Tautomeric and microscopic protonation equilibria of some alpha-amino acids

Although there is a wealth of structural and theoretical data relating to palindromic sequences in genomes, the mechanisms of extrusion of cruciform structures during various biological processes in the presence of intercalating agents are still poorly understood. The current study addresses the effects of temperature and intercalator on cruciform extrusion from plasmids and also considers the effects of divalent metal ions on cruciform extrusion. It presents evidence that the cytotoxic effects of certain DNA binding drugs in vivo occur over concentration ranges corresponding to those that modulate cruciform extrusion in vitro. The results confirm earlier studies showing an inverse relationship between the effects of negative superhelicity and temperature on cruciform extrusion. By extrapolation, divalent metal ions facilitate cruciform extrusion by increasing superhelicity. The results allow the concentrations that preclude cruciform extrusion in DNA to be determined, and these are potentially informative about the relationships among temperature, DNA helical winding, cruciform formation, and intercalation. Overall, we provide new and interesting insights into the potential role of cruciform structures in biology and, by implication, cancer therapy.     

A novel type of interaction between cruciform DNA and a cruciform binding protein from HeLa cells.

We recently identified and enriched a protein (CBP) from HeLa cells with binding specificity for cruciform-containing DNA. We have now studied the interaction of CBP with stable cruciform DNA molecules containing the 27 bp palindrome of SV40 on one strand and an unrelated 26 bp palindrome on the other strand by hydroxyl radical footprinting. The CBP-DNA interaction is localized to the four-way junction at the base of the cruciforms. CBP appears to interact with the elbows of the junctions in an asymmetric fashion. Upon CBP binding, structural distortions were observed in the cruciform stems and in a DNA region adjacent to the junction. These features distinguish CBP from other cruciform binding proteins, which bind symmetrically and display exclusively either contacts with the DNA backbone or structural alterations in the DNA.     

Interactions between structure transitions in a torsionally constrained DNA

We used S1 nuclease cleavage in conjunction with gel electrophoresis to evaluate torsion-induced cruciform extrusion at two inverted repeat sequences, IRS-B and IRS-C of plasmid pUC12. These structure transitions affect each other through competition for the available torsional free energy according to their relative energies of activation and the magnitude of DNA duplex unwinding associated with each transition. They can be modulated by the level of DNA negative torsion. Interplays between transition sequences occur over long distances and are independent of relative orientation of transition sites. DNA binding factors that enhance or repress structural transitions of specific sequences may, thus, regulate the structural and functional properties of torsionally coupled, distal sequences.     

14-3-3s are DNA-replication proteins

14-3-3 proteins are conserved multifunctional molecules, involved in many biological processes. Several 14-3-3 isoforms were recently shown to be cruciform DNA-binding proteins, which is a new activity ascribed to the 14-3-3 family. As cruciform-binding proteins, 14-3-3 proteins are putatively involved in the regulation of DNA replication. Inverted repeat sequences that are able to extrude into cruciform structures are a common feature of replication origins in both prokaryotes and eukaryotes. The involvement of cruciform structures in the initiation of DNA replication has been demonstrated. A leading model of 14-3-3 function proposes that they facilitate critical protein-protein interactions, thus serving as a central component of a wide variety of cellular processes.     

Rh(DIP)3(3+): a shape-selective metal complex which targets cruciforms.

The coordination complex tris(4,7-diphenylphenanthroline)rhodium(III), Rh(DIP)3(3+), binds to and, upon photoactivation, cleaves both DNA strands near the base of a DNA cruciform. Sites of photoinduced double-stranded DNA cleavage by the rhodium complex map to regions containing cruciforms on closed circular pBR322, pColE1 and phi X174 (replicative form) DNAs. Neither cleavage nor binding by the metal complex, assayed using S1 nuclease, is found on the linear plasmid which lacks the extruded cruciform. High resolution mapping experiments reveal that Rh(DIP)3(3+) cleaves at a specific AT-rich site neighboring the stem of the minor cruciform on pBR322. The primary site of cleavage is found at position 3238 on the 3'-strand and 3250 on the 5'-strand and is remarkably specific. The pattern of cleavage, to one side only of the cruciform stem, indicates an asymmetry in the cruciform structure recognized by the complex. These results suggest that Rh(DIP)3(3+) may provide a useful reagent to probe cruciform sites. In addition, the high degree of specificity found in targeting the cruciform structure with this simple metal complex underscores the utility of shape-selection for the recognition of specific sites on a DNA strand.     

Effect of DNA supercoiling on the geometry of holliday junctions

Unusual DNA conformations including cruciforms play an important role in gene regulation and various DNA transactions. Cruciforms are also the models for Holliday junctions, the transient DNA conformations critically involved in DNA homologous and site-specific recombination, repair, and replication. Although the conformations of immobile Holliday junctions in linear DNA molecules have been analyzed with the use of various techniques, the role of DNA supercoiling has not been studied systematically. We utilized atomic force microscopy (AFM) to visualize cruciform geometry in plasmid DNA with different superhelical densities at various ionic conditions. Both folded and unfolded conformations of the cruciform were identified, and the data showed that DNA supercoiling shifts the equilibrium between folded and unfolded conformations of the cruciform toward the folded one. In topoisomers with low superhelical density, the population of the folded conformation is 50-80%, depending upon the ionic strength of the buffer and a type of cation added, whereas in the sample with high superhelical density, this population is as high as 98-100%. The time-lapse studies in aqueous solutions allowed us to observe the conformational transition of the cruciform directly. The time-dependent dynamics of the cruciform correlates with the structural changes revealed by the ensemble-averaged analysis of dry samples. Altogether, the data obtained show directly that DNA supercoiling is the major factor determining the Holliday junction conformation.     

The effect of supercoil and temperature on the recognition of palindromic and non-palindromic regions in phi X174 replicative form DNA by S1 and Bal31

The effect of supercoil and temperature on the topology of phi X174 replicative form (RF) DNA was studied using single-strand specific endonucleases S1 and Bal31 as probes for cruciform extrusion and other structural perturbations of the B-helix. Both enzymes were found to recognize specifically and reproducibly over 30 sites, most of which were cleaved by both enzymes independent of the superhelicity of the genome. A negative superhelical density exceeding 0.06 stabilized a transition in the DNA conformation that generated several new cleavage sites for Bal31. The underlying structures appeared to be only transiently stable and were lost from in vitro supercoiled DNA during brief incubation at 65 degrees C. They were generally absent from in vivo supercoiled RF DNA of equal superhelicity as a consequence of the extraction and storage procedure. Mapping of the cleavage sites suggested that they were preferentially located near the beginnings and ends of genes and that the structural basis for at least some of them was the extrusion of relatively small palindromes into the cruciform state. Insertion of a short synthetic palindromic sequence into the phi X174 genome generated a supercoil-dependent, temperature-sensitive secondary structure that was cleaved in the Bal31 but not the S1 reaction, further supporting the hypothesis that even small cruciforms with stem size of 7 or less base pairs may be transiently stable. Subjecting supercoiled RF DNA to the typical S1 reaction conditions induced a topological shift that diminished all but one of the supercoil-induced Bal31 recognition sites and promoted the formation of one major new site.     

Nucleosome phasing on a DNA fragment from the replication origin of simian virus 40 and rephasing upon cruciform formation of the DNA.

Nucleosomes were reconstituted in vitro from a fragment of DNA spanning the simian virus 40 minimal replication origin. The fragment contains a 27-base-pair palindrome (perfect inverted repeat). DNA molecules with stable cruciform structures were generated by heteroduplexing this DNA fragment with mutants altered within the palindromic sequence (C. Nobile and R. G. Martin, Int. Virol., in press). Analyses of the structural features of the reconstituted nucleosomes by the DNase I footprint technique revealed two alternative DNA-histone arrangements, each one accurately phased with respect to the uniquely labeled DNA ends. As linear double-stranded DNA, a unique core particle was formed in which the histones strongly protected the regions to both sides of the palindrome. The cruciform structure seemed to be unable to associate with core histones and, therefore, an alternative phasing of the histone octamer along the DNA resulted. Thus, nucleosome positioning along a specific DNA sequence appears to be influenced in vitro by the secondary structure (linear or cruciform) of the 27-base-pair palindrome. The formation of cruciform structures in vivo, if they occur, might therefore represent a molecular mechanism by which nucleosomes are phased.     

Hierarchy of binding sites for chromosomal proteins HMG 1 and 2 in supercoiled deoxyribonucleic acid

The interaction of chromosomal proteins HMG 1 and 2 with various DNA structures has been examined with plasmid pPst-0.9, which contains DNA sequences that can form the Z-DNA conformation and palindromic sequences that can form cruciform structures. Direct binding and competition experiments with 32P-labeled plasmid indicated that proteins HMG 1 and 2 preferentially bind to supercoiled form I DNA as compared to double-stranded linear DNA. The preferential binding to form I is due to the presence of single-stranded regions in this DNA. The binding of HMG 1 and 2 to the form I plasmid results in inhibition of S1 nuclease digestion in a selective manner. The B-Z junction is preferentially protected as compared to the cruciform, which in turn is more protected than other minor S1-sensitive structures present in pPst-0.9. Our results indicate that the binding of HMG 1 and 2 proteins to DNA is not random in that HMG 1 and 2 can distinguish between various S1 nuclease sensitive sites in the plasmid. The existence of a hierarchy of DNA binding sites for these proteins suggests that they can selectively affect the structure of distinct regions in the genome.     

Supercoiling-induced DNA bending

Local DNA bending is a critical factor for numerous DNA functions including recognition of DNA by sequence-specific regulatory binding proteins. Negative DNA supercoiling increases both local and global DNA dynamics, and this dynamic flexibility can facilitate the formation of DNA-protein complexes. We have recently shown that apexes of supercoiled DNA molecules are sites that can promote the formation of an alternative DNA structure, a cruciform, suggesting that these positions in supercoiled DNA are under additional stress and perhaps have a distorted DNA geometry. To test this hypothesis, we used atomic force microscopy to directly measure the curvature of apical positions in supercoiled DNA. The measurements were performed for an inherently curved sequence formed by phased A tracts and a region of mixed sequence DNA. For this, we used plasmids in which an inverted repeat and A tract were placed at precise locations relative to each other. Under specific conditions, the inverted repeat formed a cruciform that was used as a marker for the unambiguous identification of the A tract location. When the A tract and cruciform were placed diametrically opposite, this yielded predominantly nonbranched plectonemic molecules with an extruded cruciform and A tract localized in the terminal loops. For both the curved A tract and mixed sequence nonbent DNA, their localization to an apex increased the angle of bending compared to that expected for DNA unconstrained in solution. This is consistent with increased helical distortion at an apical bend.     

The Roles of CRE, TRE, and TRE-Adjacent S1 Nuclease Sensitive Element in the Regulation of Tyrosine Hydroxylase Gene Promoter Activity by Angiotensin II

Abstract: The cis elements mediating activation of the tyrosine hydroxylase gene by angiotensin II were examined by transfecting tyrosine hydroxylase promoter-luciferase constructs into cultured bovine adrenal medullary cells. Angiotensin II-responsive elements are located within −54/+25-bp and −269/−55-bp promoter regions and were identified, respectively, as cyclic AMP (CRE)- and 12- O -tetradecanoylphorbol 13-acetate responsive element (TRE)-like sequences. Unlike CRE, TRE also supports basal promoter activity. Mutations of TRE or CRE that reduced angiotensin II stimulation abolished in vitro binding of nuclear proteins to those elements, suggesting that proteins forming CRE- and TRE-inducible complexes may mediate angiotensin II stimulation. The TRE is adjacent to a dyad symmetry element. Those two sites form a common regulatory unit in which the dyad symmetry element acts as a repressor of the TRE site. Isolated dyad symmetry element did not bind nuclear proteins in vitro. In supercoiled DNA it exhibited S1 nuclease sensitivity and was recognized by a DNA cruciform-specific antibody consistent with the extrusion of a cruciform structure that overlaps with the TRE. A mutation that abolished formation of the cruciform correlated with a loss of repressor activity. We propose a novel model of tyrosine hydroxylase gene regulation in which functions of the TRE are modulated via structural transition in the adjacent DNA.     

Purification and properties of a nuclease from Saccharomyces cerevisiae that cleaves DNA at cruciform junctions

An endonuclease specific for cruciform junctions has been purified from yeast cells treated with a DNA-damaging agent. The activity was followed through five chromatographic steps by assaying for the linearization of supercoiled plasmid DNA, which extrudes cruciform structures in vitro. The sites of cleavage on pColIR215 were sequenced, and nicks were located to positions symmetrically opposed across the cruciform junction. The products of cleavage were unit length linear duplexes that contained terminal hairpin loops. In contrast to pColIR215, the cleavage patterns of pXG540 plasmid DNA were found to be complex, and cuts were found up to 40 bases from an (A-T)34 sequence that extrudes into a cruciform. Little or no activity could be detected on single-stranded DNA, linear duplex DNA, or nicked circular duplex DNA. The nuclease was insensitive to RNase but was inactivated by treatment with proteinase K. Mg2+ was required as cofactor and could not be replaced by Mn2+, Ca2+, Co2+, or Cu2+. The native molecular weight of the activity was approximately 200,000 as estimated by gel filtration.     

Interaction of clinically important human DNA topoisomerase I poison, topotecan, with double-stranded DNA

Topotecan (TPT), a water-soluble derivative of camptothecin, is a potent antitumor poison of human DNA topoisomerase I (top1) that stabilizes the cleavage complex between the enzyme and DNA. The role of the recently discovered TPT affinity to DNA remains to be defined. The aim of this work is to clarify the molecular mechanisms of the TPT-DNA interaction and to propose the models of TPT-DNA complexes in solution in the absence of top1. It is shown that TPT molecules form dimers with a dimerization constant of (4.0 +/- 0.7) x 10(3) M(-1) and the presence of DNA provokes more than a 400-fold increase of the effective dimerization constant. Flow linear dichroism spectroscopy accompanied by circular dichroism, fluorescence, and surface-enhanced Raman scattering experiments provide evidence that TPT dimers are able to bind DNA by bridging different DNA molecules or distant DNA structural domains. This effect may provoke modification of the intrinsic geometry of the cruciform DNA structures, leading to the appearance of new crossover points that serve as the sites of the top1 loading position. The data presume the hypothesis of TPT-mediated modulation of top1-DNA recognition before ternary complex formation.