ALLOSTERIC INTERACTIONS BETWEEN DNA STRANDS AND MONOVALENT CATIONS INDNA QUADRUPLEX ASSEMBLY - THERMODYNAMIC EVIDENCE FOR 3 LINKED ASSOCIATION PATHWAYS

The series of cooperative transitions that: lead to [d(TG(4))(4).(K+)( m)] quadruplex assembly upon rapid addition of KCI to d(TG(4)) strands were studied, Quadruplex samples were dialyzed against KCI then LI-ET )TA and round to retain between three and five strongly bound potassiu ms with affinities > 10(6) M-2. Absorbance thermal denaturation (melt) and circular dichroism (CD) equilibrium binding data were obtained. T he latter were analyzed using two classes of binding models to simulat e the effects of the assumed intermolecular interactions on the bindin g curves (isotherms). The melt experiments yielded equilibrium dissoci ation constants (K-d) ranging from 10(-11) to 10(-12) M-3 the melting temperatures. Extrapolating these values to 23 degrees C predicts K-d values in the 10(-28) M-3 range if the heat capacity (C-p) is not stro ngly dependent upon temperature changes over this range. Assuming K-a is equal to 1/K-d (from melting analyses), very large association free energies stabilize the quadruplex at 23 degrees C in 100 mM KCl (Delt a G(a) = -43 kcal mol(-1)). Plots of the differential melt curve peak half-widths, a measure of cooperativity, versus d(TG(4)) concentration showed that quadruplex dissociation is much more cooperative at 400 m M KCI than at 100 mM KCL. Forty-eight hour quadruplex assembly time co urses were monitored by CD at 264 nm, Equilibrium quadruplex accumulat ion generally required over 10 h, and net reaction extents were in the 10-85% range. Hill plots of the data show that initial steps in the m ultistep pathway are positively cooperative, presumably due to strong strand-cation and strand-strand binding interactions in duplex and tri plex assembly reactions, then negatively cooperative in quadruplex for mation. Models were developed to rationalize the experimental observat ions in terms of consecutive cooperative allosteric transitions from c ation-deficient relaxed (R) strand-aggregates to cation-containing ten se (T) structures, driven by the allosteric effector K+., Quantitative mappings of positive and then negative cooperativity were obtained by fitting the results as a function of strand number incorporated durin g quadruplex assembly. Surprisingly, models fur reactions involving in corporation of five and six strands fit the data better than models in volving only four strands. The 5-step ''induced fit'' model fits the d ata as well as or better than 3- and 4-step models and better than all of the strand aggregation models that were devised and investigated, Net association free energies (Sigma(i)=(l,n)Delta G(i)') ranged from -20 to -26 kcal mol(-1), approximately half the magnitude of the appar ent stabilities measured by absorbance melts. Likely explanations for this discrepancy involve hysteresis and errors due to inadequate equil ibration in the melt experiments. Hysteresis is thought to be produced by irreversibility due to different predominant mechanisms in absorba nce (dissociation) and CD (association) experiments, The kinetic block to quadruplex assembly can be unambiguously attributed to quadruplex formation and not intermediate steps in the assembly mechanism. On the basis of these results we propose that, in addition to the more conve ntional assembly mechanisms involving duplex dimerization and stepwise strand addition, quadruplex formation can also proceed by tripler-tri pler disproportionation. Interaction statistics arguments that support the energetic feasibility of the disproportionation pathway are prese nted, The allosteric quadruplex assembly model provides 3 mechanism wh ich could be used bq the cell to simultaneously modulate DNA structure and activity within telomeres, transcriptional promoters, recombinati on-prone chromatin, and other G-rich DNAs. As a result of this alloste rism, cation and strand availability and strand-pairing capabilities c ould profoundly influence the functional capacity of a particular stra nd over a relatively narrow range of effector concentration changes. T his is analogous to the control of enzymes in intermediary metabolism by the availability of allosteric effecters. By extension, allosteric effectors in quadruplex assembly can be classified as both facilitory and inhibitory, Future reports present examples of each. These relatio nships might contribute to understanding how cellular and nuclear home ostasis and genetic functions are linked.