Chemical Probing of the B-Z Transition in Negatively Supercoiled DNA

Abstract

An analysis of the B-to-Z transition as a function of supercoiling for a natural Z-DNA- forming sequence found in plasmid pBR322 is presented at nucleotide resolution. The analysis is based on reactivity to four chemical probes which exhibit hyperreactivity in the presence of Z-DNA: hydroxylamine, osmium tetroxide, diethyl pyrocarbonate and dimethyl sulfate. We find that the initial transition occurs largely within a 14 base pair region which is mostly alternating purines and pyrimidines. With increasing negative supercoiling, Z-DNA extends into flanking regions having less and less alternating character, first in one direction and then in the other. Evidence of B-Z junctions is seen at four sites bracketing these three adjacent regions. One of these Z-forming regions contains the non-alternating sequence CTCCT, suggesting that such sequences can form Z-DNA without great difficulty if they are adjacent to alternating sequences. A plasmid containing three copies of a 61 base pair fragment bearing the entire Z-forming region shows equal reactivity of all three copies at any given superhelical density, implying that they compete equally and independently for the torsional strain energy which promotes the B-Z transition, and are unaffected by adjacent sequences more than 20–30 base pairs away.     

Recognition of B and Z forms of DNA by Escherichia coli DNA polymerase I

Since the substrate binding domain of the large proteolytic fragment of Escherichia coli DNA polymerase I has been shown to interact with the B forms of DNA, we have studied the ability of this enzyme to recognize structures other than the B form. The polymerase activity has been used to evaluate the degree of recognition of the B and Z forms of DNA. The Z form was found to promote less activity, indicating the probable inability of the polymerase to move along the conformationally rigid form of the template. The present study indicates that the Z-DNA found in vivo may have a role in the control of replication.     

Recognition of the structural distortions at the junctions between B and Z segments in negatively supercoiled DNA by osmium tetroxide

It has been shown for the first time that conformational junction between contiguous right-handed B and left-handed Z segments can be recognized by a chemical probe. Plasmid pRW751 containing (dC-dG)13 and (dC-dG)16 blocks was treated with osmium tetroxide, pyridine (a reagent known to be single-strand selective) at physiological ionic conditions (0.1 and 0.2 M NaCl) and neutral pH. Mapping of the osmium binding sites by restriction enzyme digestion followed by nuclease S1 cleavage has revealed selective binding of osmium at, or near to, the end of the (dC-dG)n segments proximal to the 95 bp lac sequence. The junction of the shorter (dC-dG)13 segment was modified to a substantially greater extent than that of the longer segment. Partial inhibition of DNA cleavage by BamHI was observed at the restriction sites neighbouring to the both (dC-dG)n segments as a result of DNA modification by osmium tetroxide. The site-selective modification occurred only in supercoiled and not in relaxed molecules. Differences in the sensitivity of the B/Z junctions in pRW751 to the osmium tetroxide were explained by different structural features of these junctions.     

Increase in the Calcium Content of Cardiac Tissue after Postfixation with Osmium Tetroxide

The concentration of osmium has been measured by destructive chemical analysis in glutaraldehyde fixed heart tissue postfixed with osmium tetroxide and embedded in epoxy resin. After such treatment, the mean atomic number of the specimen (Z) is close to 10, which permits a quantitative analysis of calcium (Ca) by the continuum method, using Z²/A as a correcting factor (A: atomic weight). Wavelength-dispersive X-ray microanalysis has been used to determine the Ca concentration of frog cardiac tissue fixed in glutaraldehyde and embedded in resin. These measurements have been repeated on tissue postfixed in osmium tetroxide; contrary to expectations, the apparent Ca concentration is much higher in osmium-treated than in nontreated tissue. However, this result is observed with OsO₄ solutions prepared in glass, not with solutions prepared in plastic. It is shown by energy dispersive X-ray analysis of droplets that OsO₄ solutions prepared in glass contain large amounts of calcium, potassium and silicon. Care must be taken in preparing OsO₄ fixatives when the fixed tissues are to be subjected to X-ray microanalysis of such elements as Ca or Si.     

Structural Insight Into hnRNP A2/B1 Homodimerization and DNA Recognition

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) has been identified as a nuclear DNA sensor. Upon viral infection, hnRNP A2/B1 recognizes pathogen-derived DNA as a homodimer, which is a prerequisite for its translocation to the cytoplasm to activate the interferon response. However, the DNA binding mechanism inducing hnRNP A2/B1 homodimerization is unknown. Here, we show the crystal structure of the RNA recognition motif (RRM) of hnRNP A2/B1 in complex with a U-shaped ssDNA, which mediates the formation of a newly observed protein dimer. Our biochemical assays and mutagenesis studies confirm that the hnRNP A2/B1 homodimer forms in solution by binding to pre-generated ssDNA or dsDNA with a U-shaped bulge. These results depict a potential functional state of hnRNP A2/B1 in antiviral immunity and other cellular processes.     

Improved Intracellular Morphology of Pneumocystis Carinii From Rat Lung by Postfixation with a Mixture of Potassium Ferrocyanide and Osmium Tetroxide

Pneumocystis carinii infected rat lungs were postfixed with a mixture of OsO₄ and K₄Fe(CN)₆. A marked improvement in staining of cell membranes, endoplasmic reticulum, nuclear membranes and glycogen was observed. These improvements were seen in both the trophic and cystic forms of the organisms. The addition of K₄Fe(CN)₆ did not improve the staining of cell walls, microtubules or ribosomes. Trophozoites were seen attached to both type 1 and type 2 pneumocytes by filopodia and/or intercalation of the cell body of P. carinii with the host lung cells. It is expected that the improvement in ultrastructural detail will allow better understanding of the ultrastructure of P. carinii and provide insights into the modes of action of various antimicrobial compounds on this organism.     

The Mechanism of the Supercoiled DNA Recognition by the Eukaryotic Type I Topoisomerases: I. The Enzyme Interaction with Nonspecific Oligonucleotides

Interaction of the DNA type I topoisomerases from the murine and human placenta cells with nonspecific oligonucleotides was analyzed. The contributions of strong and week nonspecific electrostatic, van der Waals's, and hydrophobic interactions, and hydrogen bonding of the enzymes to the complex formation with the single- and double-stranded DNAs were determined. The factors that determine the top-priority recognition of the topologically stressed DNA were revealed. The results were interpreted in comparison with the X-ray analysis data for human DNA topoisomerase I.     

Time-Lapse Imaging of Conformational Changes in Supercoiled DNA by Scanning Force Microscopy

Most of the scanning force microscopy (SFM) images of supercoiled DNA on untreated mica thus far reported have not shown tight plectonemic structure seen by electron microscopy, but instead less coiled molecules and sometimes a partly "condensed" state with intimate chain-chain interactions. By observing time-lapse images of conformational changes of DNA induced by decreasing ionic strength of imaging buffer in solution SFM, we could show that the process of water rinsing, an indispensable step for preparation of dried samples, may be responsible for some of the conformational anomalies in the images previously reported. We have studied several protocols to observe supercoiled DNA molecules by SFM and discuss the merits and the demerits. Images obtained following uranyl acetate treatment may be ideal for the detection of DNA damage, as the supercoiled and nicked forms are easily distinguishable.     

Theoretical Study of Ethidium Intercalation in Triple-Stranded DNA and at Triplex-Duplex Junctions

Abstract

The contribution of different factors in the interaction of ethidium intercalated into various sequences of a triple helix, or in the region of the junction between the double- and triple-stranded DNA has been studied by energy minimization. It is found that in the total energy of the ethidium - triple helix complexes, a particular electrostatic contribution emerges due to the presence of protonated cytosines in the triple helix. This parameter is determinant in the sequence-specificity of ethidium binding to the triple helix. The preferred intercalation sites of ethidium in the triple helix are proposed. The interaction of ethidium at the triplex-duplex junction, and its effects are also discussed. This study is aimed at searching for new drugs specific for the triple helix, or for the triplex-duplex junctions.     

The Mechanism of Supercoiled DNA Recognition by Eukaryotic Type I Topoisomerases: II. A Comparison of the Enzyme Interaction with Specific and Nonspecific Oligonucleotides

Data on the interaction of DNA type I topoisomerases from the murine and human placenta cells with specific and nonspecific oligonucleotides of various structures and lengths are summarized. The relative contributions of various contacts between the enzymes and DNA that have previously been detected by X-ray analysis to the total affinity of the topoisomerases for DNA substrates are estimated. Factors that determine the differences in the enzyme interactions with specific and nonspecific single- and double-stranded DNAs are revealed. The results of the X-ray analysis of human DNA topoisomerase I are interpreted taking into account data on the comprehensive thermodynamic and kinetic analysis of the enzyme interaction with the specific and nonspecific DNAs.     

Base sequence criteria and Cartesian coordinates for stable B/Z and B/Z/B junctions in relaxed DNA.

It seems increasingly evident that if the Z form of DNA exists in the genome it must exist as short sections of alternating pyrimidine-purine sequences in the midst of very long sections of B-form DNA. We have determined the minimum length of a string of alternating CG base pairs that can go into the Z form in the middle of a long B form. Self-complimentary oligomers of the form T(M)(CG)(N)A(M) were synthesized. The conformation of the resulting duplex was determined in 6M aqueous NaCl solution by Raman scattering. We have found that 12 alternating CG base pairs is the minimum length required to form a stable Z form of DNA inside of a long B form section. Only the 4 center CG base pairs go into the Z form. These 4 CG base pairs in the Z form are flanked on each side by 4 CG base pairs in a non-Z (probably B) form as well as the ..TT.. ..AA.. sequences in the B form. We propose a model of the B/Z junction in which the double helix flips directly from the B form to the Z form so that there are no base pairs in the junction. In this model the B form is nucleated in the AT base pairs on each end and is propagated into the CG base pairs in the center. This model is supported by isotopic H/D exchange experiments that shows that the H/D exchange of the non-Z form CG base pairs is highly retarded and indicates that they remain in the B form. A Thermodynamic analysis of the concentration dependence of the melting point of the duplexes in both low and high salt, supports our model and rules out the possibility of hairpin formation. The enthalpy for the formation of a B/Z junction is determined to be about +16 kcal/junction. A comparison of these results with recent results on B/Z junctions in super-coiled DNA is given. Molecular modeling calculations permit us to obtain values for the coordinates and torsional angles of the oligomers showing both B/Z and B/Z/B junctions. The Cartesian coordinates for these oligomers as well as stereo figures of these models in color are available from the authors.     

CAP binding to B and Z forms of DNA.

We have examined the interaction between the cyclic AMP receptor protein (CAP) and a small DNA fragment containing its specific recognition sequence by circular dichroism spectroscopy. The binding of CAP to this fragment induces a B to "C-like" change in the CD spectrum, which is different from that observed for non-specific binding. A one-to-one (CAP dimer to DNA) binding stoichiometry was deduced from spectroscopic titration data, as was a non-specific binding site size of 17 bp/dimer. In addition, we have compared the non-specific binding affinity of CAP for the B and Z forms of synthetic DNA copolymers. A slight preference for the B form was found. These results do not support the recent specific suggestion that CAP binds to a left-handed form of DNA (1), but indicate more generally that an optically detectable conformational change takes place in DNA on binding CAP.     

Effects of Glutaraldehyde and Osmium Tetroxide on Hypotrichous Ciliates, and Determination of the Most Satisfactory Fixation Methods for Electron Microscopy

SYNOPSIS. In electron microscope studies on hyppotrichous ciliates, cytolysis and/or body deformation–resulting from insufficient contact with glutaraldehyde and poor infiltration of Epon 812, particularly into the buccal cavity, usually were observed. Fixation experiments were carried out to examine the effects of some fixatives on Euplotes eurystomus, Oxytricha bifaria and Stylonychia mytilus to establish the best fixation technic applicable to all species of hypotrichous ciliates. Although the effects of fixation varied considerably with the species, 2 fully satisfactory fixation methods were developed by using OsO₄ and glutaraldehyde. In one, a mixture of both fixatives was employed; in the other a very short application of OsO₄ was followed by glutaraldehyde. The problem of infiltration was solved by using Spurr's low-viscosity embedding medium in place of Epon 812.     

Recognition of the pro-mutagenic base uracil by family B DNA polymerases from archaea

Archaeal family B DNA polymerases contain a specialised pocket that binds tightly to template-strand uracil, causing the stalling of DNA replication. The mechanism of this unique "template-strand proof-reading" has been studied using equilibrium binding measurements, DNA footprinting, van't Hoff analysis and calorimetry. Binding assays have shown that the polymerase preferentially binds to uracil in single as opposed to double-stranded DNA. Tightest binding is observed using primer-templates that contain uracil four bases in front of the primer-template junction, corresponding to the observed stalling position. Ethylation interference analysis of primer-templates shows that the two phosphates, immediately flanking the uracil (NpUpN), are important for binding; contacts are also made to phosphates in the primer-strand. Microcalorimetry and van't Hoff analysis have given a fuller understanding of the thermodynamic parameters involved in uracil recognition. All the results are consistent with a "read-ahead" mechanism, in which the replicating polymerase scans the template, ahead of the replication fork, for the presence of uracil and halts polymerisation on detecting this base. Post-stalling events, serving to eliminate uracil, await full elucidation.     

The Interactions of Enzyme and Chemical Probes with Inverted Repeats in Supercoiled DNA

Abstract

In negatively supercoiled DNA molecules some inverted repeat sequences adopt a perturbed conformation which is characterised by the following properties. They are centrally hypersensitive to single-strand-specific nucleases such as SI, and to a much lower extent the flanking regions may also be sensitive. They are also hypersensitive to modification by bromoacetaldehyde, particularly in their flanking region. They may be resistant to endo- nucleolysis by restriction enzymes and are cleaved (resolved) by a T4 resolving enzyme. All these properties can only be consistently explained by a model in which the inverted repeat adopts a cruciform structure. This property has been shown to depend sharply on a superhelix density, and the transition to nuclease sensitivity is accompanied by a marked alteration in the overall molecular geometry as judged by frictional properties. The probable dynamics of these structures are discussed.     

Structural studies of DNA fragments: the G.T wobble base pair in A, B and Z DNA; the G.A base pair in B-DNA

The crystal structures of five double helical DNA fragments containing non-Watson-Crick complementary base pairs are reviewed. They comprise four fragments containing G.T base pairs: two deoxyoctamers d(GGGGCTCC) and d(GGGGTCCC) which crystallise as A type helices; a deoxydodecamer d(CGCGAATTTGCG) which crystallises in the B-DNA conformation; and the deoxyhexamer d(TGCGCG), which crystallises as a Z-DNA helix. In all four duplexes the G and T bases form wobble base pairs, with bases in the major tautomer forms and hydrogen bonds linking N1 of G with O2 of T and O6 of G with N3 of T. The X-ray analyses establish that the G.T wobble base pair can be accommodated in the A, B or Z double helix with minimal distortion of the global conformation. There are, however, changes in base stacking in the neighbourhood of the mismatched bases. The fifth structure, d(CGCGAATTAGCG), contains the purine purine mismatch G.A where G is in the anti and A in the syn conformation. The results represent the first direct structure determinations of base pair mismatches in DNA fragments and are discussed in relation to the fidelity of replication and mismatch recognition.     

Recognition and cleavage at the DNA major groove.

DNA recognition agents based on the indole-based aziridinyl eneimine and the cyclopent[b]indole methide species were designed and evaluated. The recognition process involved either selective alkylation or intercalating interactions in the major groove. DNA cleavage resulted from phosphate backbone alkylation (hydrolytic cleavage) and N(7) -alkylation (piperidine cleavage). The formation and fate of the eneimine was studied using enriched 13C NMR spectra and X-ray crystallography. The aziridinyl eneimine specifically alkylates the N(7) position of DNA resulting in direction of the aziridinyl alkylating center to either the 3'- or 5'-phosphate of the alkylated base. The eneimine species forms dimers and trimers that appear to recognize DNA at up to three base pairs. The cyclopent[b]indole quinone methide recognizes the 3'-GT-5' sequence and alkylates the guanine N(7) and the thymine 6-carbonyl oxygen causing the hydrolytic removal of these bases. In summary, new classes of DNA recognition agents are described and the utility of 13C-enrichment and 13C NMR to study DNA alkylation reactions is illustrated.