Theory of friction-limited DNA unwinding

The theory of friction-limited DNA unwinding is developed explicitly for moderate tind large perturbations. This extension of the earlier theory of the relaxation kinetics is necessary because of the complex nature of the rate limitation for small perturbations. The assumption of the theory that is violated under relaxation conditions is that base pairing reactions occurring at a constant local degree of twist of the strands are fast compared to the net unwinding of the molecule. However, these reactions that are slow for small perturbations have a large activation energy, and become faster than friction-limited un winding for large enough temperature jumps and sufficiently large DXA molecules. Thus only the rate for moderate and large perturbations is clearly limited by frictional resistance to turning the molecule in solution. The model used is a diffusional unwinding of the two strands, driven by the accompanying decrease in free energy. For large perturbations a numerical solution of the diffusion equation is required, since the diffusion coefficient is not constant. Two new parameters must be introduced into the equilibrium statistical theory to describe friction-limited unwinding kinetics. These are the force constant b, for winding up coil regions and the frictional coefficient per base pair β_cfor rotating coil regions in solution. We find by fitting the theory to experiment that b = 1.8 × 10^?13 ergs/ rad²- and β_c = 3.5 × 10^?21 erg see/base pair, both for DNA melted in alkali at 0.4.M Na ⁺ and ～30 °C. The latter value is in agreement with predictions based on the viscosity of single stranded DNA in alkali. The quoted value of bcan be interpreted to mean that the number of conformational states of a nucleolide is reduced by an average factor of 1.55 when it is wound around another strand to the degree of twist in a double helix, but without forming a base pair.     

Interactions of DNA with divalent metal ions. I. 31P-nmr studies

³¹P-nmr has been used to investigate the specific interaction of three divalent metal ions, Mg²⁺, Mn²⁺, and Co⁺², with the phosphate groups of DNA. Mg²⁺ is found to have no significant effect on any of the ³¹P-nmr parameters (chemical shift, line-width, T₁, T₂, and NOE) over a concentration range extending from 20 to 160 mM. The two paramagnetic ions, Mn²⁺ and Co²⁺, on the other hand, significantly change the ³¹P relaxation rates even at very low levels. From an analysis of the paramagnetic contributions to the spin–lattice and spin–spin relaxation rates, the effective internuclear metal–phosphorus distances are found to be 4.5 ± 0.5 and 4.1 ± 0.5 Å for Mn²⁺ and Co²⁺, respectively, corresponding to only 15 ± 5% of the total bound Mn²⁺ and Co²⁺ being directly coordinated to the phosphate groups (inner-sphere complexes). This result is independent of any assumptions regarding the location of the remaining metal ions which may be bound either as outer-sphere complexes relative to the phosphate groups or elsewhere on the DNA, possibly to the bases. Studies of the temperature effects on the ³¹P relaxation rates of DNA in the absence and presence of Mn²⁺ and Co²⁺ yielded kinetic and thermodynamic parameters which characterize the association and dissociation of the metal ions from the phosphate groups. A two-step model was used in the analysis of the kinetic data. The lifetimes of the inner-sphere complexes are 3 × 10^?7 and 1.4 × 10^?5 s for Mn²⁺ and Co²⁺, respectively. The rates of formation of the inner-sphere complexes with the phosphate are found to be about two orders of magnitude slower than the rate of the exchange of the water of hydration of the metal ions, suggesting that expulsion of water is not the rate-determining step in the formation of the inner-sphere complexes. Competition experiments demonstrate that the binding of Mg²⁺ ions is 3–4 times weaker than the binding of either Mn²⁺ or Co²⁺. Since the contribution from direct phosphate coordination to the total binding strength of these metal ion complexes is small (～15%), the higher binding strength of Mn²⁺ and Co²⁺ may be attributed either to base binding or to formation of stronger outer-sphere metal–phosphate complexes. At high levels of divalent metal ions, and when the metal ion concentration exceeds the DNA–phosphate concentration, the fraction of inner-sphere phosphate binding increases. In the presence of very high levels of Mg²⁺ (e.g., 3.1M), the inner-sphere ? outer-sphere equilibrium is shifted toward ～100% inner-sphere binding. A comparison of our DNA results and previous results obtained with tRNA indicates that tRNA and DNA have very similar divalent metal ion binding properties. A comparison of the present results with the predictions of polyelectrolyte theories is presented.     

Metal ion requirement by glutamine synthetase of Escherichia coli in catalysis of gamma-glutamyl transfer.

The requirement for metal ions by glutamine synthetase of Escherichia coli in catalyzing the γ-glutamyl transfer reaction has been investigated. In order of decreasing V at pH 7.0, Cd²⁺, Mn²⁺, Mg²⁺, Ca²⁺, Co²⁺, or Zn²⁺ will support the activity of the unadenylylated enzyme in the presence of ADP. With AMP substituted for ADP to satisfy the nucleotide requirement, only Mn²⁺ or Cd²⁺ will support the activity of the unadenylylated enzyme. Kinetic and equilibrium binding measurements show a 1:1 interaction between the nonconsumable substrate ADP and each enzyme subunit of the dodecamer. (To obtain this result, each enzyme subunit must be active in catalyzing γ-glutamyl transfer.) The stability constant of the unadenylylated subunit for ADP-Mn is 3.5 × 10⁵m^?1, or ～2.86 × 10⁷m^?1 under assay conditions, with arsenate, Mn²⁺, and glutamine being responsible for this large affinity increase. Saturation of two Mn²⁺ ion-binding sites per enzyme subunit is absolutely required for activity expression. While apparently not affecting the affinity of the first Mn²⁺ bound (K′ = 1.89 × 10⁶ M^?1), glutamine increases the stability constant for the second Mn²⁺ bound from 2 × 10⁴ to 5.9 × 10⁵m^?1. Reciprocally, increasing Mn²⁺ concentrations decreases the apparent K_m′ value for glutamine. Glutamine (by producing a net uptake of protons in binding to the enzyme) is responsible for changing the proton release from 3 to about 1 for 2 Mn²⁺ bound per enzyme subunit, with ～0.5 H⁺ displaced in both fast and slow processes. The uv spectral change induced by the binding of the first Mn²⁺ to each enzyme subunit remains unchanged by the presence of glutamine. However, glutamine reduces the half-time of the spectral change or slow proton release from ～30 to ～20 sec at 37 °C. Binding and kinetic results indicate a mechanism involving a random addition of Mn²⁺ to two subunit sites. Saturation of the high-affinity site with Mn²⁺ induces a conformational change to an active configuration, while activity expression depends also on the saturation of a second Mn²⁺ binding site (at or near the catalytic site). Once the first Mn²⁺ binding site of the subunit is saturated, an active enzyme complex can be formed either by the sequential binding of Mn²⁺ and ADP at the second site or by the binding of ADP-Mn complex directly to this site if the concentration of ADP-Mn is greater than 10^?8m in the assay. Some additional observations on the binding of Mg²⁺, Ba²⁺, Ca²⁺, and Zn²⁺ to the enzyme are presented.     

Hysteresis and partial irreversibility of denaturation of DNA as a means of investigating the topology of base distribution constraints: application to a yeast rho- (petite) mitochondrial DNA

Low salt concentrations prevent reassociation of separated single strands of DNA, but not the renaturation of partially melted molecules. Rewinding, however, may be delayed (hysteresis) and/or incomplete (partial irreversibility). Long-range fluctuations in base compositioncould account for these observations: (a) the “zippering-up” of a denatured (G + C)-rich section may have to await that of one of its neighbouring (A + T)-rich sections, hence a temperature lag in rewinding; (b) the removal of intramolecular heterogeneities in base composition by fragmentation will give rise to a dispersal of strand-separation temperatures. Conversely, it is shown how a considerable amount of information about the topology of base distribution constraints could be derived from these phenomena.Some yeast ρ^? (petite) mitochondrial DNAs, the melting of which is quasidiscontinuous, provide an excellent opportunity for testing the applicability of this new approach to denaturation mapping. Alternating partial denaturation and renaturation with a low rate of temperature change were followed by high-frequency recording of absorbance at 260 nm. A typical experiment (counterion concentration 0.015 m-Na⁺) carried out on a low-complexity (length of repetitive unit about 3000 base-pairs) ρ^? DNA is reported in full detail. Analysis of the data disclosed the existence of two relatively (G + C)-rich clusters separated by long homogeneous stretches of high (A + T) content.The rewinding of ρ^? DNAs is a discontinuous process. Both equilibrium and non-equilibrium melting processes were observed. Hysteresis in rewinding, which is restricted to the melting range, increases discontinuously with the extent of unwinding reached prior to cooling. Results are shown to be fully consistent with a model that presupposes that nucleation does not play any part in the renaturation process. They are briefly discussed further in the light of current concepts in the theory of helix-coil transitions of DNA.     

Metal ion dependence of DNA cleavage by SepMI and EhoI restriction endonucleases

Most of type II restriction endonucleases show an absolute requirement for divalent metal ions as cofactors for DNA cleavage. While Mg²⁺ is the natural cofactor other metal ions can substitute it and mediate the catalysis, however Ca²⁺ (alone) only supports DNA binding. To investigate the role of Mg²⁺ in DNA cleavage by restriction endonucleases, we have studied the Mg²⁺ and Mn²⁺ concentration dependence of DNA cleavage by SepMI and EhoI. Digestion reactions were carried out at different Mg²⁺ and Mn²⁺ concentrations at constant ionic strength. These enzymes showed different behavior regarding the ions requirement, SepMI reached near maximal level of activity between 10 and 20 mM while no activity was detected in the presence of Mn²⁺ and in the presence of Ca²⁺ cleavage activity was significantly decreased. However, EhoI was more highly active in the presence of Mn²⁺ than in the presence of Mg²⁺ and can be activated by Ca²⁺. Our results propose the two-metal ion mechanism for EhoI and the one-metal ion mechanism for SepMI restriction endonuclease. The analysis of the kinetic parameters under steady state conditions showed that SepMI had a K_m value for pTrcHisB DNA of 6.15 nM and a V_max of 1.79 × 10^?2 nM min^?1, while EhoI had a K_m for pUC19 plasmid of 8.66 nM and a V_max of 2 × 10^?2 nM min^?1.     

Effects of calcium and manganese ions on the helix–coil transition of DNA

The thermal denaturation method was employed to study the effect of Ca²⁺ and Mn²⁺ ions on the DNA helix–coil transition parameters at Na⁺ concentrations of 10^?3–10^?1M. At low ion concentrations, thermal stability increases, the melting range passes through a maximum, and the denaturation curves become asymmetric. These changes are quantitatively similar for Mn²⁺ and Ca²⁺ ions. With a further increase in the concentration of bivalent ions, the conformational transition temperatures pass through a maximum, and the melting range first tends to saturation and then rapidly decreases to 1–2°C. The Mn²⁺ concentrations, at which the above effects occur, are an order of magnitude lower than the Ca²⁺ concentrations. Comparison of experimental results and calculation in terms of the ligand theory permitted estimation of binding constants characterizing association between Mn²⁺ and Ca²⁺ ions and bases of native and denatured DNA. We show that, unlike the interaction with phosphates, bivalent ion–DNA base binding is weakly dependent on monovalent ion concentration in the solution.     

Helix-coil transition in nucleoprotein: renaturation of polylysine-DNA and polylysine-nucleohistone complexes

Thermal denaturation and renaturation of directly mixed and reconstituted polylysine–DNA, directly mixed polylysine–nucleohistone complexes, and NaCl-treated nucleohistones in 2.5 × 10^?4 M EDTA, pH 8.0 have been studied. At the same input ratio of polylysine to DNA, the percent of renaturation of free base pairs in a directly mixed polylysine–DNA complex is higher than that in a reconstituted complex. For a directly mixed complex, the renaturation of free base pairs is proportional to the fraction of DNA bound by polylysine or inversely proportional to the sizes of free DNA loops. A of large amount of renaturation of free base pairs has also been observed for 0.6 M and 1.6 M NaCl-treated nucleohistones. The binding of polylysine to nucleohistone enhances the renaturation of histone-bound base pairs. The percent of renaturation of polylysine–bound base pairs is high and is approximately independent of the extent of binding on DNA by polylysine. This is true in polylysine–DNA complexes prepared either by reconstitution or by directly mixing. It also applies for polylysine–nucleohistone complexes. The model where polylysine-bound base pairs collapse at T_m′ with two complementary strands still bound by polylysine is favored over the model where polylysine is dissociated from DNA during melting. The low renaturation of histone-bound base pairs in nucleo-histone indicates that either histones do not hold two complementary strands of DNA tightly or that histones are fully or partially dissociated from DNA when the nucleo-histone is fully denatured.     

Kinetics of DNA Unwinding by the RecD2 Helicase from Deinococcus radiodurans

RecD2 from Deinococcus radiodurans is a superfamily 1 DNA helicase that is homologous to the Escherichia coli RecD protein but functions outside the context of RecBCD enzyme. We report here on the kinetics of DNA unwinding by RecD2 under single and multiple turnover conditions. There is little unwinding of 20-bp substrates by preformed RecD2-dsDNA complexes when excess ssDNA is present to trap enzyme molecules not bound to the substrate. A shorter 12-bp substrate is unwound rapidly under single turnover conditions. The 12-bp unwinding reaction could be simulated with a mechanism in which the DNA is unwound in two kinetic steps with rate constant of k_unw = 5.5 s⁻¹ and a dissociation step from partially unwound DNA of k_off = 1.9 s⁻¹. These results indicate a kinetic step size of about 3–4 bp, unwinding rate of about 15–20 bp/s, and low processivity (p = 0.74). The reaction time courses with 20-bp substrates, determined under multiple turnover conditions, could be simulated with a four-step mechanism and rate constant values very similar to those for the 12-bp substrate. The results indicate that the faster unwinding of a DNA substrate with a forked end versus only a 5′-terminal single-stranded extension can be accounted for by a difference in the rate of enzyme binding to the DNA substrates. Analysis of reactions done with different RecD2 concentrations indicates that the enzyme forms an inactive dimer or other oligomer at high enzyme concentrations. RecD2 oligomers can be detected by glutaraldehyde cross-linking but not by size exclusion chromatography.     

Conformation and reactivity of DNA. IV. Base binding ability of transition metal ions to native DNA and effect on helix conformation with special reference to DNA-Zn(II) complex

The effects of metal ions of the first-row transition and of alkaline earth metals on the DNA helix conformation have been studied by uv difference spectra, circular dichroism, and sedimentation measurements. At low ionic strength (10^?3 M NaClO₄) DNA shows a maximum in the difference absorption spectra in the presence of Zn²⁺, Mn²⁺, Co²⁺, Cd²⁺, and Ni²⁺ but not with Mg²⁺ or Ca²⁺. The amplitude of this maximum is dependent on GC content as revealed by detailed studies of the DNA-Zn²⁺ complex of eight different DNA's. Pronounced changes also occur in the CD spectra of DNA transition metal complexes. A transition appears up to a total ratio of approximately 1 Zn²⁺ per DNA phosphate at 10^?3 M NaClO₄; then no further change was observed up to high concentrations. The characteristic CD changes are strongly dependent on the double-helical structure of DNA and on the GC content of DNA. Differences were also observed in hydrodynamic properties of DNA metal complexes as revealed by the greater increase of the sedimentation coefficient of native DNA in the presence of transition metal ions. Spectrophotometric acid titration experiments and CD measurements at acidic pH clearly indicate the suppression of protonation of GC base-pair regions on the addition of transition metal ions to DNA. Similar effects were not observed with DNA complexes with alkaline earth metal ions such as Mg²⁺ or Ca²⁺. The data are interpreted in terms of a preferential interaction of Zn²⁺ and of other transition metal ions with GC sites by chelation to the N-7 of guanine and to the phosphate residue. The binding of Zn²⁺ to DNA disappears between 0.5 M and 1 M NaClO₄, but complex formation with DNA is observable again in the presence of highly concentrated solutions of NaClO₄ (3?7.2 M NaClO₄) or at 0.5 to 2 M Mn²⁺. At relatively high cation concentration Mg²⁺ is also effective in changing the DNA comformation. These structural alterations probably result from both the shielding of negatively charged phosphate groups and the breakdown of the water structure along the DNA helix. Differential effects in CD are also observed between Mn²⁺, Zn²⁺ on one hand and Mg²⁺ on the other hand under these conditions. The greater sensitivity of the double-helical conformation of DNA to the action of transition metal ions is due to the affinity of the latter to electron donating sites of the bases resulting from the d electronic configuration of the metal ions. An order of the relative phosphate binding ability to base-site binding ability in native DNA is obtained as follows: Mg²⁺, Ba²⁺, < Ca²⁺ < Fe²⁺, Ni²⁺, Co²⁺ < Mn²⁺, Zn²⁺ < Cd²⁺ < Cu²⁺. The metal-ion induced conformational changes of the DNA are explained by alternation of the winding angle between base pairs as occurs in the transition from B to C conformation. These findings are used for a tentative molecular interpretation of some effects of Zn²⁺ and Mn²⁺ in DNA synthesis reported in the literature.     

Studies on the activation of isocitrate dehydrogenase kinase/phosphatase (AceK) by Mn<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript>

Isocitrate dehydrogenase kinase/phosphatase (AceK) is a bifunctional enzyme with both kinase and phosphatase activities that are activated by Mg²⁺. We have studied the interactions of Mn²⁺and Mg²⁺ with AceK using isothermal titration calorimetry (ITC) combined with molecular docking simulations and show for the first time that Mn²⁺ also activates the enzyme activities. However, Mn²⁺ and Mg²⁺ exert their effects by different mechanisms. Although they have similar binding constants (of 1.11?×?10⁵ and 0.98?×?10⁵ M^?1, respectively) for AceK and induce conformational changes of the enzyme, they do not compete for the same binding site. Instead Mn²⁺ appears to bind to the regulatory domain of AceK, and its effect is transmitted to the active site of the enzyme by the conformational change that it induces. The information in this study should be very useful for understanding the molecular mechanism underlying the interaction between AceK and metal ions, especially Mn²⁺ and Mg²⁺.     

DNA conformational kinetics

A model for the time dependence of DNA conformational state probabilities is formulated in the form of first-order differential equations. This model is applied to investigate the renaturation and denaturation rates for T2 and T7 DNA as reported in the series of experiments by Record and Zimm. Qualitative agreement is found in denaturation and for series of renaturation experiments with the same initial condition. However, partial agreement with series of renaturation experiments having the same final condition is obtained only by including an initial bimolecular step with properly matched pairs of strands. Comparison of all experiments with the calculated rates yields 5 × 10⁴ min^?1 as the step rate for melting a single base pair.     

Interactions of DNA with divalent metal ions. III. Extent of metal binding: Experiment and theory

DNA with Mn²⁺ as the only counterion has been prepared, and the extent of the Mn²⁺ binding was determined under a variety of conditions through measurements of the proton relaxation enhancement of water. The total extent of Mn²⁺ binding per DNA phosphate is found to be 0.43 ± 0.04, independent of the metal ion concentration in the experimental range of 2.8 × 10^?5 to 2.1 × 10^?3M. The predictions of Manning's condensation theory and those obtained from solution of the generalized Poisson-Boltzmann equation regarding the extent of divalent ion binding to polyelectrolytes, in the presence and absence of monovalent counterions, are compared with one another and with the experimental results. Good agreement between the two theoretical approaches is found, with less than 14% variance in the predicted extent of binding over a large range of mono- and divalent ion concentrations. While the predictions of both theoretical approaches generally agree with the experimental results, some discrepancies are noted and their possible origins discussed.     

Interactions of DNA with divalent metal ions. II. Proton relaxation enhancement studies

The extent and modes of binding of the divalent metal ions Mn²⁺ and Co²⁺ to DNA and the effects of salt on the binding have been studied by measurements of the effects of these paramagnetic metal ions on the longitudinal and transverse relaxation rates of the protons of the solvent water molecules, a technique that is sensitive to overall binding. The number of water molecules coordinated to the DNA–bound Mn²⁺ and Co²⁺ is found to be between five and six, and the electron spin relaxation times and the electron-nuclear hyperfine constants associated with Mn²⁺ and Co²⁺ are little or not affected by the binding. These observations indicate little disturbance of the hydration sphere of Mn²⁺ and Co²⁺ upon binding to DNA. An average 2–3-fold reduction in the exchange rate of the water of hydration of the bound metal ions and an order-of-magnitude increase in their rotational correlation time are attributed to hydrogen-bond formation with the DNA. The binding constants of Mn²⁺ to DNA, at metal concentrations approaching zero, are found to be inversely proportional to the second power of the salt concentration, in agreement with the predictions of Manning's polyelectrolyte theory. A remarkable quantitative agreement with the polyelectrolyte theory is also obtained for the anticooperativity in the binding of Mn²⁺ to DNA, although the experimental results can be well accounted for by another simple electrostatic model. The various modes of binding of divalent metal ions to DNA are discussed.     

Degradation of DNA into 5′-Monodeoxyribonucleotides in the Presence of Mn2+ Ions

DNA is known to be aggregated by metal ions including Mn²⁺ ions, but analysis of the aggregation process from a chemical viewpoint, which means identification of the product yielded during the process, has not been performed yet. On examination of the kinds of degraded materials that were in the supernatant obtained on centrifugation of a DNA mixture aggregated under conditions of 10 mM Mn²⁺ ions ([Mn]/[P] = 46.3) at 70 °C for 1 h, the degradation products were found to be dAMP, dCMP, dGMP, and TMP. These dNMPs were purified by HPLC on TSKgel ODS-80Ts and identified by LC-TOF/MS. The degradation activity was lost on pretreatment of the DNA with a phenol–chloroform mixture, and the activity was recovered by pretreatment with a mixture of DMSO and a buffer containing surfactants. Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, and Cd²⁺, as transition element metal ions, were effective as to the degradation into dNMP. Mg²⁺, Ca²⁺, Sr²⁺, and Ba²⁺, as alkali earth element metal ions, were not effective as to the degradation. Monovalent anions such as Cl^?, CH₃OO^?, and NO₃ ^? were found to increase the degradation rate. Sixty μg of the 120 μg of the starting DNA in 450 μl was degraded into dNMP on reaction for 1 h in the presence of 100 mM NaCl and 10 mM Mn²⁺ ions. In this process, aggregation did not occur, and thus was not considered to be necessary for degradation. The degradation was found not to occur at pH 7.0, and to be very sensitive to pH. The OH^? ion should have a critical role in cleavage of the phosphodiester linkages in this case. The dNMP obtained in the degradation process was found to be only 5′-NMP, based on the H¹NMR spectra. This prosess should prove to be a new process for the production of 5′-dNMP in addtion to the exonuclease.     

Mechanism and substrate specificity of telomeric protein POT1 stimulation of the Werner syndrome helicase

Loss of the RecQ helicase WRN protein causes the cancer-prone progeroid disorder Werner syndrome (WS). WS cells exhibit defects in DNA replication and telomere preservation. The telomeric single-stranded binding protein POT1 stimulates WRN helicase to unwind longer telomeric duplexes that are otherwise poorly unwound. We reasoned that stimulation might occur by POT1 recruiting and retaining WRN on telomeric substrates during unwinding and/or by POT1 loading on partially unwound ssDNA strands to prevent strand re-annealing. To test these possibilities, we used substrates with POT1-binding sequences in the single-stranded tail, duplex or both. POT1 binding to ssDNA tails did not alter WRN activity on nontelomeric duplexes or recruit WRN to telomeric ssDNA. However, POT1 bound tails inhibited WRN activity on telomeric duplexes with a single 3'-ssDNA tail, which mimic telomeric ends in the open conformation. In contrast, POT1 bound tails stimulated WRN unwinding of forked telomeric duplexes. This indicates that POT1 interaction with the ssDNA/dsDNA junction regulates WRN activity. Furthermore, POT1 did not enhance retention of WRN on telomeric forks during unwinding. Collectively, these data suggest POT1 promotes the apparent processivity of WRN helicase by maintaining partially unwound strands in a melted state, rather than preventing WRN dissociation from the substrate.     

Changes in Conformation,Stability and Condensation of DNA by Univalent and Divalent Cations in Methanol-Water Mixtures

Abstract

Circular dichroism spectroscopy, absorption spectroscopy, measurements of T_m values, sedimentation analysis and electron microscopy were used to study properties of calf thymus DNA in methanol-water mixtures as a function of monovalent cation (Na⁺ or Cs⁺) concentration and also in the presence of divalent cations Ca²⁺, Mg²⁺, and Mn²⁺. In the absence of divalent cations only slight conformational changes occured and no condensation and/or aggregation could be detected. The T_m values depend on the amount of methanol and on the nature and concentration of cations. In methanol-water mixtures higher thermal stability was observed in solutions containing Cs⁺ ions. Up to 40% (v/v) methanol the addition of divalent ions leads to DNA stabilization. At methanol concentration higher than 50% the presence of divalent cations causes DNA condensation and denaturation even at room temperature. The denaturation is reversible with respect to EDTA addition indicating that no separation of complementary strands occured and the resulting form of DNA is probably similar to the P form. DNA destacking appears to be a direct consequence of stronger cation binding by the condensed DNA in methanol-water mixtures.     

Electron microscopic studies of the binding of Escherichia coli RNA polymerase to DNA. I. Characterization of the non-specific interactions of holoenzyme with a restriction fragment of bacteriophage T7 DNA

Non-specific interactions between a 3800 base-pair restriction fragment of bacteriophage T7 DNA (MboI-C) and Escherichia coli RNA polymerase holoenzyme have been examined by electron microscopy. Holoenzyme displays a relatively weak and rapidly reversible binding to DNA that is only slightly reduced at elevated salt concentrations. As the concentration of NaCl is increased from 50 mm to 200 mm, the binding constant decreases from 2 × 10⁴m^?1to 4 × 10³m^?1. It is concluded that only 1 to 2 sodium ions are released from the DNA when holoenzyme binds non-specifically.The validity of the electron microscopic technique for determining binding constants has been investigated by varying aspects of the grid surface and by examining the non-specific interactions of lac repressor with DNA.     

The non-specific role of Mg2+ in ribosomal subunit association: kinetics and equilibrium in the presence of other divalent metal ions.

The effects of Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺, Mn²⁺, Co²⁺, Ni²⁺ and Zn²⁺ on the kinetics and equilibrium of the association of vacant “tight” ribosomal subunits from Escherichia coli were studied. Increments of Mg²⁺, Ca²⁺, Sr²⁺ and, by and large, Ba²⁺, to ribosomes dissociated to 30 S and 50 S particles at 1.2 mm-Mg²⁺ (60 mm-M²⁺, pH 7.5, 25°C) produce nearly indistinguishable association curves, with midpoints at 1.8 mm total M²⁺ and complete association to 70 S particles at 4 to 5 mm total M²⁺ . The association rate constants at 1 mm-Mg²⁺, 2 mM-M²⁺ are similar (0.5 × 10⁶ to 0.9 × 10⁶m^?1s^?1), as are the dissociation rate constants at 1 mm-(Mg²⁺ + M²⁺) (0.2 to 0.4 s^?1). Mn²⁺ and Zn²⁺ increase the degree of association, as well as further aggregation (Zn²⁺ especially), at lower concentrations than the alkaline earth ions. Co²⁺ and Ni²⁺ produce lower degrees of association, by promoting dissociation of the 70 S particle : the association rate constants at 1 mm-Mg²⁺, 2 mm-M²⁺ for the transition metal ions are all grouped at 2 × 10⁶ to 3 × 10⁶m^?1s^?1. Ni²⁺ also causes a slower inactivation of one or both subunits.The results are compatible with the view that the effects on the rate and equilibrium constants arise from decreases in the electrostatic free energies of the 30 S, 50 S and 70 S particles produced by large-scale, relatively indiscriminate, charge-neutralization “binding” of M²⁺ , and are difficult if not impossible to reconcile with a specific-sites mode of action of M²⁺.     

Heterogeneity of mitochondrial DNA from Saccharomyces carlsbergensis: renaturation and sedimentation studies

The renaturation kinetics of mitochondrial DNA from the yeast Saccharomyces carlsbergensis have been studied at different temperatures and molecular weights. At renaturation temperatures 25 deg. C below the mean denaturation temperature (T_m) in 1 M-sodium chloride the renaturation rate constant is found to decrease with increasing molecular weight of the reacting strands. This unusual molecular weight dependency gradually disappears with an increase in the renaturation temperature. At a temperature 10 deg. C below the melting point, the rate constant shows the normally expected increase with the square root of the molecular weight. From the renaturation data at this temperature, the molecular weight of the mitochondrial genome is estimated to be about 5·0 × 10⁷. The same size of genome was found from renaturation at low molecular weight and 25 deg. C below the T_m.The sedimentation properties of denatured mitochondrial DNA at pH values 7·0 to 12·5 were used to study the conformation of this DNA in 1 M-sodium chloride. The results obtained support the conclusion from the renaturation studies: that the pieces of denatured mitochondrial DNA with a molecular weight above 2 × 10⁵ to 3 × 10⁵, in 1 M-sodium chloride at 25 deg. C below the mean denaturation temperature are not fully extended random coils. Presumably, interaction between adenine and thymine-rich sequences, which are clustered at certain distances within the molecules, is the molecular basis for these observations.     

Magnetic resonance studies of the anthranilate synthetase-phosphoribosyltransferase enzyme complex from Salmonella typhimurium.

The binding of Mn²⁺ to the anthranilate synthetase-phosphoribosyltransferase enzyme complex from Salmonella typhimurium was examined by electron paramagnetic resonance studies. Two types of binding sites were observed: one to two tight sites with a dissociation constant of 3–5 μm and five to six weaker sites with a dissociation constant of 40–70 μm. The activator constant for Mn²⁺ was found to be 9 μm for the glutamine-linked anthranilate synthetase activity and 4 μm for the phosphoribosyltransferase activity. These values are both in the range of the dissociation constant for the tight sites. Water proton relaxation rate measurements showed that the binary enhancement values for both classes of sites were equivalent, ?_b = 10.7 ± 2.0. The addition of chorismate to the Mn²⁺-enzyme complexes when predominantly the tight Mn²⁺ sites were occupied resulted in a large decrease in the observed enhancement (?_T = 2.0). Addition of 5-phosphoribosyl-1-pyrophosphate to the enzyme-Mn²⁺ complexes caused large decreases in the water proton relaxation rate (?_T = 1.5) when tight or tight plus weaker Mn²⁺ sites were occupied. No changes in the water proton relaxation rate were observed when glutamine, pyruvate, or anthranilate were added; a small decrease was observed when enzyme-Mn²⁺ was titrated with tryptophan. Tryptophan significantly altered the effect of the binding of chorismate but not of 5-phosphoribosyl-1-pyrophosphate. The effect of tryptophan on the water proton relaxation rate of a Mn²⁺-enzyme-chorismate complex using a variant enzyme complex which is tryptophan hypersensitive (P. D. Robison, and H. R. Levy, 1976, Biochim. Biophys. Acta. 445, 475–485) occurred at lower concentrations than for the normal enzyme complex. The uncomplexed anthranilate synthetase subunit was titrated with Mn²⁺ and found to have one to two binding sites with a dissociation constant of 300 ± 100 μm. This dissociation constant is much larger than the activator constant for Mn²⁺ for uncomplexed anthranilate synthetase which was determined to be 4 μm. These results indicate that the Mn²⁺-binding sites on anthranilate synthetase are altered when the enzyme complex is formed and that both chorismate and 5-phosphoribosyl-1-pyrophosphate interact closely with enzyme-bound Mn²⁺ or cause a large effect upon its environment.