Condensation of DNA by multivalent cations: experimental studies of condensation kinetics

DNA in viruses and cells exists in highly condensed, tightly packaged states. We have undertaken an in vitro study of the kinetics of DNA condensation by the trivalent cation hexaammine cobalt (III) with the aim of formulating a quantitative, mechanistic model of the condensation process. Experimental approaches included total intensity and dynamic light scattering, electron microscopy, and differential sedimentation. We determined the average degree of condensation, the distribution of condensate sizes, and the fraction of uncondensed DNA as a function of reaction time for a range of [DNA] and [Co(NH(3))(3+)(6)]. We find the following: (1) DNA condensation occurs only above a critical [Co(NH(3))(3+)(6)] for a given DNA and salt concentration. At the onset of condensation, [Co(NH(3))(3+)(6)]/[DNA-phosphate] is close to the average value of 0.54, which reflects the 89-90% charge neutralization criterion for condensation. (2) The equilibrium weight average hydrodynamic radius of the condensates first decreases, then increases with increasing [Co(NH(3))(3+)(6)] as they undergo a transition from intramolecular (monomolecular) to intermolecular (multimolecular) condensation. However, is insensitive to [DNA]. (3) The uncondensed DNA fraction decays approximately exponentially with time. The equilibrium uncondensed DNA fraction and relaxation time decrease with increasing [Co(NH(3))(3+)(6)] but are insensitive to [DNA]. (4) The condensation rate in its early stages is insensitive to [DNA] but proportional to [Co(NH(3))(3+)(6)](xs) = [Co(NH(3))(3+)(6)] - [Co(NH(3))(3+)(6)](crit). (5) Data for low [DNA] and low [Co(NH(3))(3+)(6)] at early stages of condensation are most reliable for kinetic modeling since under these conditions there is minimal clumping and network formation among separate condensates. A mechanism with initial monomolecular nucleation and subsequent bimolecular association and unimolecular dissociation steps with rate constants that depend on the number of DNA molecules in the condensate, accounts reasonably well for these observations.     

Competitive substitution of hexammine cobalt(III) for Na+ and K+ ions in oriented DNA fibers

Competition of the trivalent cation, Co(NH3)(3+)(6), with K+ and Na+ ions in binding to DNA was studied by equilibrating oriented DNA fibers with ethanol/water solutions (65 and 52% v/v EtOH), containing different combinations and concentrations of KCl and NaCl and constant concentration (0.8 mM) of Co(NH3)(6)Cl(3). The degree of Co(NH3)(3+)(6) binding to DNA does not depend significantly on the ethanol concentration or on the kind of univalent cation (Na+ or K+). The ion exchange selectivity coefficient of monovalent-trivalent ion competition, D(1)(c3), increases with the concentration of Me+, C(o)(+), and the monotonic dependence of log D(1)(c3) vs log C(o)(+) has an inflection between 100 and 300 mM that is caused by a structural transformation of DNA from A- to B-form. The ion exchange experimental data are compared with results of grand canonical Monte Carlo (GCMC) simulations of systems of parallel and hexagonally ordered, discretely charged polyions with density and spatial distribution of the charged groups modeling B- and A-forms of DNA. The GCMC method for discretely charged models of the DNA polyion produces a quantitative agreement with experimental data on trivalent-monovalent ion competition in dependence on DNA structural state and salt concentration. Based on this and previous studies it is concluded that the affinity of DNA for the cations decreases in the order Co(NH3)(3+)(6) > Ca2+ > Mg2+ > Na+ approximately K+ > Li+. DNA does not exhibit selectivity for Na+ or K+ in ethanol/water solutions either in the absence or in the presence of Co(NH3)(3+)(6), Ca2+, and Mg2+.     

Structure and dynamics of condensed DNA probed by 1,1'-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)bis[4-[[3- methylbenz-1,3-oxazol-2-yl]methylidine]-1,4-dihydroquinolinium] tetraiodide fluorescence

Information on the structure and dynamics of condensed forms of DNA is important in understanding both natural situations such as DNA packaging and artificial systems such as gene delivery complexes. We have established the fluorescence of bisintercalator 1,1'-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)bis[4-[[3-methylbenz-1,3-oxazol-2-yl]methylidine]-1,4-dihydroquinolinium] tetraiodide (YOYO-1) as a novel probe for DNA condensation. When the level of DNA-bound YOYO-1 is sufficiently large, condensation by either polyethylenimine (PEI) or the cationic detergent cetyltrimethylammonium bromide (CTAB) leads to electronic interaction among YOYO-1 molecules bound on the same DNA molecule. This interaction results in an excitonic blue shift of the absorption spectra of YOYO-1 and dramatic decrease in the fluorescence quantum yield. These observations constitute a signature of the condensation of DNA. We further examined the comparative properties of DNA condensed by PEI, CTAB, or Co(NH(3))(6)(3+) through the steady-state and dynamic fluorescence of YOYO-1. Condensation by either PEI or CTAB was associated with a blue shift in the absorption spectra of YOYO-1, although the magnitude of the shift was larger in the case of PEI when compared to that of CTAB. In contrast, condensation by Co(NH(3))(6)(3+) was not associated with a measurable shift in the absorption spectra. These results were interpreted as signifying the varying level of compactness of the DNA condensates. Quenching of fluorescence by acrylamide showed that condensation by all three agents led to an increase in the level of solvent exposure of the base pairs. Observation of the decay of fluorescence intensity and anisotropy of DNA-bound YOYO-1 showed that while condensation by either PEI or CTAB froze the segmental mobility of the helix, condensation by Co(NH(3))(6)(3+) enhanced the flexibility of DNA. The relevance of our findings to functions such as efficiency of gene delivery is discussed.     

Hexaamminecobalt(III) binding environments on double-helical DNA.

Previous cation nmr evidence suggests that univalent cations such as Na+ bind to DNA in a diffuse, nonspecific manner, whereas di- and trivalent cations show distinct binding heterogeneity. Here are reported 59Co- and 23Na-nmr measurements of the %GC dependence of the DNA binding behavior of the trivalent hexaamminecobalt(III) cation. When Co(NH3)6Cl3 titrations are performed on one mammalian and three bacterial DNAs, evidence is found for at least three distinct classes of bound Co(NH3)6(3+). A comparison of titration curves for all four DNAs demonstrates that an increase in GC content correlates with an increase in the fraction of specific Co(NH3)6(3+). binding sites. For M. lysodeikticus DNA (72% GC), a slowly exchanging class of bound 59Co(NH3)6(3+) is apparent. This class of sites is saturated at very low binding densities (between 0.02 and 0.03 cobalt cations per DNA phosphate). At higher binding densities (greater than 0.03), the signal due to slowly exchanging 59Co(NH3)6(3+) disappears into the noise, and a single 59Co(NH3)6(3+) signal is observed. Within the sensitivity limitations of these measurements, no evidence for slowly exchanging bound 59Co(NH3)6(3+) could be found for any of the other DNAs, for which a single, rapidly exchanging 59Co(NH3)6(3+) signal is observed at all binding densities. For this rapidly exchanging signal, for all four DNAs, the measured 59Co(NH3)6(3+) nmr parameters depend significantly on (a) binding density and (b) GC content of the DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Atomic force microscopy analysis of intermediates in cobalt hexammine-induced DNA condensation

The packaging pathway of cobalt hexammine-induced DNA condensation on the surface of mica was examined by varying the concentration of Co(NH3)6(3+) in a dilute DNA solution and visualizing the condensates by atomic force microscopy (AFM). Images reveal that cobalt hexammine-induced DNA condensation on mica involves well-defined structures. At 30 microM Co(NH3)6(3+), prolate ellipsoid condensates composed of relatively shorter rods with linkages between them are formed. At 80 microM Co(NH3)6(3+), the condensed features include toroids with average diameter of approximately 240 nm as well as U-shaped and rod-like condensates with nodular appearances. The results imply that the condensates, whether toroids, U-shaped or rod-like structures have similar intermediate state which includes relatively shorter rod-like segments. The average size of the condensed toroids after incubated at room temperature for 5 h (approximately 240 nm) is much larger than that incubated for 0.5 h (approximately 100 nm). The results indicate that the condensation of DNA by Co(NH3)6(3+) is a kinetic-controlled process.     

The iso-competition point for counterion competition binding to DNA: calculated multivalent versus monovalent cation binding equivalence.

In this paper we introduce an important parameter called the iso-competition point (ICP), to characterize the competition binding to DNA in a two-cation-species system. By imposing the condition of charge neutralization fraction equivalence theta1 = ZthetaZ upon the two simultaneous equations in Manning's counterion condensation theory, the ICPs can be calculated. Each ICP, which refers to a particular multivalent concentration where the charge fraction on DNA neutralized from monovalent cations equals that from the multivalent cations, corresponds to a specific ionic strength condition. At fixed ionic strength, the total DNA charge neutralization fractions thetaICP are equal, no matter whether the higher valence cation is divalent, trivalent, or tetravalent. The ionic strength effect on ICP can be expressed by a semiquantitative equation as ICPZa/ICPZb = (Ia/Ib)Z, where Ia, Ib refers to the instance of ionic strengths and Z indicates the valence. The ICP can be used to interpret and characterize the ionic strength, valence, and DNA length effects on the counterion competition binding in a two-species system. Data from our previous investigations involving binding of Mg2+, Ca2+, and Co(NH3)63+ to lambda-DNA-HindIII fragments ranging from 2.0 to 23.1 kbp was used to investigate the applicability of ICP to describe counterion binding. It will be shown that the ICP parameter presents a prospective picture of the counterion competition binding to polyelectrolyte DNA under a specific ion environment condition.     

Polyamine-DNA interactions. Condensation of chromatin and naked DNA

We have used flow linear dichroism (LD) and light scattering at 90 degrees to study the condensation of both DNA and calf thymus chromatin by polyamines, such as spermine, spermidine and its analogs designated by formula NH3+(CH2)iNH2+(CH2)jNH3+, where i = 2,3 and j = 2,3, putrescine, cadaverine and MgCl2. It has been found that the different polyamines affect DNA and chromatin in a similar way. The level of compaction of the chromatin fibers induced by spermine, spermidine and the triamines NH3+(CH2)3NH2+(CH2)3NH3+ and NH3+(CH2)3NH2+(CH2)2NH3+ and MgCl2 is found to be identical. The triamine NH3+(CH2)3NH2+(CH2)2NH3+ and the diamines studied condense neither chromatin nor DNA. This drastic difference in the action of the triamines indicates that not only the charge, but also the structure of the polycations might play essential roles in their interactions with DNA and chromatin. It is shown that a mixture of mono- and multivalent cations affect DNA and chromatin condensation competitively, but not synergistically, as claimed in a recent report by Sen and Crothers (Biochemistry 25, 1495-1503, 1986). We have also estimated the extent of negative charge neutralization produced by some of the polyamines on their binding to chromatin fibers. The stoichiometry of polyamine binding at which condensation of chromatin is completed is found to be two polyamine molecules per DNA turn. The extent of neutralization of the DNA phosphates by the histones in these compact fibers is estimated to be about 55%. The model of polyamine interaction with chromatin is discussed.     

Metal ion stabilization of the U-turn of the A37 N6-dimethylallyl-modified anticodon stem-loop of Escherichia coli tRNAPhe

Nucleoside base modifications can alter the structures, dynamics, and metal ion binding properties of transfer RNA molecules and are important for accurate aminoacylation and for maintaining translational fidelity and efficiency. The unmodified anticodon stem-loop from Escherichia coli tRNA(Phe) forms a trinucleotide loop in solution, but Mg(2+) and dimethylallyl modification of A(37) N6 disrupt the loop conformation and increase the mobility of the loop and loop-proximal nucleotides. We have used NMR spectroscopy to investigate the binding and structural effects of multivalent cations on the unmodified and dimethylallyl-modified anticodon stem-loops from E. coli tRNA(Phe). The divalent cation binding sites were probed using Mn(2+) and Co(NH(3))(6)(3+). These ions bind along the major groove of the stem and associate with the anticodon loop on the major groove side in a nonspecific manner. Co(NH(3))(6)(3+) stabilizes the U-turn conformation of the loop in the dimethylallyl-modified molecule, and the chemical shift changes that accompany Co(NH(3))(6)(3+) binding are similar to those observed with the addition of Mg(2+). The base-phosphate and base-2'-OH hydrogen bonds that characterize the UNR U-turn motif lead to spectral signatures in the form of unusual (15)N and (1)H chemical shifts and reduced solvent exchange of the U(33) 2'-OH and N3H protons. The unmodified molecule also displays spectral features of the U-turn fold in the presence of Co(NH(3))(6)(3+), but the loop has additional conformations and is dynamic. The results indicate that charge neutralization by a polyvalent cation is sufficient to promote formation of the U-turn fold. However, base modification is necessary to destabilize competing alternative conformers even for a purine-rich loop sequence that is predicted to have strongly favorable base stacking energy.     

Comparative study of the condensation of chicken erythrocyte and calf thymus chromatins by di- and multivalent cations

The condensation of chicken erythrocyte (CE) and calf thymus (CT) chromatins upon addition of di- and multivalent cations has been studied using turbidity, precipitation and electric dichroism measurements. For all the cations investigated (Mg2+, Tb3+, Co(NH3)6(3+), spermidine Spd2+ and spermine Sp4+) condensation of CE chromatin occurred before the onset of aggregation, while aggregation of CT chromatin started before condensation with all cations except Mg2+ and Tb3+. Precipitation of CE chromatin required lower di- and multivalent cations concentrations than CT chromatin. The electric dichroism data for both chromatins, at low ionic strength in the absence of di- or multivalent cations, indicated that the nucleoprotein molecules were not totally decondensed but that a "precondensed" state was already present. A positive electric dichroism was observed for the most condensed chromatin fibers, in agreement with the "cross-linker" models. Tb3+ led to less compact condensed particles as judged from the electric dichroism observations, but electron microscopy revealed that "30 nm fibers" were formed. Very little aggregation was produced by Tb3+. On the contrary, spermine produced very large networks of condensed molecules, but large spheroidal particles were also observed. The condensation of CE chromatin happened without changes of solution conductivity upon cation salt addition, regardless of the condensing cation, indicating a cooperative uptake of the ions during this process.     

Interplay of ion binding and attraction in DNA condensed by multivalent cations

We have measured forces generated by multivalent cation-induced DNA condensation using single-molecule magnetic tweezers. In the presence of cobalt hexammine, spermidine, or spermine, stretched DNA exhibits an abrupt configurational change from extended to condensed. This occurs at a well-defined condensation force that is nearly equal to the condensation free energy per unit length. The multivalent cation concentration dependence for this condensation force gives the apparent number of multivalent cations that bind DNA upon condensation. The measurements show that the lower critical concentration for cobalt hexammine as compared to spermidine is due to a difference in ion binding, not a difference in the electrostatic energy of the condensed state as previously thought. We also show that the resolubilization of condensed DNA can be described using a traditional Manning–Oosawa cation adsorption model, provided that cation–anion pairing at high electrolyte concentrations is taken into account. Neither overcharging nor significant alterations in the condensed state are required to describe the resolubilization of condensed DNA. The same model also describes the spermidine³⁺/Na⁺ phase diagram measured previously.     

Removal of PCR inhibitors from soil DNA by chemical flocculation

Extracting high-purity DNA directly from soil has become essential for the study of microorganisms in environmental samples. However, many soils contain compounds that inhibit enzymes involved in manipulating DNA. In this study, chemical flocculation using multivalent cations was investigated as a potential method for eliminating soil-based inhibitors during the extraction process. The addition of AlNH(4)(SO(4))(2) during extraction significantly reduced the co-purification of PCR inhibitors with minimal loss of DNA yield.     

Metal binding induces conversion of B- to the hybrid B-Z-form in natural DNA

Highly polymerized herring testis DNA of the random nucleotide sequence has been studied in solution by circular dichroism and ultra-violet absorption spectrometry under various experimental conditions. At low temperature upon addition of 0.05M NaCl or 1.15M MgSO(4) the DNA formed a helix that belonged to the B-family. As the temperature was increased a transition from the pure B- to the hybrid B-Z-form occurred in the presence of 1.15M MgSO(4). This transition occurred over a large range of temperatures and corresponded to a non-cooperative conformational change. A similar DNA transition was induced with 0.098mM Co(NH(3))(6)Cl(3). However, in the presence of 5.3M NaCl the DNA conformation was not similar to that observed in 1.15M MgSO(4) or 0.098mM Co(NH(3))(6)Cl(3) independently on the environmental temperature. In 5.3M NaCl the DNA is thought to undergo a transition from one to another right-handed conformation that could be intermediate partially dehydrated conformer arising on the first step in the sequential transition to the dehydration of the polynucleotide. Our results show that a realistic model of native DNA, bearing Z-tracts embedded in B-helixes, can be obtained upon binding of alkaline earth or transition metals.     

Enthalpy of the B-to-Z conformational transition of a DNA oligonucleotide determined by isothermal titration calorimetry

The influence of high concentrations of Na(+) or [Co(NH(3))(6)](3+) on the conformation of two related DNA oligomers was investigated by circular dichroism spectropolarimetry (CD), isothermal titration calorimetry (ITC), and differential scanning calorimetry (DSC). As revealed by CD, DNA oligomers, (dC-dG)(4) and (dm(5)C-dG)(4), both form right-handed double helical structures (B-DNA) in standard phosphate buffer with 115 mM Na(+) at 25 degrees C. However, at 2.0 M Na(+) or 200 microM [Co(NH(3))(6)](3+), (dm(5)C-dG)(4) assumes a left-handed double helical structure (Z-DNA), whereas the unmethylated (dC-dG)(4) analog remains right-handed under those conditions. ITC was then used to determine the enthalpy change upon increasing the concentration of either Na(+) or [Co(NH(3))(6)](3+) for both DNA oligomers at 25 degrees C. The titration with Na(+) resulted in endothermic isotherms with (dm(5)C-dG)(4) being more endothermic than (dC-dG)(4) by 700 cal/mol basepair. In contrast, titration with [Co(NH(3))(6)](3+) resulted in exothermic isotherms with (dC-dG)(4) being more exothermic than (dm(5)C-dG)(4) by 720 cal/mol basepair. We attribute the enthalpy difference to the conformational transition from B-form DNA to Z-form DNA for (dm(5)C-dG)(4), a transition which does not occur for the unmethylated (dC-dG)(4). The value of approximately 700 cal/mol basepair for the enthalpy of the B-Z transition compares favorably with previously published results obtained by different techniques. DSC was used to monitor the duplex to single strand transitions for both oligomers under the different concentrations. These results indicated that methylation of the cytidine destabilizes (dm(5)C-dG)(4) relative to (dC-dG)(4). Coupling the DSC data with the ITC data allowed construction of a thermodynamic cycle which gives insight into the influence of both temperature and ionic strength on the heat content of the two DNA systems studied. Further, this study reveals the utility of using ITC for determinations of transition enthalpies with the appropriate choice of control.     

Neomycin, spermine and hexaamminecobalt (III) share common structural motifs in converting B- to A-DNA.

The (dG)n.(dC)n-containing 34mer DNA duplex [d(A2G15C15T2)]2 can be effectively converted from the B-DNA to the A-DNA conformation by neomycin, spermine and Co(NH3)6(3+). Conversion is demonstrated by a characteristic red shift in the circular dichroism spectra and dramatic NMR spectral changes in chemical shifts. Additional support comes from the substantially stronger CH6/GH8-H3'NOE intensities of the ligand-DNA complexes than those from the native DNA duplex. Such changes are consistent with a deoxyribose pucker transition from the predominate C2'-endo (S-type) to the C3'-endo (N-type). The changes for all three ligand-DNA complexes are identical, suggesting that those three complex cations share common structural motifs for the B- to A-DNA conversion. The A-DNA structure of the 4:1 complex of Co(NH3)6(3+)/d(ACCCGCGGGT) has been analyzed by NOE-restrained refinement. The structural basis of the transition may be related to the closeness of the two negatively charged sugar-phosphate backbones along the major groove in A-DNA, which can be effectively neutralized by the multivalent positively charged amine functions of these ligands. In addition, ligands like spermine or Co(NH3)6(3+) can adhere to guanine bases in the deep major groove of the double helix, as is evident from the significant direct NOE cross-peaks from the protons of Co(NH3)6(3+) to GH8, GH1 (imino) and CH4 (amino) protons. Our results point to future directions in preparing more potent derivatives of Co(NH3)6(3+) for RNA binding or the induction of A-DNA.     

Evidence that both kinetic and thermodynamic factors govern DNA toroid dimensions: effects of magnesium(II) on DNA condensation by hexammine cobalt(III)

Millimolar concentrations of divalent cations are shown to affect the size of toroids formed when DNA is condensed by multivalent cations. The origins of this effect were explored by varying the order in which MgCl(2) was added to a series of DNA condensation reactions with hexammine cobalt chloride. The interplay between Mg(II), temperature, and absolute cation concentration on DNA condensation was also investigated. These studies reveal that DNA condensation is extremely sensitive to whether Mg(II) is associated with DNA prior to condensation or Mg(II) is added concurrently with hexammine cobalt(III) at the time of condensation. It was also found that, in the presence of Mg(II), temperature and dilution can have opposite effects on the degree of DNA condensation. A systematic comparison of DNA condensates observed in this study clearly illustrates that, under our low-salt conditions, toroid size is determined by the kinetics of toroid nucleation and growth. However, when Mg(II) is present during condensation, toroid size can also be limited by a thermodynamic parameter (e.g., undercharging). The path dependence of DNA condensation presented here illustrates that regardless of which particular factors limit toroid growth, toroids formed under the various conditions of this study are largely nonequilibrium structures.     

Interaction of polyamines with chromatin and DNA: formation of compact structures

We have used flow linear dichroism (LD) and light scattering at 90 degrees to study the condensation of both DNA and calf thymus chromatin induced by spermine, triamines NH3+(CH2)iNH+(CH2)jNH3+, designated as much less than i, j much greater than: much less than 3, 4 much greater than (spermidine), much less than 3, 3 much greater than, much less than 2, 3 much greater than, much less than 2, 2 much greater than; the diamines putrescine and cadaverine and MgCl2. It is found that the different polyamines affected DNA and chromatin in a similar way. The degree of compaction of the chromatin fibers induced by spermine, triamines except much less than 2, 2 much greater than and Mg2+ has been found to be identical. The triamine much less than 2, 2 much greater than and the diamines studied do not condense either chromatin of DNA. Such a big difference in the action of the triamines indicates that not only the charge, but also the structure of the polycations are important for their interactions with DNA and chromatin. The stoichiometry of polyamine binding to chromatin at which condensation occurred is found to be 2 polyamine molecules per DNA helical turn. Polyamines are supposed to bind to the exposed sites of core DNA every 10 b.p. The extent of DNA phosphate neutralization by the histones is estimated to be about 55%. It has been shown that a mixture of mono- and multivalent cations affected DNA and chromatin condensation competitively and not synergistically, as claimed in a recent report by Sen and Crothers.     

Synthesis and DNA conformational changes of non-covalent polynuclear platinum complexes

Polynuclear platinum compounds demonstrate many novel phenomena in their interactions with DNA and proteins as well as novel anti-cancer activities. Previous studies indicated that the high positive charge and the non-coordinated "central linker" of the polynuclear compounds could have major contributions to these features. Therefore, a series of non-covalent polynuclear platinum complexes, [[Pt(NH(3))(3)](2)-mu-Y](n+) (Y=polyamine linker or [trans-Pt(NH(3))(2)(H(2)N(CH(2))(6)NH(2))(2)]) was synthesized and the DNA interactions of these platinum complexes were investigated. The conformational changes induced by these compounds in polymer DNA were studied by circular dichroism and the reversibility of the transition was tested by subsequent titration with the DNA intercalating agent ethidium bromide (EtBr). Fluorescent quenching was also used to assess the ability of EtBr to intercalate into A and Z-DNA induced by the compounds. The non-covalent polynuclear platinum complexes induced both B-->A and B-->Z conformational changes in polymer DNA. These conformational changes were partially irreversible. The platinum compound with the spermidine linker, [[Pt(NH(3))(3)](2)-mu-spermidine-N(1),N(8)]Cl(5).2H(2)O, is more efficient in inducing the conformational changes of DNA and it is less reversible than complexes with other linkers. The melting point study showed that the non-covalent polynuclear platinum complexes stabilized the duplex DNA and the higher the electrical charge of the complexes the greater the stabilization observed.     

Metal ion requirements for structure and catalysis of an RNA ligase ribozyme

The class I ligase, a ribozyme previously isolated from random sequence, catalyzes a reaction similar to RNA polymerization, positioning its 5'-nucleotide via a Watson-Crick base pair, forming a 3',5'-phosphodiester bond between its 5'-nucleotide and the substrate, and releasing pyrophosphate. Like most ribozymes, it requires metal ions for structure and catalysis. Here, we report the ionic requirements of this self-ligating ribozyme. The ligase requires at least five Mg(2+) for activity and has a [Mg(2+)](1/2) of 70-100 mM. It has an unusual specificity for Mg(2+); there is only marginal activity in Mn(2+) and no detectable activity in Ca(2+), Sr(2+), Ba(2+), Zn(2+), Co(2+), Cd(2+), Pb(2+), Co(NH(3))(6)(3+), or spermine. All tested cations other than Mg(2+), including Mn(2+), inhibit the ribozyme. Hill analysis in the presence of inhibitory cations suggested that Ca(2+) and Co(NH(3))(6)(3+) inhibit by binding at least two sites, but they appear to productively fill a subset of the required sites. Inhibition is not the result of a significant structural change, since the ribozyme assumes a nativelike structure when folded in the presence of Ca(2+) or Co(NH(3))(6)(3+), as observed by hydroxyl-radical mapping. As further support for a nativelike fold in Ca(2+), ribozyme that has been prefolded in Ca(2+) can carry out the self-ligation very quickly upon the addition of Mg(2+). Ligation rates of the prefolded ribozyme were directly measured and proceed at 800 min(-1) at pH 9.0.     

Circular dichroism study of the irreversibility of conformational changes induced by polyamine-linked dinuclear platinum compounds

In this work, the reversibility of both the B-->Z and B-->A conformational change in polymer DNA induced by polynuclear platinum compounds was studied. The compounds examined were: [[trans-PtCl(NH(3))(2)](2)[NH(2) (CH(2))(6)NH(2)]](2+) (BBR3005); [[trans-PtCl(NH(3))(2)](2)[mu-spermine-N1,N12]](4+) (BBR3535); [[trans-PtCl(NH(3))(2)](2)[mu-spermidine-N1,N8]](3+) (BBR3571); [[trans-PtCl(NH(3))(2)](2)[mu-BOC-spermidine]](2+) (BBR3537); and [[trans-PtCl(NH(3))(2)](2)[mu-trans-Pt(NH(3))(2)(H(2)N(CH(2))(6)NH(2))(2)]](4+) (BBR3464). The conformational changes were assessed by circular dichroism and the reversibility of the transitions was tested by subsequent titration with the DNA intercalator ethidium bromide (EtBr). Fluorescent quenching was also used to assess the ability of ethidium bromide to intercalate into A and/or Z-DNA induced by the compounds. The results were compared with those produced by the simple hexamminecobalt cation [Co(NH(3))(6)](3+). The data suggest that while conformational changes induced by electrostatic interactions are confirmed to be reversible, covalent binding induces irreversible changes in both the A and Z conformation. The relevance of these changes to the novel biological action of polynuclear platinum compounds is discussed.