Experimental Study of Antitumor Activity of Pefagtal Addressed to αvß3 Integrins.

We studied the effect of a new targeted drug Pefagtal that represents a conjugate in which the MS2 phage filled with a substance toxic to cells (thallium salts) is covalently linked to peptides containing the RGD motif. The antitumor and pronounced antimetastatic effects of Pefagtal were demonstrated on transplanted mouse tumors differing in histological type and status of metastasis: Krebs-2 ascites adenocarcinoma of the mammary gland, Lewis lung adenocarcinoma, hepatoma-29, and lung adenocarcinoma. It is assumed that the RGD motif mediates primary binding of the construct to αvß3 and αvß5 integrins that are predominantly overexpressed in the endothelial cells of tumor blood vessels and in tumor and metastatic cells.

Braiding DNA: experiments, simulations, and models.

DNA encounters topological problems in vivo because of its extended double-helical structure. As a consequence, the semiconservative mechanism of DNA replication leads to the formation of DNA braids or catenanes, which have to be removed for the completion of cell division. To get a better understanding of these structures, we have studied the elastic behavior of two braided nicked DNA molecules using a magnetic trap apparatus. The experimental data let us identify and characterize three regimes of braiding: a slightly twisted regime before the formation of the first crossing, followed by genuine braids which, at large braiding number, buckle to form plectonemes. Two different approaches support and quantify this characterization of the data. First, Monte Carlo (MC) simulations of braided DNAs yield a full description of the molecules' behavior and their buckling transition. Second, modeling the braids as a twisted swing provides a good approximation of the elastic response of the molecules as they are intertwined. Comparisons of the experiments and the MC simulations with this analytical model allow for a measurement of the diameter of the braids and its dependence upon entropic and electrostatic repulsive interactions. The MC simulations allow for an estimate of the effective torsional constant of the braids (at a stretching force F = 2 pN): C(b) approximately 48 nm (as compared with C approximately 100 nm for a single unnicked DNA). Finally, at low salt concentrations and for sufficiently large number of braids, the diameter of the braided molecules is observed to collapse to that of double-stranded DNA. We suggest that this collapse is due to the partial melting and fraying of the two nicked molecules and the subsequent right- or left-handed intertwining of the stretched single strands.

Cooperative helix stabilization by complex Arg-Glu salt bridges.

Among the interactions that stabilize the native state of proteins, the role of electrostatic interactions has been difficult to quantify precisely. Surface salt bridges or ion pairs between acidic and basic side chains have only a modest stabilizing effect on the stability of helical peptides or proteins: estimates are roughly 0.5 kcal/mol or less. On the other hand, theoretical arguments and the occurrence of salt bridge networks in thermophilic proteins suggest that multiple salt bridges may exert a stronger stabilizing effect. We show here that triads of charged side chains, Arg(+)-Glu(-)-Arg(+) spaced at i,i+4 or i,i+3 intervals in a helical peptide stabilize alpha helix by more than the additive contribution of two single salt bridges. The free energy of the triad is more than 1 kcal/mol in excess of the sum of the individual pairs, measured in low salt concentration (10 mM). The effect of spacing the three groups is severe; placing the charges at i,i+4 or i,i+3 sites has a strong effect on stability relative to single bridges; other combinations are weaker. A conservative calculation suggests that interactions of this kind between salt bridges can account for much of the stabilization of certain thermophilic proteins.

[Distributions of circular DNA according its topological states]. / Raspredelenie kil'tsevykh DNK po topologicheskim sostoianiiam.

A distinctive feature of closed circular DNA molecules is their particular topological state, which cannot be altered by any conformational rearrangement short of breaking at least one strand. This topological constraint opens unique possibilities for experimental studies of the distributions of topological states created in different ways. Primarily, the equilibrium distributions of topological properties are considered in the review. It is described how such distributions can be obtained and measured experimentally, and how they can be computed. Comparison of the calculated and measured equilibrium distributions over the linking number of complementary strands, equilibrium fractions of knots and links formed by circular molecules has provided much valuable information about the properties of the double helix. Study of the steady-state fraction of knots and links created by type II DNA topoisomerases has revealed a surprising property of the enzymes: their ability to reduce these fractions considerably below the equilibrium level.

Mechanism of topology simplification by type II DNA topoisomerases.

Type II DNA topoisomerases actively reduce the fractions of knotted and catenated circular DNA below thermodynamic equilibrium values. To explain this surprising finding, we designed a model in which topoisomerases introduce a sharp bend in DNA. Because the enzymes have a specific orientation relative to the bend, they act like Maxwell's demon, providing unidirectional strand passage. Quantitative analysis of the model by computer simulations proved that it can explain much of the experimental data. The required sharp DNA bend was demonstrated by a greatly increased cyclization of short DNA fragments from topoisomerase binding and by direct visualization with electron microscopy.

Dynamics of site juxtaposition in supercoiled DNA.

Juxtaposition kinetics between specific sites in supercoiled DNA is investigated at close to physiological ionic conditions by Brownian dynamics simulations. At such conditions, supercoiled DNA is interwound, and the probability of spatial site juxtaposition is much higher than in relaxed DNA. We find, however, that supercoiling does not correspondingly increase the rate of juxtaposition at these physiological conditions. An explanation to this unexpected finding emerges on analysis of the juxtaposition dynamics. We note that although a particular site i(1) in supercoiled DNA is often in close proximity (juxtaposed) to another site i(2), the change of i(2) occurs very slowly and depends largely on internal slithering of opposite segments of the DNA superhelix. Such slithering results in long correlations between successive values of i(2); these correlations increase the average time of juxtaposition between two DNA sites. Random collisions between sites located on different superhelix branches-although increasing in importance with DNA size-contribute less substantially to site juxtaposition at high salt than slithering for DNA up to 6 kb in length.

Positive torsional strain causes the formation of a four-way junction at replication forks.

The advance of a DNA replication fork requires an unwinding of the parental double helix. This in turn creates a positive superhelical stress, a (+)-DeltaLk, that must be relaxed by topoisomerases for replication to proceed. Surprisingly, partially replicated plasmids with a (+)-DeltaLk were not supercoiled nor were the replicated arms interwound in precatenanes. The electrophoretic mobility of these molecules indicated that they have no net writhe. Instead, the (+)-DeltaLk is absorbed by a regression of the replication fork. As the parental DNA strands re-anneal, the resultant displaced daughter strands base pair to each other to form a four-way junction at the replication fork, which is locally identical to a Holliday junction in recombination. We showed by restriction endonuclease digestion that the junction can form at either the terminus or the origin of replication and we visualized the structure with scanning force microscopy. We discuss possible physiological implications of the junction for stalled replication in vivo.

Multimerization-cyclization of DNA fragments as a method of conformational analysis.

Ligation of short DNA fragments results in the formation of linear and circular multimers of various lengths. The distribution of products in such a reaction is often used to evaluate fragment bending caused by specific chemical modification, by bound ligands or by the presence of irregular structural elements. We have developed a more rigorous quantitative approach to the analysis of such experimental data based on determination of j-factors for different multimers from the distribution of the reaction products. j-Factors define the effective concentration of one end of a linear chain in the vicinity of the other end. To extract j-factors we assumed that kinetics of the reaction is described by a system of differential equations where j-factors appear as coefficients. The assumption was confirmed by comparison with experimental data obtained here for DNA fragments containing A-tracts. At the second step of the analysis j-factors are used to determine conformational parameters of DNA fragments: the equilibrium bend angle, the bending rigidity of the fragment axis, and the total twist of the fragments. This procedure is based on empirical equations that connect the conformational parameters with the set of j-factors. To obtain the equations, we computed j-factors for a large array of conformational parameters that describe model fragments. The approach was tested on both simulated and actual experimental data for DNA fragments containing A-tracts. A-tract DNA bend angle determined here is in good agreement with previously published data. We have established a set of experimental conditions necessary for the data analysis to be successful.

Tightness of random knotting.

Long polymers in solution frequently adopt knotted configurations. To understand the physical properties of knotted polymers, it is important to find out whether the knots formed at thermodynamic equilibrium are spread over the whole polymer chain or rather are localized as tight knots. We present here a method to analyze the knottedness of short linear portions of simulated random chains. Using this method, we observe that knot-determining domains are usually very tight, so that, for example, the preferred size of the trefoil-determining portions of knotted polymer chains corresponds to just seven freely jointed segments.

Experimental and theoretical analysis of the invasive signal amplification reaction.

The invasive signal amplification reaction is a sensitive method for single nucleotide polymorphism detection and quantitative determination of viral load and gene expression. The method requires the adjacent binding of upstream and downstream oligonucleotides to a target nucleic acid (either DNA or RNA) to form a specific substrate for the structure-specific 5' nucleases that cleave the downstream oligonucleotide to generate signal. By running the reaction at an elevated temperature, the downstream oligonucleotide cycles on and off the target leading to multiple cleavage events per target molecule without temperature cycling. We have examined the performance of the FEN1 enzymes from Archaeoglobus fulgidus and Methanococcus jannaschii and the DNA polymerase I homologues from Thermus aquaticus and Thermus thermophilus in the invasive signal amplification reaction. We find that the reaction has a distinct temperature optimum which increases with increasing length of the downstream oligonucleotide. Raising the concentration of either the downstream oligonucleotide or the enzyme increases the reaction rate. When the reaction is configured to cycle the upstream instead of the downstream oligonucleotide, only the FEN1 enzymes can support a high level of cleavage. To investigate the origin of the background signal generated during the invasive reaction, the cleavage rates for several nonspecific substrates that arise during the course of a reaction were measured and compared with the rate of the specific reaction. We find that the different 5' nuclease enzymes display a much greater variability in cleavage rates on the nonspecific substrates than on the specific substrate. The experimental data are compared with a theoretical model of the invasive signal amplification reaction.

The kinetics of oligonucleotide replacements.

The formation of a duplex between two nucleic acid strands is restricted if one of the strands forms an intra- or intermolecular secondary structure. The formation of the new duplex requires the dissociation and replacement of the initial structure. To understand the mechanism of this type of kinetics we studied the replacement of a labeled DNA oligonucleotide probe bound to a complementary DNA target with an unlabeled probe of the same sequence. The replacement kinetics were measured using a gel-shift assay for 12, 14 and 16-nucleotide probes as a function of temperature and concentration of the unlabeled probe. The results demonstrate that the overall replacement rate is a combination of two kinetic pathways: dissociative and sequential displacement. The dissociative pathway occurs by the spontaneous dissociation of the initial duplex followed by association of the target and unlabeled probe. The sequential displacement pathway requires only the partial melting of the initial duplex to allow for the formation of a branched nucleation complex with the unlabeled probe, followed by the complete displacement of the labeled probe by migration of the branch point. The contribution from the dissociative pathway is predominant at temperatures close to the melting point of the labeled probe, whereas the contribution from the displacement pathway prevails at lower temperatures and when the concentration of the replacing unlabeled probe is high. The results show that at physiological conditions, duplex formation between a single-stranded oligonucleotide probe and a structured region of a target molecule occurs mainly by the sequential-displacement mechanism.

Equilibrium distributions of topological states in circular DNA: interplay of supercoiling and knotting.

Two variables define the topological state of closed double-stranded DNA: the knot type, K, and DeltaLk, the linking number difference from relaxed DNA. The equilibrium distribution of probabilities of these states, P(DeltaLk, K), is related to two conditional distributions: P(DeltaLk|K), the distribution of DeltaLk for a particular K, and P(K|DeltaLk) and also to two simple distributions: P(DeltaLk), the distribution of DeltaLk irrespective of K, and P(K). We explored the relationships between these distributions. P(DeltaLk, K), P(DeltaLk), and P(K|DeltaLk) were calculated from the simulated distributions of P(DeltaLk|K) and of P(K). The calculated distributions agreed with previous experimental and theoretical results and greatly advanced on them. Our major focus was on P(K|DeltaLk), the distribution of knot types for a particular value of DeltaLk, which had not been evaluated previously. We found that unknotted circular DNA is not the most probable state beyond small values of DeltaLk. Highly chiral knotted DNA has a lower free energy because it has less torsional deformation. Surprisingly, even at |DeltaLk| > 12, only one or two knot types dominate the P(K|DeltaLk) distribution despite the huge number of knots of comparable complexity. A large fraction of the knots found belong to the small family of torus knots. The relationship between supercoiling and knotting in vivo is discussed.

Large-scale effects of transcriptional DNA supercoiling in vivo.

The scale of negative DNA supercoiling generated by transcription in Top(+) Escherichia coli cells was assessed from the efficiency of cruciform formation upstream of a regulated promoter. An increase in negative supercoiling upon promoter induction led to cruciform formation, which was quantitatively measured by chemical probing of intracellular DNA. By placing a cruciform-forming sequence at varying distances from the promoter, we found that the half-dissociation length of transcription supercoiling wave is approximately 800 bp. This is the first proof that transcription can affect DNA structure on such a remarkably large scale in vivo. Moreover, cooperative binding of the cI repressor to the upstream promoter DNA did not preclude efficient diffusion of transcriptional supercoiling. Finally, our plasmids appeared to contain discrete domains of DNA supercoiling, defined by the features and relative orientation of different promoters.

Effect of magnesium on cruciform extrusion in supercoiled DNA.

Recently, it was reported that Mg2+greatly facilitates cruciform extrusion in the short palindromes of supercoiled DNA, thereby allowing the formation of cruciform structures in vivo. Because of the potential biological importance of this phenomenon, we undertook a broader study of the effect of Mg2+on a cruciform extrusion in supercoiled DNA. The method of two-dimensional gel electrophoresis was used to detect the cruciform extrusion both in the absence and in the presence of these ions. Our results show that Mg2+shifts the cruciform extrusion in the d(CCC(AT)16GGG) palindrome to a higher, rather than to a lower level of supercoiling. In order to study possible sequence-specific properties of the short palindromes for which the unusual cruciform extrusion in the presence Mg2+was reported, we constructed a plasmid with a longer palindromic region. This region bears the same sequences in the hairpin loops and four-arm junction as the short palindrome, except that the short stems of the hairpins are extended. The extension allowed us to overcome the limitation of our experimental approach which cannot be used for very short palindromes. Our results show that Mg2+also shifts the cruciform extrusion in this palindrome to a higher level of supercoiling. These data suggest that cruciform extrusion in the short palindromes at low supercoiling is highly improbable. We performed a thermodynamic analysis of the effect of Mg2+on cruciform extrusion. The treatment accounted for the effect of Mg2+on both free energy of supercoiling and the free energy of cruciform structure per se. Our analysis showed that although the level of supercoiling required for the cruciform extrusion is not reduced by Mg2+, the ions reduce the free energy of the cruciform structure.

Mechanisms of separation of the complementary strands of DNA during replication.

This article is a perspective on the separation of the complementary strands of DNA during replication. Given the challenges of DNA strand separation and its vital importance, it is not surprising that cells have developed many strategies for promoting unlinking. We summarize seven different factors that contribute to strand separation and chromosome segregation. These are: (1) supercoiling promotes unlinking by condensation of DNA; (2) unlinking takes place throughout a replicating domain by the complementary action of topoisomerases on precatenanes and supercoils; (3) topological domains isolate the events near the replication fork and permit the supercoiling-dependent condensation of partially replicated DNA; (4) type-II topoisomerases use ATP to actively unlink DNA past the equilibrium position; (5) the effective DNA concentration in vivo is less than the global DNA concentration; (6) mechanical forces help unlink chromosomes; and (7) site-specific recombination promotes unlinking at the termination of replication by resolving circular dimeric chromosomes.

Internal motion of supercoiled DNA: brownian dynamics simulations of site juxtaposition.

Thermal motions in supercoiled DNA are studied by Brownian dynamics (BD) simulations with a focus on the site juxtaposition process. It had been shown in the last decade that the BD approach is capable of describing actual times of large-scale DNA motion. The bead model of DNA used here accounts for bending and torsional elasticity as well as the electrostatic repulsion among DNA segments. The hydrodynamic interaction among the beads of the model chain and the aqueous solution is incorporated through the Rotne-Prager tensor. All simulations were performed for the sodium ion concentration of 0.01 M. We first showed, to test our BD procedure, that the same distributions of equilibrium conformational properties are obtained as by Monte Carlo simulations for the corresponding DNA model. The BD simulations also predict with accuracy published experimental values of the diffusion coefficients of supercoiled DNA. To describe the rate of conformational changes, we also calculated the autocorrelation functions for the writhe and radius of gyration for the supercoiled molecules. The rate of site juxtaposition was then studied for DNA molecules up to 3000 bp in length. We find that site juxtaposition is a very slow process: although accelerated by a factor of more than 100 by DNA supercoiling, the times of juxtaposition are in the range of ms even for highly supercoiled DNA, about two orders of magnitude higher than the relaxation times of writhe and the radius of gyration for the same molecules. By inspecting successive simulated conformations of supercoiled DNA, we conclude that slithering of opposing segments of the interwound superhelix is not an efficient mechanism to accomplish site juxtaposition, at least for conditions of low salt concentration. Instead, transient distortions of the interwound superhelix, followed by continuous reshaping of the molecule, contribute more significantly to site juxtaposition kinetics.

Sedimentation and electrophoretic migration of DNA knots and catenanes.

Various site-specific recombination enzymes produce different types of knots or catenanes while acting on circular DNA in vitro and in vivo. By analysing the types of knots or links produced, it is possible to reconstruct the order of events during the reaction and to deduce the molecular "architecture" of the complexes that different enzymes form with DNA. Until recently it was necessary to use laborious electron microscopy methods to identify the types of knots or catenanes that migrate in different bands on the agarose gels used to analyse the products of the reaction. We reported recently that electrophoretic migration of different knots and catenanes formed on the same size DNA molecules is simply related to the average crossing number of the ideal representations of the corresponding knots and catenanes. Here we explain this relation by demonstrating that the expected sedimentation coefficient of randomly fluctuating knotted or catenated DNA molecules in solution shows approximately linear correlation with the average crossing number of ideal configurations of the corresponding knots or catenanes.

Non-complementary DNA helical structure induced by positive torsional stress.

We have induced a local conformational transition by positive torsional stress in small synthetic circular DNA molecules containing cruciforms with immobile or tetramobile branched junctions. The immobile species correspond to the extruded and intruded extrema of the tetramobile junction. Under normal conditions the sequences of all the branched species prevent them from being re-absorbed into the circle. We have induced positive stress by addition of ethidium to the circle, in a low ionic strength medium. Alterations in gel electrophoretic mobility under increasing concentrations of ethidium suggest that the cruciforms undergo a transition under torsional stress. The product of this transition contains mispaired nucleotides, but interwound backbones. By comparing the electrophoretic mobilities of circles containing these structures with that of a completely complementary circle of the same length, we conclude that the twist in the mispairing region is similiar to that of completely paired species.

Torsional control of double-stranded DNA branch migration.

DNA branched junctions are analogues of Holliday junction recombination intermediates. Partially mobile junctions contain a limited amount of homology flanking the branch point. A partially mobile DNA branched junction has been incorporated into a synthetic double-stranded circular DNA molecule. The junction is flanked by four homologous nucleotide pairs, so that there are five possible locations for the branch point. Two opposite arms of the branched junction are joined to form the circular molecule, which contains 262 nucleotides to the base of the junction. This molecule represents a system whereby torque applied to the circular molecule can have an impact on the junction, by relocating its branch point. Ligation of the molecule produces two topoisomers; about 87% of the product is a relaxed molecule, and the rest is a molecule with one positive supercoil. The position of the branch point is assayed by cleaving the molecule with endonuclease VII. We find that the major site of the branch point in the relaxed topoisomer is at the maximally extruded position in the relaxed molecule. Upon the addition of ethidium, the major site of the branch point migrates to the minimally extruded position.