loop. These DNA structures, referred to as insertion/deletion mismatches (IDM), are abnormal intermediate structures generated during replication of repetitive DNA sequences. In addition, the enzyme also cleaved the 5'-single-stranded tails of flap and pseudo-Y DNA structures, suggesting that deoxyinosine 3'-endonuclease is a bacterial functional homologue of human FEN1 and yeast RTH1 nucleases. These biochemical properties suggest that deoxyinosine 3'-endonuclease might be important in the repair of IDM structures generated in lagging strand during DNA replication.
DNA Repair↗
Analysis of DNA structure in the human insulin gene-linked polymorphic region in vivo.
An altered DNA structure exists within the hypervariable region located 360 bp upstream of the human insulin gene. The aim of the present study was to determine whether this structure exists in the insulin gene in vivo, and whether its presence is related to the expression of the insulin gene. However, since there were no clonal human beta-cell lines available for such studies, the human insulin gene was transfected into a rat insulinoma-derived beta-cell line and several human insulin-expressing clones were selected. One such cell line was treated in vivo with the DNA structural probe bromoacetaldehyde and the chromosomal DNA was extracted. Following digestion with TaqI and subsequent digestion with S1-nuclease to cleave at the bromoacetaldehyde-reactive sites, the DNA was subjected to agarose gel electrophoresis, and insulin gene fragments were detected by Southern blot analysis. Bromoacetaldehyde generated subfragments of 2500, 1700 and 800 bp in the human insulin gene isolated from the rat beta-cell line, while the human insulin gene in the non-expressing HeLa cell line was unreactive to bromoacetaldehyde. These results suggest that an altered structure might exist in the insulin gene-linked polymorphic region of the human insulin gene in vivo, and that this structure may play a role in the expression of the insulin gene.
Animals↗
[Trivaline initiates formation of homo- and heteroquadruplex DNA structures].
Formation of heterologous (calf thymus double-stranded DNA) and homologous (linearized pBR322 plasmid double-stranded DNA) quadruplexes upon binding with the simple aliphatic tripeptide derivative (dansyl hydrazide trivaline) was examined by fluorimetry, flow linear and circular dichroism and electron microscopy. The morphology of the rod-like compact particles formed due to the association of double-stranded DNA segments proved to be the same for both DNAs, whereas the stability of the compact DNA structure upon tripeptide removal from the complex with DNA differed substantially for homologous versus non-homologous double-stranded DNA used. The increase of NaCl concentration in the solution up to 30 mM removes the peptide from both types of the complexes completely. At the same time at 20 mM NaCl calf thymus DNA quadruplexes readily dissociate, whereas the structures formed by plasmid DNA retain their morphology in the solution containing NaCl with concentrations up to 40 mM and are only partially disrupted at even higher NaCl concentration. These results provide the analogy between trivaline-DNA model complexes and RecA-DNA binding.
Animals↗
Osmium tetroxide reactivity of DNA bases in nucleotide sequencing and probing of DNA structure.
Osmium tetroxide, 2,2'-bipyridine (Os,bipy) has been widely applied as a probe of the DNA structure. To obtain information about reactivity of DNA bases toward this probe synthetic homopolynucleotides poly(dT), poly(dC), poly(dG) and poly(dA) were treated with Os,bipy and the content of modified bases measured by stripping voltammetry and absorption spectrophotometry. After 20 hours' treatment strong modification of poly(dT) and poly(dC) and weak modification of poly(dG) were observed, while no modification was detected in poly(dA). At short incubation times under conditions close to those usually used in probing the DNA structure the extent of poly(dT) modification was more than 10 times higher than that of poly(dC). Thus, in single-stranded DNA Os,bipy reacts with T much greater than C and G. Due to the fast reaction of thymines with Os,bipy (and osmium tetroxide, pyridine) these chemicals can be applied in Maxam-Gilbert nucleotide sequencing as agents specific for thymines in single-stranded DNA.
2,2'-Dipyridyl↗
The non-B-DNA structure of d(CA/TG)n does not differ from that of Z-DNA.
A number of recent studies have shown that simple repetitive d(CA/TG) dinucleotide sequences adopt a left-handed non-B-DNA structure under negative superhelical stress. The pattern of chemical reactivities and the helical parameters observed for these sequences differ significantly from those of standard Z-DNA. In this study, the data for two naturally occurring d(CA/TG)n sequences are reevaluated by a statistical mechanics treatment of the B- to Z-DNA transition. The behavior of these sequences under negative superhelical stress is accurately simulated by this model, including the multiple and discrete transitions observed for the rat prolactin promoter. Furthermore, the average helical twist for the left-handed structure of d(CA/TG)n deviates < 2% from that expected for standard Z-DNA. Finally, the predicted distribution of the junctions between B- and Z-DNA are shown to account for differences observed in the patterns of chemical reactivity of d(CA/TG)n and d(CG)n. Thus, no new left-handed structure that differs from Z-DNA is needed to describe the supercoil-induced conformation in d(CA/TG)n sequences.
Animals↗
A DNA structural atlas for Escherichia coli.
We have performed a computational analysis of DNA structural features in 18 fully sequenced prokaryotic genomes using models for DNA curvature, DNA flexibility, and DNA stability. The structural values that are computed for the Escherichia coli chromosome are significantly different from (and generally more extreme than) that expected from the nucleotide composition. To aid this analysis, we have constructed tools that plot structural measures for all positions in a long DNA sequence (e.g. an entire chromosome) in the form of color-coded wheels (http://www.cbs.dtu. dk/services/GenomeAtlas/). We find that these "structural atlases" are useful for the discovery of interesting features that may then be investigated in more depth using statistical methods. From investigation of the E. coli structural atlas, we discovered a genome-wide trend, where an extended region encompassing the terminus displays a high of level curvature, a low level of flexibility, and a low degree of helix stability. The same situation is found in the distantly related Gram-positive bacterium Bacillus subtilis, suggesting that the phenomenon is biologically relevant. Based on a search for long DNA segments where all the independent structural measures agree, we have found a set of 20 regions with identical and very extreme structural properties. Due to their strong inherent curvature, we suggest that these may function as topological domain boundaries by efficiently organizing plectonemically supercoiled DNA. Interestingly, we find that in practically all the investigated eubacterial and archaeal genomes, there is a trend for promoter DNA being more curved, less flexible, and less stable than DNA in coding regions and in intergenic DNA without promoters. This trend is present regardless of the absolute levels of the structural parameters, and we suggest that this may be related to the requirement for helix unwinding during initiation of transcription, or perhaps to the previously observed location of promoters at the apex of plectonemically supercoiled DNA. We have also analyzed the structural similarities between groups of genes by clustering all RNA and protein-encoding genes in E. coli, based on the average structural parameters. We find that most ribosomal genes (protein-encoding as well as rRNA genes) cluster together, and we suggest that DNA structure may play a role in the transcription of these highly expressed genes.
Bacterial Proteins↗
Recognition of distorted DNA structures by HMG domains.
Recent biochemical and structural studies have shown that the preferential recognition of distorted DNA structures, including DNA bulges, four-way junctions and cis-platinated DNA, by HMG domains is dependent on residues immediately preceding the second alpha helix of the L-shaped HMG domain.
Amino Acid Sequence↗
Multiple transitions to non-B-DNA structures occur in the distal regulatory region of the rat prolactin gene.
The developmentally regulated rat prolactin (rPRL) gene presents a promising model system toward understanding the biological role of non-B-DNA structural elements. Two predominantly alternating purine-pyrimidine (APP) (dA-dC)n.(dG-dT)n repeats of 58 and 178 base-pairs flank the (A + T)-rich distal regulatory region. We have characterized several transitions to non-B-DNA structures within this region in negatively supercoiled plasmids by utilizing high resolution chemical probing. Each repeat undergoes a full-length conversion to a novel left-handed helical structure via the stepwise nucleation and propagation of discrete "segments". These segments are delimited by out-of-alternation bases that are susceptible to attack by potassium permanganate and thus appear to be significantly unstacked within the left-handed helices. Moreover, the spatial order of successive right- to left-handed DNA transitions within each repeat exhibits a clear polarity toward the distal regulatory region of the rPRL gene. An additional transition involving the long-range unpairing of (A + T)-rich sequences establishes a directional propagation toward the regulatory region. These data demonstrate a complex series of quasi-independent transitions to non-B-DNA structures that impinge upon a known regulatory control region.
Animals↗
The effects of N7-methylguanine on duplex DNA structure.
BACKGROUND: Non-enzymatic methylation of DNA by endogenous and exogenous agents produces a variety of adducts, of which the predominant one is N7-methyl-2'-deoxyguanosine (m7dG). Although it is known that living organisms counter the deleterious effects of m7dG by producing adduct-specific DNA repair proteins, the molecular basis for specific recognition and catalysis by these proteins is poorly understood. In addition to its role as an endogenous DNA adduct, m7dG is also widely used as an in vitro probe of protein-DNA interactions. We set out to examine whether incorporation of m7dG into DNA affects duplex DNA structure. RESULTS: We carried out a large-scale synthesis of a dodecamer containing the m7dG adduct at a single, defined position. Because the instability of m7dG precludes its incorporation into oligonucleotides by standard solid-phase methods, a novel strategy employing chemical and enzymatic synthesis was used. Characterization of the m7dG-containing dodecamer by NMR reveals no structural distortion; indeed, m7dG appears to encourage a modest shift toward a more characteristically B-form duplex. CONCLUSIONS: These results argue strongly against induced DNA distortion as a mechanism for specific recognition of m7dG by adduct-specific repair proteins. The broad substrate specificity of these repair proteins disfavors a model involving direct recognition of aberrantly placed methyl groups; hence, it may be that m7dG is recognized indirectly, perhaps by its effects on the dynamics of DNA. On the other hand, the evidence presented here suggests that m7dG interferes directly with sequence-specific recognition by DNA-binding proteins by steric blockage or by masking of required contact functionalities. The synthetic methodology used here should be generally applicable to high-resolution structural studies of oligonucleotides bearing adducts that are unstable to the conditions of solid-phase DNA synthesis.
DNA↗
Importance of stereospecific positioning of the upstream cis-acting DNA element containing a curved DNA structure for the functioning of the Escherichia coli proV promoter.
The mechanism by which the Escherichia coli proV promoter is activated more than 100-fold in response to the medium osmolarity, without the help of any known trans-acting activators, is not yet fully understood. In this context, it has recently begun to be realized that structural features, not the primary sequences, of cis-acting DNA elements may be important for transcriptional regulation in prokaryotes. From this point of view, in this study the proV promoter was characterized by constructing a series of spacer-insertion mutants in a proV-lacZ fusion on the chromosome. Here it was found that the upstream cis-acting sequence must be positioned stereospecifically with respect to the principal -35 and -10 regions for the proV promoter to be fully activated. In this regard, it was suggested that an overall DNA structure, particularly DNA curvature, is an important cis-acting parameter for activation of the proV promoter.
Bacterial Proteins↗
Double-strand break formation by the RAG complex at the bcl-2 major breakpoint region and at other non-B DNA structures in vitro.
The most common chromosomal translocation in cancer, t(14;18) at the 150-bp bcl-2 major breakpoint region (Mbr), occurs in follicular lymphomas. The bcl-2 Mbr assumes a non-B DNA conformation, thus explaining its distinctive fragility. This non-B DNA structure is a target of the RAG complex in vivo, but not because of its primary sequence. Here we report that the RAG complex generates at least two independent nicks that lead to double-strand breaks in vitro, and this requires the non-B DNA structure at the bcl-2 Mbr. A 3-bp mutation is capable of abolishing the non-B structure formation and the double-strand breaks. The observations on the bcl-2 Mbr reflect more general properties of the RAG complex, which can bind and nick at duplex-single-strand transitions of other non-B DNA structures, resulting in double-strand breaks in vitro. Hence, the present study reveals novel insight into a third mechanism of action of RAGs on DNA, besides the standard heptamer/nonamer-mediated cleavage in V(D)J recombination and the in vitro transposase activity.
Animals↗
Structure calculations for single-stranded DNA complexed with the single-stranded DNA binding protein GP32 of bacteriophage T4: a remarkable DNA structure.
In this study it is established by calculation which regular conformations single-stranded DNA and RNA can adopt in the complex with the single-stranded DNA binding protein GP32 of bacteriophage T4. In order to do so, information from previous experiments about base orientations and the length and diameter of the complexes is used together with knowledge about bond lengths and valence angles between chemical bonds. It turns out that there is only a limited set of similar conformations which are in agreement with experimental data. The arrangement of neighboring bases is such that there is ample space for aromatic residues of the protein to partly intercalate between the bases, which is in agreement with a previously proposed model for the binding domain of the protein [Prigodich, R. V., Shamoo, Y., Williams, K. R., Chase, J. W., Konigsberg, W. H., & Coleman, J. E. (1986) Biochemistry 25, 3666-3671]. Both C2'endo and C3'endo sugar conformations lead to calculated DNA conformations that are consistent with experimental data. The orientation of the O2' atoms of the sugars in RNA can explain why the binding affinity of GP32 for polyribonucleotides is lower than for polydeoxyribonucleotides.
Binding Sites↗