Duration time of a one-dimensional random walk as a function of the energies of the intermediate states: application for dissociation and relaxation processes in DNA hybrids.

Kinetic parameters of macromolecular systems are important for their function in vitro and in vivo. These parameters describe how fast the system dissociates (the characteristic dissociation time), and how fast the system reaches equilibrium (characteristic relaxation time). For many macromolecular systems, the transitions within the systems are described as a random walk through a number of states with various free energies. The rate of transition between two given states within the system is characterized by the average time which passes between starting the movement from one state, and reaching the other state. This time is referred to as the mean first-passage time between two given states. The characteristic dissociation and relaxation times of the system depend on the first-passages times between the states within the system. Here, for a one-dimensional random walk we derived an equation, which connects the mean first-passage time between two states with the free energies of the states within the system. We also derived the general equation, which is not restricted to one-dimensional systems, connecting the relaxation time of the system with the first-passage times between states. The application of these equations to DNA branch migration, DNA structural transitions and other processes is discussed.

Theoretical analysis of DNA branch migration in the presence of a slow reversible initiation step.

Branched DNA structures include several DNA regions connected by three- or four-way DNA junctions. Branched DNAs can be intermediates in DNA replication and recombination in living organisms and in sequence-specific DNA targeting in vitro. Branched DNA structures are usually metastable and irreversibly dissociate to non-branched products via a DNA strand exchange process commonly known as DNA branch migration. The key parameter in the DNA dissociation process is its characteristic time, which depends on the length of the dissociating DNA structure. Here, we predict that the presence of a slow reversible initiation step, which precedes DNA branch migration, can alter, to almost linear dependence, the "classic" quadratic dependence of the dissociation time on the length of the dissociating DNA structure. This prediction can be applied to dissociation of Y-like DNA structures and double D-loop DNA hybrids, which are DNA structures similar to replication bubbles. In addition, the slow initiation step can increase the effect of DNA sequence heterologies within the structure on its kinetic stability. Applications of our analysis for genetic manipulations with branched DNA structures are discussed.

DNA hybrids stabilized by heterologies.

The double D-loop DNA hybrid contains four DNA strands following hybridization of two RecA protein coated complementary single-stranded DNA probes with a homologous region of a double-stranded DNA target. A remarkable feature of the double D-loop DNA hybrids is their kinetic stabilities at internal sites within linear DNA targets after removal of RecA protein from hybrids. We report here that heterologous DNA inserts in one or both probe strands affect the kinetic stability of protein-free double D-loop hybrids. DNA heterologies normally distort DNA-DNA hybrids and consequently accelerate hybrid dissociation. In contrast, heterologous DNA inserts impede dissociation of double D-loops, especially when the insert sequences interact with each other by DNA base pairing. We propose a mechanism for this kinetic stabilization by heterologous DNA inserts based on the hypothesis that the main pathway of dissociation of double D-loop DNA hybrids is a DNA branch migration process involving the rotation of both probe-target duplexes in the hybrids. Heterologous DNA inserts constrain rotation of probe-target duplexes and consequently impede hybrid dissociation. Potential applications of the stabilized double D-loops for gene targeting are discussed.

A random-walk model for retardation of interacting species during gel electrophoresis: implications for gel-shift assays.

We recently showed that intermolecular DNA triplexes can form during gel electrophoresis when a faster migrating single strand overtakes a slower migrating band containing a duplex of appropriate sequence. We proposed a model to account for the resulting apparent comigration of triplexes with the duplex band when the lifetime of the triplex is much shorter than the time of electrophoresis. The model predicts that short-lived complexes can be detected by a gel-shift assay if the faster migrating component of the complex is labeled, a slower migrating component is in excess, and the complex itself migrates more slowly than either of the components. In this case the labeled component, after dissociation from the complex, overtakes a slower migrating band of the free, unlabeled second component and can be captured by the unlabeled component and again retarded; after dissociation of the newly formed complex the cycle is repeated. If the concentration of unlabeled component in the band is larger than some critical value (c(cr)), most of the labeled component becomes trapped in this band during the entire time of gel electrophoresis, thus effectively comigrating with the slower migrating unlabeled component. We call this mechanism of comigration "cyclic capture and dissociation" (CCD). Here we present a quantitative analysis of the model of CCD comigration which predicts that CCD comigration can be used not only for the detection of relatively short-lived complexes, but also for estimation of the specificity of complex formation.

Denaturation and association of DNA sequences by certain polypropylene surfaces.

We observed that DNA fragments in room temperature solution undergo low levels of denaturation in the presence of certain types of polypropylene tube surfaces. If the fragments contain (GT)n.(CA)n or (GA)n.(CT)n sequences, multimeric complexes are also formed. This surface activity is inhibited by addition of micromolar concentrations of an oligodeoxyribonucleotide of arbitrary sequence to the tube prior to adding the double-stranded DNA. The reaction was not observed in tubes made of borosilicate glass or in polypropylene-based tubes designed to have low-binding properties. In the case of the DNA fragments that form surfaced-induced multimers, similar complexes can be obtained by denaturation and renaturation of the fragment ("induced" association) without regard to the type of tube surface. However, induced association requires the presence of magnesium ions or polyethylene glycol (or concentration by evaporation) for efficient formation of complexes, whereas surface-dependent dissociation has no such requirements. This difference in buffer requirement suggests that association as well as denaturation takes place on the surface. We suggest models for the formation and structure of these complexes based on surface-dependent denaturation followed by misaligned renaturation of repeated sequences and intermolecular pairing of unpaired regions. This denaturation and complex formation may be important for the interpretation of protein-DNA binding experiments and might be related to hydrophobic interactions of DNA in vivo.

Capture in the gel: intermoleculare triplex formation during gel electrophoresis.

Analysis of unusual gel mobility patterns formed by certain DNA triplexes has revealed that intermolecular triplex formation can occur during gel electrophoresis when a faster migrating single strand overtakes a slower migrating band containing a duplex of appropriate sequence. Control experiments showed that this capture of the third strand occurs by sequence-specific hybridization rather than some nonspecific retardation. This phenomenon can be used to detect triplexes by a gel-shift assay even if their lifetime is much shorter than the time of gel electrophoresis.

Alternate-strand triplex formation: modulation of binding to matched and mismatched duplexes by sequence choice in the Pu-Pu-Py block.

In double-stranded DNA, tandem blocks of purines (Pu) and pyrimidines (Py) can form triplexes by pairing with oligonucleotides which also consist of blocks of purines and pyrimidines, using both Py.Pu.Py (Y-type) and Pu.Pu.Py (R-type) pairing motifs in a scheme called "alternate-strand recognition," or ASR [Jayasena, S. D., & Johnston, B. H. (1992) Biochemistry 31, 320-327; Beal P. A., & Dervan, P. B. (1992) J. Am. Chem. Soc. 114, 1470-1478]. We investigated the relative contributions of the Py.Pu.Py and Pu.Pu.Py blocks in the 16-bp duplex sequence 5'-AAGGAGAATTCCCTCT-3' paired with the third-strand oligonucleotides 5'-TTCCTCTTXXGGGZGZ-3' (XZ-16), where X and Z are either T or A and C is 5-methylcytosine, using chemical footprinting and get electrophoretic mobility shift measurements. We found that the left-hand, pyrimidine half (Y-block) of the third strand (TTCCTCTT, Y-8) forms a Py.Pu.Py triplex as detected by both dimethyl sulfate (DMS) probing and a gel-shift assay; in contrast, the triplex formed by the right-hand half alone (R-block) with X = T (TTGGGTGT, R-8) is not detectable under the conditions tested. However, when tethered to the Y-block (i.e., as XZ-16), the R-block contributes greatly increased specificity of target recognition and confers protection from DMS onto the duplex even under conditions unfavorable for Pu-Pu-Py triplexes (lack of divalent cations). In general, the 16-mer (XZ-16) can bind with apparent strength either greater or lesser than Y-8, depending on whether X and Z are A or T. The order of apparent binding strength, as measured by the target duplex concentration necessary to cause retardation of the third strand during gel electrophoresis, is TT-16 approximately AT-16 > Y-8 > AA-16 > TA-16. Chemical probing experiments showed that both halves of the triplex form even for AA-16, which binds with less apparent binding strength than the pyrimidine block alone (Y-8). The presence of the right half of the 16-mers, although detracting from affinity in cases of AA-16 and TA-16, provides strong specificity for the correct target compared to a target incapable of forming the Pu.Pu.Py part of the triplex. We discuss possible explanations for these observations in terms of alternate oligonucleotide conformations and suggest practical applications of affinity modulation by A-to-T replacements.

Kinetics and mechanism of polyamide ("peptide") nucleic acid binding to duplex DNA.

To elucidate the mechanism of recognition of double-stranded DNA (dsDNA) by homopyrimidine polyamide ("peptide") nucleic acid (PNA) leading to the strand-displacement, the kinetics of the sequence-specific PNA/DNA binding have been studied. The binding was monitored with time by the gel retardation and nuclease S1 cleavage assays. The experimental kinetic curves obey pseudo-first-order kinetics and the dependence of the pseudo-first-order rate constant, kps, on PNA concentration, P, obeys a power law kps approximately P gamma with 2 < gamma < 3. The kps values for binding of decamer PNA to dsDNA target sites with one mismatch are hundreds of times slower than for the correct site. A detailed kinetic scheme for PNA/DNA binding is proposed that includes two major steps of the reaction of strand invasion: (i) a transient partial opening of the PNA binding site on dsDNA and incorporation of one PNA molecule with the formation of an intermediate PNA/DNA duplex and (ii) formation of a very stable PNA2/DNA triplex. A simple theoretical treatment of the proposed kinetic scheme is performed. The interpretation of our experimental data in the framework of the proposed kinetic scheme leads to the following conclusions. The sequence specificity of the recognition is essentially provided at the "search" step of the process, which consists in the highly reversible transient formation of duplex between one PNA molecule and the complementary strand of duplex DNA while the other DNA strand is displaced. This search step is followed by virtually irreversible "locking" step via PNA2/DNA triplex formation. The proposed mechanism explains how the binding of homopyrimidine PNA to dsDNA meets two apparently mutually contradictory features: high sequence specificity of binding and remarkable stability of both correct and mismatched PNA/DNA complexes.

Study of pH-dependent structural transition in telomeric sequences by two-dimensional gel electrophoresis.

We have applied the method of two-dimensional gel electrophoresis to study a pH-dependent structural transition in plasmid carrying the Tetrahymena telomeric sequence, (G4T2)n. A study of the two-dimensional patterns makes it possible to obtain energetic characteristics of the transition. We have obtained the nucleation energy of the structure. It proved to be two times lower than the nucleation energy of H-DNA determined before for a d(AG)n insert. The finding partly explains the formation of an eclectic structure in telomeric sequences. We also studied the effect of Zn++ ions on the two-dimensional patterns of gel electrophoresis. Our data show that Zn++ ions can significantly affect the transitions themselves. This finding leads to reinterpretation of numerous data on probing of structural transitions in DNA by the S1 nuclease.

DNA unwinding upon strand-displacement binding of a thymine-substituted polyamide to double-stranded DNA.

It was recently found that polyamide nucleic acid (PNA) analogues consisting of thymines attached to an aminoethylglycine backbone bind strongly and sequence-selectively to adenine sequences of oligonucleotides and double-stranded DNA [Nielsen, P. E., Egholm, M., Berg, R. H. & Buchardt, O. (1991) Science 254, 1497-1500]. It was concluded that the binding to double-stranded DNA was accomplished via strand displacement, in which the PNA bound to the Watson-Crick complementary adenine-containing strand, whereas the thymine-containing strand was extruded in a virtually single-stranded conformation. This model may provide a general way in which to obtain sequence-specific recognition of any sequence in double-stranded DNA by Watson-Crick hydrogen-bonding base-pair recognition, and it is thus paramount to rigorously establish this binding mode for synthetic DNA-binding ligands. We now report such results from electron microscopy. Furthermore, we show that binding of PNA to closed circular DNA results in unwinding of the double helix corresponding to approximately one turn of the double helix per 10 base pairs. The DNA.PNA complex, which is formed at low salt concentration (only a small portion of DNA molecules show complex formation at NaCl concentration higher than 40 mM), is exceptionally kinetically stable and cannot be dissociated by increasing salt concentration up to 500 mM.

Kinetic trapping of H-DNA by oligonucleotide binding.

Homopurine-homopyrimidine mirror repeats are known to adopt the H form under acidic pH and/or negative supercoiling. In H-DNA, one half of the purine strand enters the triplex whereas the second half is unstructured and can form duplex with complementary oligonucleotide. However, because the same oligonucleotide can form triplex with the homopurine-homopyrimidine insert, one could expect that oligonucleotide would make H-DNA thermodynamically less favorable, as was claimed by Lyamichev et al. Nucl. Acids Res. 16, 2165-2178 (1988). Now we show that complex between oligonucleotide and H-DNA, formed under conditions favorable for the H-form extrusion, is kinetically trapped in superhelical DNA and remains stable up much higher pH values than H-DNA alone. Experiments on chemical probing show that such complex exists for a plasmid with native superhelical density at pH7. We have also used this approach to demonstrate a pH-dependent structural transition in yeast telomeric sequence, d(CACACCCA)16.

An eclectic DNA structure adopted by human telomeric sequence under superhelical stress and low pH.

We have found, with the aid of 2-D gel electrophoresis, that double-stranded human telomeric repeat, (T2AG3)12.(C3TA2)12, being cloned within a plasmid, forms a protonated superhelically-induced structure. Experiments on chemical and enzymatic probing also indicate that the human telomeric repeats adopt an unusual structure. We have proposed an eclectic model for this structure in which four different elements coexist: a non-orthodox intramolecular triplex stabilized by the canonical protonated C.G*C+ base-triads and highly enriched by noncanonical base-triads; the intramolecular quadruplex formed by a portion of the G-rich strand; the single-stranded region encompassing a portion of the G-rich strand and, probably, the (C,A)-hairpin formed by a portion of the C-rich strand.

Formation of intramolecular triplex in homopurine-homopyrimidine mirror repeats with point substitutions.

We have used two-dimensional gel electrophoresis to study the structural transition to the triplex H form of sequences 5'-AAGGGAGAAXGGGGTATAGGGGYAAGAGGGAA-3' where X and Y are any DNA bases. The transition was observed at acid pH under superhelical stress. For X = Y = A or X = Y = G the sequences corresponded to homopurine-homopyrimidine mirror repeats (H-palindrome) which are known to adopt the H form under acid pH and superhelical stress. We have shown that the H form is actually formed for all X and Y, though in cases other than X = Y = A and X = Y = G the transition requires larger negative superhelical stress. Different substitutions require different superhelicity levels for the transition to occur. Theoretical analysis of the data obtained made it possible to estimate the energy cost of triplex formation due to all possible mismatched base triads.

Chemical probing of homopurine-homopyrimidine mirror repeats in supercoiled DNA.

We have recently shown that under superhelical stress and/or acid pH the homopurine-homopyrimidine tracts conforming to the mirror symmetry (H palindromes) form a novel DNA structure, the H form. According to our model, the H form includes (1) a triplex formed by half of the purine strand and by the homopyrimidine hairpin and (2) the unstructured other half of the purine strand. We used four specially designed sequences to demonstrate that, in accordance with our model, the mirror symmetry is essential for facile formation of the H form detected by two-dimensional gel electrophoresis. Here we report that, under conditions favouring the H-form extrusion, guanines of the 3' half of the purine strand are protected against alkylation by dimethylsulphate, whereas adenines of the 5' half of the purine strand react with diethyl pyrocarbonate. These data indicate that the 3' half of the homopurine strand is within the triplex whereas the 5' half is unstructured, in full agreement with our model.