Real time fluorescence analysis of the RecA filament: implications of base pair fluidity in repeat realignment

During recombination, when Escherichia coli RecA mediates annealing across DNA repeats, Watson-Crick chemistry can only specify the complementarity of pairing, but not the most optimal frame of alignment. We describe that although stochastic alignments across poly(dA) and poly(dT) can lead to sub-optimally annealed duplexes containing ssDNA gaps/overhangs, the same are realigned into an optimal frame by a putative motor activity of RecA [Sen et al. (2000) Biochemistry 39, 10196-10206]. In the present study, we analyze the nature of realignment intermediates in real time, by employing a fluorescent probe, 2-aminopurine (2AP), which can not only report the status of RecA on the unstacked duplex, but also the fluidity of bases in such a filament. Although known to display a lower affinity for duplex DNA, RecA seems to remain functionally associated with these sub-optimally aligned repeat duplexes, until the realignment approaches completion. Moreover, a comparison of 2AP fluorescence in repeat versus mixed sequences indicates that bases in a RecA repetitive DNA filament exhibit higher degrees of freedom that might mediate a 'non-planar hydrogen bonding cross talk' across the bases on either strand. We discuss a model to explain the mechanistic basis of realignment and its implications in signaling the end of homology maximization, which triggers RecA fall off.     

RecA protein reinitiates strand exchange on isolated protein-free DNA intermediates. An ADP-resistant process

Efficient homologous pairing de novo of linear duplex DNA with a circular single strand (plus strand) coated with RecA protein requires saturation and extension of the single strand by the protein. However, strand exchange, the transfer of a strand from duplex DNA to the nucleoprotein filament, which follows homologous pairing, does not require the stable binding of RecA protein to single-stranded DNA. When RecA protein was added back to isolated protein-free DNA intermediates in the presence of sufficient ADP to inhibit strongly the binding of RecA protein to single-stranded DNA, strand exchange nonetheless resumed at the original rate and went to completion. Characterization of the protein-free DNA intermediate suggested that it has a special site or region to which RecA protein binds. Part of the nascent displaced plus strand of the deproteinized intermediate was unavailable as a cofactor for the ATPase activity of RecA protein, and about 30% resisted digestion by P1 endonuclease, which acts preferentially on single-stranded DNA. At the completion of strand exchange, when the distal 5' end of the linear minus strand had been fully incorporated into heteroduplex DNA, a nucleoprotein complex remained that contained all three strands of DNA from which the nascent displaced strand dissociated only over the next 50 to 60 minutes. Deproteinization of this intermediate yielded a complex that also contained three strands of DNA in which the nascent displaced strand was partially resistant to both Escherichia coli exonuclease I and P1 endonuclease. The deproteinized complex showed a broad melting transition between 37 degrees C and temperatures high enough to melt duplex DNA. These results show that strand exchange can be subdivided into two stages: (1) the exchange of base-pairs, which creates a new heteroduplex pair in place of a parental pair; and (2) strand separation, which is the physical displacement of the unpaired strand from the nucleoprotein filament. Between the creation of new heteroduplex DNA and the eventual separation of a third strand, there exists an unusual DNA intermediate that may contain three-stranded regions of natural DNA that are several thousand bases in length.     

Sequence dependent effects in methylphosphonate deoxyribonucleotide double and triple helical complexes.

Deoxyribooligonucleotides containing 19 repeating bases of A, T or U were prepared with normal phosphodiester (dA19, dT19, dU19) or methylphosphonate (dA*19, dT*19, dU*19) linkages. Complexes of these strands have been investigated at 1:1 and 1:2 molar ratios (purine:pyrimidine) by thermal melting and gel electrophoresis. There are dramatic sequence dependent differences in stabilities of complexes containing methylphosphonate strands. Duplexes of dA*19 with dT19 or dU19 have sharp melting curves, increased Tm values, and slopes of Tm versus log (sodium ion activity) plots reduced by about one half relative to their unmodified 'parent' duplexes. Duplexes of dA19 with either dT*19 or dU*19, however, have broader melting curves, reduced Tm values at most salt concentrations and slopes of less than one tenth the values for the unmodified duplexes. Duplex stabilization due to reduced phosphate charge repulsion is offset in the pyrimidine methylphosphonate complexes by steric and other substituent effects. Triple helical complexes with dA19 + 2dT19 and dA19 + 2dU19, which can be detected by biphasic melting curves and gel electrophoresis, are stable at increased Na+ or Mg+2 concentrations. Surprisingly, however, no triple helix forms, even at very high salt concentrations, when any normal strand(s) is replaced by a methylphosphonate strand. Since triple helical complexes with methylphosphonates have been reported for shorter oligomers, inhibition with larger oligomers may vary due to their length and extent of substitution.     

Magnesium ion-dependent activation of the RecA protein involves the C terminus

Optimal conditions for RecA protein-mediated DNA strand exchange include 6-8 mm Mg(2+) in excess of that required to form complexes with ATP. We provide evidence that the free magnesium ion is required to mediate a conformational change in the RecA protein C terminus that activates RecA-mediated DNA strand exchange. In particular, a "closed" (low Mg(2+)) conformation of a RecA nucleoprotein filament restricts DNA pairing by incoming duplex DNA, although single-stranded overhangs at the ends of a duplex allow limited DNA pairing to occur. The addition of excess Mg(2+) results in an "open" conformation, which can promote efficient DNA pairing and strand exchange regardless of DNA end structure. The removal of 17 amino acid residues at the Escherichia coli RecA C terminus eliminates a measurable requirement for excess Mg(2+) and permits efficient DNA pairing and exchange similar to that seen with the wild-type protein at high Mg(2+) levels. Thus, the RecA C terminus imposes the need for the high magnesium ion concentrations requisite in RecA reactions in vitro. We propose that the C terminus acts as a regulatory switch, modulating the access of double-stranded DNA to the presynaptic filament and thereby inhibiting homologous DNA pairing and strand exchange at low magnesium ion concentrations.     

UV resonance Raman and circular dichroism studies of a DNA duplex containing an A(3)T(3) tract: evidence for a premelting transition and three-centered H-bonds.

The presence of A(n) and A(n)T(n) tracts in double-helical sequences perturbs the structural properties of DNA molecules, resulting in the formation of an alternate conformation to standard B-DNA known as B'-DNA. Evidence for a transition occurring prior to duplex melting in molecules containing A(n) tracts was previously detected by circular dichroism (CD) and calorimetric studies. This premelting transition was attributed to a conformational change from B'- to B-DNA. Structural features of A(n) and A(n)T(n) tracts revealed by X-ray crystallography include a large degree of propeller twisting of adenine bases, narrowed minor grooves, and the formation of three-centered H-bonds between dA and dT bases. We report UV resonance Raman (UVRR) and CD spectroscopic studies of two related DNA dodecamer duplexes, d(CGCAAATTTGCG)(2) (A(3)T(3)) and d(CGCATATATGCG)(2) [(AT)(3)]. These studies address the presence of three-centered H-bonds in the B' conformation and gauge the impact of these putative H-bonds on the structural and thermodynamic properties of the A(3)T(3) duplex. UVRR and CD spectra reveal that the premelting transition is only observed for the A(3)T(3) duplex, is primarily localized to the dA and dT bases, and is associated with base stacking interactions. Spectroscopic changes associated with the premelting transition are not readily detectable for the sugar-phosphate backbone or the cytosine and guanosine bases. The temperature-dependent concerted frequency shifts of dA exocyclic NH(2) and dT C4=O vibrational modes suggest that the A(3)T(3) duplex forms three-centered hydrogen bonds at low temperatures, while the (AT)(3) duplex does not. The enthalpy of this H-bond, estimated from the thermally induced frequency shift of the dT C4=O vibrational mode, is approximately 1.9 kJ/mol or 0.46 kcal/mol.     

Solution structure of the (+)-cis-anti-benzo[a]pyrene-dA ([BP]dA) adduct opposite dT in a DNA duplex.

Minor adducts, derived from the covalent binding of anti-benzo[a]pyrene-7,8-dihydroxy-9,10-epoxide to cellular DNA, may play an important role in generating mutations and initiating cancer. We have applied a combined NMR-computational approach including intensity based refinement to determine the solution structure of the minor (+)-cis-anti-[BP]dA adduct positioned opposite dT in the d(C1-T2-C3-T4-C5-[BP]A6-C7-T8-T9-C10-C11). (d(G12-G13-A14-A15-G16-T17-G18-A19-G20+ ++-A21-G22) 11-mer duplex. The BP ring system is intercalated toward the 5'-side of the [BP]dA6 lesion site without disrupting the flanking Watson-Crick dC5.dG18 and [BP]dA6.dT17 base pairs. This structure of the (+)-cis-anti-[BP]dA.dT 11-mer duplex, containing a bay region benzo[a]pyrenyl [BP]dA adduct, is compared with the corresponding structure of the (+)-trans-anti-[BPh]dA.dT 11-mer duplex (Cosman et al., Biochemistry 32, 12488-12497, 1993), which contains a fjord region benzo[c]phenanthrenyl [BPh]dA adduct with the same R stereochemistry at the linkage site. The carcinogen intercalates toward the 5'-direction of the modified strand in both duplexes (the adduct is embedded within the same sequence context) with the buckling of the Watson-Crick [BP]dA6.dT17 base pair more pronounced in the (+)-cis-anti-[BP]dA.dT 11-mer duplex compared to its Watson-Crick [BPh]dA.dT17 base pair in the (+)-trans-anti-[BPh]dA.dT 11-mer duplex. The available structural studies of covalent polycyclic aromatic hydrocarbon (PAH) carcinogen-DNA adducts point toward the emergence of a general theme where distinct alignments are adopted by PAH adducts covalently linked to the N(6) of adenine when compared to the N(2) of guanine in DNA duplexes. The [BPh]dA and [BP]dA N(6)-adenine adducts intercalate their polycyclic aromatic rings into the helix without disruption of their modified base pairs. This may reflect the potential flexibility associated with the positioning of the covalent tether and the benzylic ring of the carcinogen in the sterically spacious major groove. By contrast, such an intercalation without modified base pair disruption option appears not to be available to [BP]dG N(2)-guanine adducts where the covalent tether and the benzylic ring are positioned in the more sterically crowded minor groove. In the case of [BP]dG adducts, the benzopyrenyl ring is either positioned in the minor groove without base pair disruption, or if intercalated into the helix, requires disruption of the modified base pair and displacement of the bases out of the helix.     

The thermodynamic contribution of the 5-methyl group of thymine in the two- and three-stranded complexes formed by poly(dU) and poly(dT) with poly(dA)

To assess the thermodynamic contribution of the 5-methyl group of thymine, we have studied the two-stranded helical complexes poly(dA).poly(dU) and poly(dA).poly(dT) and the three-stranded complexes--poly(dA).2poly(dU), poly(dA).poly(dT).poly(dU) and poly(dA).2poly(dT)--by differential scanning calorimetry, and uv optical melting experiments. The thermodynamic quantities associated with the 3 --> 2, 2 --> 1, and 3 --> 1 melting transitions are found to vary with salt concentration and temperature in a more complex manner than commonly believed. The transition temperatures, T(m), are generally not linear in the logarithm of concentration or activity of NaCl. The change in enthalpy and in entropy upon melting varies with salt concentration and temperature, and a change in heat capacity accompanies each transition. The poly(dA).2poly(dU) triple helix is markedly different from poly(dA).2poly(dT) in both its CD spectrum and thermodynamic behavior, while the poly(dA).poly(dT).poly(dU) triple helix resembles poly(dA).2poly(dT) in these properties. In comparing poly(dA).2poly(dT) with either the poly(dA).poly(dT).poly(dU) or the poly(dA).2poly(dU) triplexes, the substitution of thymine for uracil in the third strand results in an enhancement of stability against the 3 --> 2 dissociation of deltadeltaG degrees = -135 +/- 85 cal (mol A)(-1) at 37 degrees C. This represents a doubling of the absolute stability toward dissociation compared to the triplexes with poly(dU) as the third strand. The poly (dA).poly (dT) duplex is more stable than poly(dA).poly(dU) by deltadeltaG degrees = -350 +/- 60 cal (mol base pair)(-1) at 37 degrees C. Poly(dA).poly(dT) has 50% greater stability than poly(dA).poly(dU) as a result of the dT for dU substitution in the duplex.     

FTIR and UV spectroscopy studies of triplex formation between pyrimidine methoxyethylphosphoramidates alpha-oligodeoxynucleotides and ds DNA targets

The ability of non-ionic methoxyethylphosphoramidate (PNHME) alpha-oligodeoxynucleotides (ODNs), alpha dT(15) and alpha dCT dodecamer, to form triplexes with their double-stranded DNA targets was evaluated. Thermal stability of the formed complexes was studied by UV thermal denaturation and the data showed that these PNHME alpha-ODNs formed much more stable triplexes than phosphodiester (PO) beta-ODNs did (Delta Tm = + 20 degrees C for alpha dCT PNHME). In addition, FTIR spectroscopy was used to determine the base pairing and the strand orientations of the triplexes formed by alpha dT(15) PNHME compared to phosphodiester ODNs with beta or alpha anomeric configuration. While beta dT(15) PO failed to form a triplex with a long beta dA(n) x beta dT(n) duplex, the Tm of the Hoogsteen part of the triplex formed by alpha dT(15) PNHME reached 40 degrees C. Moreover alpha dT(15) PNHME displaced the beta dT(15) strand of a shorter beta dA(15) x beta dT(15) duplex. The alpha dCT PNHME and alpha dT(15) PNHME third strands were found antiparallel in contrast to alpha dT(15) PO which is parallel to the purine strand of their duplex target. The uniform preferential Hoogsteen pairing of the nucleotides alpha dT and alpha dC combining both replacements might contribute to the improve stability of the triplexes.     

Anchorage of oligonucleotide hybridization by tethered phenazine nucleoside analogue

UV absorption and fluorescence techniques with a thermal denaturation procedure were used in studies of the anchorage of an oligonucleotide hybridization by a covalently tethered nucleoside analogue of an intercalating imidazophenazine derivative (Pzn). The formation by the (dT)(10)Pzn conjugate of the duplex complex with (dA)(15) and the triplex complex with (dA)(15) or poly(dA).poly(dT) was studied in buffered solutions with 0.11 and/or 1M sodium ions at the oligomer strand concentration of 10 microM. Because of the Pzn emission sensitivity to the interaction with adenine bases, a fluorescence technique was found to be effective in the detection of melting transitions. The attached Pzn substantially enhanced the thermal stability of complexes formed by (dT)(10) because of the intercalation mechanism, which increased the temperature of half-dissociation of the duplex by 10-12 degrees C and of the triplexes by approximately 13 degrees C. With the assumption of a two-state model of transition, the thermodynamic parameters for duplex formations were derived. The investigated variant of conjugation has a certain advantage over the widely used attachment via a flexible linker, consisting of a predetermined location of the Pzn chromophore in target sequences that makes it useful as a fluorescent reporter of the hybridization correctness. Molecular modeling was used to construct the geometries of the intercalation sites that turned out to be in conformity with the behavior of the Pzn fluorescence.     

Thermodynamics-structure relationship of single mismatches in RNA/DNA duplexes

Thermodynamics of 66 RNA/DNA duplexes containing single mismatches were measured by UV melting methods. Stability enhancements for rG. dT mismatches were the largest of all mismatches examined here, while rU.dG mismatches were not as stable. The methyl group on C5 of thymine enhanced the stability by 0.12 approximately 0.53 kcal mol(-)(1) depending on the identity of adjacent Watson-Crick base pairs, whereas the 2'-hydroxyl group in ribouridine stabilized the duplex by approximately 0.6 kcal mol(-)(1) regardless of the adjacent base pairs. Stabilities induced by the methyl group in thymine, the 2'-hydroxyl group of ribouridine, and an nucleotide exchange at rG.dT and rU.dG mismatches were found to be independent of each other. The order for the mismatch stabilities is rG.dT > rU. dG approximately rG.dG > rA.dG approximately rG.dA approximately rA. dC > rA.dA approximately rU.dT approximately rU.dC > rC.dA approximately rC.dT, although the identity of the adjacent base pairs slightly altered the order. The pH dependence stability and structural changes were suggested for the rA.dG but not for rG.dA mismatches. Comparisons of trinucleotide stabilities for G.T and G.U pairs in RNA, DNA, and RNA/DNA duplexes indicate that stable RNA/DNA mismatches exhibit a stability similar to RNA mismatches while unstable RNA/DNA mismatches show a stability similar to that of DNA mismatches. These results would be useful for the design of antisense oligonucleotides.     

RecA protein-promoted homologous pairing between duplex molecules: functional role of duplex regions of gapped duplex DNA

RecA protein promotes homologous pairing and symmetrical strand exchange between partially single-stranded duplex DNA and fully duplex molecules. We constructed circular gapped DNA with a defined gap length and studied the pairing reaction between the gapped substrate and fully duplex DNA. RecA protein polymerizes onto the single-stranded and duplex regions of the gapped DNA to form a nucleoprotein filament. The formation of such filaments requires a stoichiometric amount of RecA protein. Both the rate and yield of joint molecule formation were reduced when the pairing reaction was carried out in the presence of a sub-saturating amount of RecA protein. The amount of RecA protein required for optimal pairing corresponds to the binding site size of RecA protein at saturation on duplex DNA. The result suggests that in the 4-stranded system the single-stranded as well as the duplex regions are involved in pairing. By using fully duplex DNA that shares different lengths and regions of homology with the gapped molecule, we directly showed that the duplex region of the gapped DNA increased both the rate and yield of joint molecule formation. The present study indicates that even though strand exchange in the 4-stranded system must require the presence of a single-stranded region, the pairing that occurs in duplex regions between DNA molecules is functionally significant and contributes to the overall activity of the gapped DNA.     

RNA-DNA hybridization promoted by E. coli RecA protein.

RecA protein of E. coli plays a central regulatory role that is induced by damage to DNA and results in the inactivation of LexA repressor. In vitro, RecA protein binds preferentially to single-stranded DNA to form a nucleoprotein filament that can recognize homology in naked duplex DNA and promote extensive strand exchange. Although RecA protein shows little tendency at neutral pH to bind to RNA, we found that it nonetheless catalyzed at 37 degrees C the hybridization of complementary RNA and single-stranded DNA sequences. Hybrids made by RecA protein at 37 degrees C appeared indistinguishable from ones prepared by thermal annealing. RNA-DNA hybridization by RecA protein at neutral pH required, as does RecA-promoted homologous pairing, optimal conditions for the formation of RecA nucleoprotein filaments. The cosedimentation of RNA with those filaments further paralleled observations made on the formation of networks of nucleoprotein filaments with double-stranded DNA, an instrumental intermediate in homologous pairing in vitro. These similarities with the pairing reaction support the view that RecA protein acts specifically in the hybridization reaction.     

Binding of unnatural alpha,beta-oligocytidylates with DNA duplexes

Binding of short fluorescently labeled AT-containing DNA duplexes with modified oligocytidylates is studied. The latter are modified to contain unnatural alpha-anomers along with natural beta-nucleotides; the nucleotide composition is selected according to putative pattern of unconventional triplex formation between duplex and oligomer bases. Nondenaturing gel electrophoresis is used to study complexation of fluorescent duplexes with cytidyl oligomers and oligocytidylate self-association at low temperatures. A DNA duplex of random AT composition is shown to bind with an excess of the corresponding oligocytidylate in 0.1 M Tris-HCl in the presence of Mg2+. Binding is observed at neutral pH values, while more basic pH (8.0) prevents complexation of the AT duplex and oligocytidylate. Contrary to oligonucleotides of irregular composition, a regular dA30:dT30 duplex does not bind with the dC strand. It is also shown that alternating self-complementary duplex d(AT)16 and oligocytidylate d(CbetaCalpha)15 do not form complexes, and poly-dC self-associates are formed instead. The effect of 2'-O-methylation of the third strand on complex formation and self-association is also analyzed. The results suggest that a modified oligocytidylate binds with a random-composition duplex, albeit with lower efficiency.     

Differential hydration of dA.dT base pairing and dA and dT bulges in deoxyoligonucleotides

The role of water in the formation of stable duplexes of nucleic acids is being studied by determining the concurrent volume change, heats, and counterion uptake that accompany the duplexation process. The variability of the volume contraction that we have observed in the formation of a variety of homoduplexes suggests that sequence and conformation acutely affect the degree of hydration. We have used a combination of densimetric and calorimetric techniques to measure the change in volume and enthalpy resulting from the mixing of two complementary strands to form (a) fully paired duplexes with 10 or 11 base pairs and (b) bulged decameric duplexes with an extra dA or dT unmatched residue. We also monitored absorbance vs temperature profiles as a function of strand and salt concentration for all four duplexes. Relative to the decamer duplex, insertion of an extra dA.dT base pair to form an undecamer duplex results in a favorable enthalpy of -5.6 kcal/mol that is nearly compensated by an unfavorable entropy term of -5.1 kcal/mol. This enthalpy difference correlates with a differential uptake of water molecules, corresponding to an additional hydration of 16 mol of water molecules/mol of base pair. Relative to the fully paired duplexes, both bulged duplexes are 12-16 degrees C less stable and exhibit marginally larger counterion uptake on forming the duplex. The enthalpy change is slightly lower for the T-bulge duplex and less still for the A-bulge duplex. The volume change results indicate that an unmatched residue increases the amount of coulombic and/or structural hydration. The combined results strongly suggest that the destabilizing forces in bulged duplexes are partially compensated by an increase in hydration levels.     

Parallel-stranded duplex DNA containing dA · dU base pairs

DNA oligonucleotides with dA and dU residues can form duplexes with trans d(A · U) base pairing and the sugar-phosphate backbone in a parallel-stranded orientation, as previously established for oligonucleotides with d(A · T) base pairs. The properties of such parallel-stranded DNA (ps-DNA) 25-mer duplexes have been characterized by absorption (uv), CD, ir, and fluorescence spectroscopy, as well as by nuclease sensitivity. Comparisons were made with duplex molecules containing (a) dT in both strands, (b) dU in one strand and dT in the second, and (c) the same base combinations in reference antiparallel-stranded (aps) structures. Thermodynamic analysis revealed that total replacement of deoxythymine by deoxyuridine was accompanied by destabilization of the ps-helix (reduction in T_m by −13°C in 2 mM MgGl₂, 10 mM Na-cacodylate). The U-containing ps-helix (U1 · U2) also melted 14°C lower than the corresponding aps-helix under the same ionic conditions; this difference was very close to that observed between ps and aps duplexes with d(A · T) base pairs. Force field minimized structures of the various ps and aps duplexes with either d(A · T) or d(A · U) base pairs ps/aps and dT/dU combinations are presented. The energy-minimized helical parameters did not differ significantly between the DNAs containing dT and dU. © 1996 John Wiley & Sons, Inc.     

Hydration and conformational transitions in DNA, RNA, and mixed DNA-RNA triplexes studied by gravimetry and FTIR spectroscopy

We have studied by gravimetric measurements and FTIR spectroscopy the hydration of duplexes and triplexes formed by combinations of dA(n), dT(n), rA(n), and rU(n) strands. Results obtained on hydrated films show important differences in their hydration and in the structural transitions which can be induced by varying the water content of the samples. The number of water molecules per nucleotide (w/n) measured at high relative humidity (98% R.H.) is found to be 21 for dA(n).dT(n) and 15 for rA(n).rU(n). Addition of a third rU(n) strand does not change the number of water molecules per nucleotide: w/n=21 for rU(n)*dA(n).dT(n) and w/n=15 for rU(n)*rA(n).rU(n). On the contrary, the addition of a third dT(n) strand changes the water content but in a different way, depending whether the duplex is DNA or RNA. Thus, a loss of four water molecules per nucleotide is measured for dT(n)*dA(n).dT(n) while an increase of two water molecules per nucleotide is observed for dT(n)*rA(n).rU(n). The final hydration is the same for both triplexes (w/n=17). The desorption profiles obtained by gravimetry and FTIR spectroscopy are similar for the rA(n).rU(n) duplex and the rU(n)*rA(n).rU(n) triplex. On the contrary, the desorption profiles of the dA(n).dT(n) duplex and the triplexes formed with it (rU(n)*dA(n).dT(n) and dT(n)*dA(n).dT(n)) are different from each other. This is correlated with conformational transitions induced by varying the hydration content of the different structures, as shown by FTIR spectroscopy. Modifications of the phosphate group hydration and of the sugar conformation (S to N type repuckering) induced by decrease of the water content are observed in the case of triplexes formed on the dA(n).dT(n) duplex.     

Solution conformation of the (+)-trans-anti-benzo[g]chrysene-dA adduct opposite dT in a DNA duplex.

The solution structure of the adduct derived from the covalent bonding of the fjord region (+)-(11S, 12R, 13R, 14S) stereoisomer of anti -11,12-dihydroxy-13,14-epoxy-11,12,13, 14-tetrahydrobenzo[g]chrysene, (+)- anti -B[g]CDE, to the exocyclic N(6)amino group of the adenine residue dA6, (designated (+)- trans-anti -(B[g]C)dA6), positioned opposite a thymine residue dT17 in the DNA sequence context d(C1-T2-C3-T4-C5-(B[g]C)A6-C7-T8-T9-C10-C11). d(G12-G13-A14-A15-G16-T17-G18-A19-G20++ +-A21-G22) (designated (B[g]C)dA. dT 11-mer duplex), has been studied using structural information derived from NMR data in combination with molecular dynamics (MD) calculations. The solution structure of the (+)- trans-anti -(B[g]C)dA.dT 11-mer duplex has been determined using an MD protocol where both interproton distance and dihedral angle restraints deduced from NOESY and COSY spectra are used during the refinement process, followed by additional relaxation matrix refinement to the observed NOESY intensities to account for spin diffusion effects. The results established that the covalently attached benzo[g]chrysene ring intercalates into the DNA helix directed towards the 5'-side of the modified strand and stacks predominantly with dT17 when intercalated between dC5.dG18 and (B[g]C)dA6.dT17 base-pairs. All base-pairs, including the modified (B[g]C)dA6.dT17 base-pair, are aligned through Watson-Crick pairing as in normal B -DNA. In addition, the potential strain associated with the highly sterically hindered fjord region of the aromatic portion of the benzo[g]chrysenyl ring is relieved through the adoption of a non-planar, propeller-like geometry within the chrysenyl ring system. This conformation shares common structural features with the related (+)- trans-anti -(B[c]Ph)dA adduct in the identical base sequence context, derived from the fjord region (+)-(1S,2R,3R,4S)-3, 4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene stereoisomer, in which intercalation is also observed towards the 5'-side of the modified dA6.dT17 base-pair.     

The mechanics of winding and unwinding helices in recombination: torsional stress associated with strand transfer promoted by RecA protein

Homologous recombination usually involves the production of heteroduplex DNA, DNA containing strands contributed from two different duplexes. RecA protein of E. coli can promote the formation of heteroduplex DNA in vitro by the exchange of DNA strands between two helical structures, duplex DNA and a helical recA nucleoprotein filament containing a single strand of DNA. Complete unwinding of the parental duplex and the rewinding of one strand with a new complement requires rotation of the helical structures about one another, or about their respective longitudinal axes. The observations described here demonstrate an association of torsional stress with strand exchange, and suggest that exchange is accomplished principally by concomitant rotation of duplex DNA and the recA nucleoprotein filament, each about its longitudinal axis.     

A physico-chemical study of triple helix formation by an oligodeoxythymidylate with N3'--> P5' phosphoramidate linkages.

Non-denaturing gel retardation assay, DNA melting experiments and FTIR spectroscopy were used to characterize the triple helix formed by a 15mer 2'-deoxythymidylate with N3'-->P5'phosphoramidate linkages with its target sequence. The results indicate that: (i) the pentadecadeoxythymidylate with phosphoramidate linkages [dT15(np)] is highly potent to form a triple helix with a dT15*dA15target duplex through Hoogsteenbase-pairing; (ii) it forms a dT15(np)*dA15xdT15(np) triplex with the single-stranded oligo-2'-deoxyadenylate (dA15) without detectable double-helical intermediate; (iii) it does not only form a triple helix on the dT15*dA15target duplex, but also partially displaces the dT15 strand from the dT15*dA15duplex to form a dT15(np)*dA15xdT15(np) complex.