Roles of Mg2+ in the mechanism of formation and dissociation of open complexes between Escherichia coli RNA polymerase and the lambda PR promoter: kinetic evidence for a second open complex requiring Mg2+.

Comparative studies of the effects of Mg2+ vs Na+ and of acetate (OAc-) vs Cl- on the kinetics of formation and dissociation of E. coli RNA polymerase (E sigma 70)-lambda PR promoter open complexes have been used to probe the mechanism of this interaction. Composite second-order association rate constants ka and first-order dissociation rate constants kd, and their power dependences on salt concentration SKa (SKa identical to d log ka/d log [salt]) and Skd (Skd identical to d log kd/d log [salt]), were determined in MgCl2 and NaOAc to compare with the results of Roe and Record (1985) in NaCl. Replacement of NaCl by MgCl2 reduces the magnitude of Ska 2-fold (Ska = -11.9 +/- 1.1 in NaCl; Ska = -5.2 +/- 0.3 in MgCl2) and (by extrapolation) drastically reduces the magnitude of ka at any specified salt concentration (e.g., approximately 10(6)-fold at 0.2 M). Replacement of NaCl by NaOAc does not significantly affect Ska (Ska = -12.0 +/- 0.7 in NaOAc) and (by extrapolation) increased ka by approximately 80-fold at any fixed [Na+]. In the absence of Mg2+, replacement of NaCl by NaOAc is found to increase the half-life of the open complex by approximately 560-fold at fixed [Na+] without affecting Skd [Skd = 7.6 +/- 0.1 in NaOAc; in NaCl, Skd = 7.7 +/- 0.2 (Roe & Record, 1985)]. Replacement of NaCl by MgCl2 drastically reduces both Skd and the half-life of the open complex at any salt concentration below approximately 0.2 M. Strikingly, Skd = 0.4 +/- 0.1 in MgCl2, indicating that the net uptake of Mg2+ ions in the kinetically significant steps in dissociation of the open complex is much smaller than that expected by analogy with the uptake of approximately 8 Na+ ions in the corresponding steps in NaCl. In NaCl/MgCl2 mixtures, at a constant [NaCl] in the range 0.1-0.2 M, initial addition of MgCl2 (0.5 mM less than or equal to [MgCl2] less than or equal to 1 mM) increases the half-life of the open complex; further addition of MgCl2 causes the half-life to decrease, though the effect of [MgCl2] on kd is always less than that predicted by a simple competitive model. The observed effects of MgCl2 on Skd and kd differ profoundly from those expected from the behavior of kd and Skd in NaCl and NaOAc and indicate that the role of Mg2+ in dissociation is not merely that of a nonspecific divalent competitor with RNAP for interactions with DNA phosphates and of a DNA helix-stabilizer, both of which should cause kd to increase monotonically with increasing [Mg2+].(ABSTRACT TRUNCATED AT 400 WORDS)     

Plasmid DNA cleavage by MunI restriction enzyme: single-turnover and steady-state kinetic analysis

Mutational analysis has previously indicated that D83 and E98 residues are essential for DNA cleavage activity and presumably chelate a Mg2+ ion at the active site of MunI restriction enzyme. In the absence of metal ions, protonation of an ionizable residue with a pKa > 7.0, most likely one of the active site carboxylates, controls the DNA binding specificity of MunI [Lagunavicius, A., Grazulis, S., Balciunaite, E., Vainius, D., and Siksnys, V. (1997) Biochemistry 36, 11093-11099.]. Thus, competition between H+ and Mg2+ binding at the active site of MunI presumably plays an important role in catalysis/binding. In the present study we have identified elementary steps and intermediates in the reaction pathway of plasmid DNA cleavage by MunI and elucidated the effect of pH and Mg2+ ions on the individual steps of the DNA cleavage reaction. The kinetic analysis indicated that the multiple-turnover rate of plasmid cleavage by MunI is limited by product release throughout the pH range 6.0-9.3. Quenched-flow experiments revealed that open circle DNA is an obligatory intermediate in the reaction pathway. Under optimal reaction conditions, open circle DNA remains bound to the MunI; however it is released into the solution at low [MgCl2]. Rate constants for the phoshodiester bond hydrolysis of the first (k1) and second (k2) strand of plasmid DNA at pH 7.0 and 10 mM MgCl2 more than 100-fold exceed the kcat value which is limited by product dissociation. The analysis of the pH and [Mg2+] dependences of k1 and k2 revealed that both H+ and Mg2+ ions compete for the binding to the same residue at the active site of MunI. Thus, the decreased rate of phosphodiester hydrolysis by MunI at pH < 7.0 may be due to the reduction of affinity for the Mg2+ binding at the active site. Kinetic analysis of DNA cleavage by MunI yielded estimates for the association-dissociation rate constants of enzyme-substrate complex and demonstrated the decreased stability of the MunI-DNA complex at pH values above 8.0.     

Magnesium binding and conformational change of DNA in chromatin

The structure of chromatin in the presence of Mg2+ ions was examined by circular dichroism and equilibrium dialysis. Circular dichroism (CD) shows that above 260 nm the intensity of the spectrum of DNA in nucleoproteins decreases as the Mg2+ concentration increases. This change is an intrinsic characteristic of DNA since it is also observed in protein-free DNA and has been attributed to a change in the winding angle of base pairs around the DNA axis. Some structural elements of the DNA in the nucleosome core, therefore, are as movable as those of protein-free DNA. The basic organization of H1-depleted chromatin, 146 base pairs (bp) of DNA wound around core histones and a residual 49 bp in the linker region in the repeating unit, is maintained both in the presence and in the absence of Mg2+ ions, as shown by the fact that the CD spectrum of H1-depleted chromatin has the same type of linear combination between the spectrum of protein-free DNA and that of the nucleosome core in 0.2 mM MgCl2-10 mM triethanolamine (pH 7.8) as it has in 1 mM ethylenediaminetetraacetic acid-10 mM tris(hydroxymethyl) aminomethane (pH 7.8). The ellipticity of chromatin shows a smaller decrease relative to the other nucleoproteins and protein-free DNA upon the addition of Mg2+ ions. Therefore, some structural elements of chromatin are apparently somewhat protected against the conformational change induced by these ions. The spectrum of chromatin becomes almost indistinguishable from that of H1-depleted chromatin in 0.2 mM MgCl2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamics of triple helix formation: spectrophotometric studies on the d(A)10.2d(T)10 and d(C+3T4C+3).d(G3A4G3).d(C3T4C3) triple helices.

We have stabilized the d(A)10.2d(T)10 and d(C+LT4C+3).d(G3A4G3).d(C3T4C3) triple helices with either NaCl or MgCl2 at pH 5.5. UV mixing curves demonstrate a 1:2 stoichiometry of purine to pyrimidine strands under the appropriate conditions of pH and ionic strength. Circular dichroic titrations suggest a possible sequence-independent spectral signature for triplex formation. Thermal denaturation profiles indicate the initial loss of the third strand followed by dissociation of the underlying duplex with increasing temperature. Depending on the base sequence and ionic conditions, the binding affinity of the third strand for the duplex at 25 degrees C is two to five orders of magnitude lower than that of the two strands forming the duplex. Thermodynamic parameters for triplex formation were determined for both sequences in the presence of 50 mM MgCl2 and/or 2.0 M NaCl. Hoogsteen base pairs are 0.22-0.64 kcal/mole less stable than Watson-Crick base pairs, depending on ionic conditions and base composition. C+.G and T.A Hoogsteen base pairs appear to have similar stability in the presence of Mg2+ ions at low pH.     

The effect of divalent cations on the mode of action of DNase I. The initial reaction products produced from covalently closed circular DNA

The effect of different divalent metal ions on the hydrolysis of DNA by DNase I was studied with an assay which distinguishes between cleavage of one or both strands of the DNA substrate during initial encounters between enzyme and DNA. Using covalently closed superhelical SV40(I) DNA as substrate, initial reaction products consisting of relaxed circles or unit-length linears are resolved by electrophoresis of radioactively labeled DNA in agarose gels. Only in the presence of a transition metal ion, such as Mn2+ or Co2+, and only under certain reaction conditions, is DNase I able to cut both DNA strands at or near the same point, generating unit-length linears. This ability to cut both DNA strands is inhibited by such factors as temperature decrease, the addition of a monovalent ion or another divalent cation which is not a transition metal ion, or a reduction in the number of superhelical turns in the DNA substrate. All of these factors lead to a winding of the duplex helix and antagonize the unwinding of the duplex promoted by transition metal ion binding. Transition metal ions may thus convert the DNA substrate locally to a form in which DNase I can introduce breaks into both strands. In the presence of Mg2+, DNase I introduces single strand nicks into SV40(I), generating exclusively the covalently open, relaxed circular SV40(II) as the initial product of the reaction. In the presence of Mn2+, DNase I generates as initial products a mixture of SV40(II) and unit-length SV40 linear DNA molecules, formed by two nicks in opposite strands at or near the same point in the duplex. These circular SV40(II) molecules consist of two types. A minority class is indistinguishable from the nicked SV40(II) produced by DNase I in the presence of Mg2+. The majority class consists of molecules containing a gap in one of the two strands, the mean length of the gap being 11 nucleotides. The SV40(L) molecules produced in the presence of Mn2+ appear to have single strand extensions at one or both ends.     

The DNA guanyl radical: kinetics and mechanisms of generation and repair

The one-electron oxidation of DNA bases and single-stranded DNA was studied by pulse radiolysis of aqueous solutions from pH 7-7.4 at 20 degrees C. Thallic ions, Tl(II), were found to rapidly oxidize the purine nucleotides, deoxyguanosine 5'-monophosphate, k[Tl(II) + dGMP2-] = 3.4.10(9) M-1.s-1, and deoxyadenosine 5'-monophosphate, k[Tl(II) + dAMP2-] = 1.3.10(8) M-1.s-1. The reactivities of Tl(II) ions with model pyrimidine DNA bases, 1-methylcytosine and 1-methylthymine, were too low to be measured by pulse radiolysis, k less than 10(7) M-1.s-1. The Tl(II)-mediated oxidation of ssDNA, k = 2.8.10(8) M-1.s-1, produces DNA-guanyl radical, DNA-G.(-H), exclusively. The DNA-guanyl radical is found to be a potent oxidant in neutral media, E7 = 1.04 +/- 0.05 V. It rapidly oxidizes the aromatic amino acids in glycyl-tryptophan and tyrosine methyl ester, k = 3.6.10(7) M-1.s-1 and k = 1.7.10(8) M-1.s-1, respectively. These electron transfer processes indicate that a positive 'hole' may be transferred from DNA to a DNA-associated protein. The positive 'hole' in DNA can also be repaired by antioxidants, which are electron donors. The chemical repair of the DNA-guanyl radical by negatively charged antioxidants is slower than that by positively charged and neutral antioxidants.     

Processivity of the DNA polymerase alpha-primase complex from calf thymus

The processivity of the DNA polymerase alpha-primase complex from calf thymus was analyzed under various conditions. When multi-RNA-primed M13 DNA was used as the substrate, the DNA polymerase alpha-primase complex was found to incorporate 19 +/- 3 nucleotides per primer binding event. This result was confirmed by product analysis on sequencing gels following DNA synthesis on poly(dT) X (rA)10. The processivity depends strongly on the assay conditions but does not correlate with enzymic activity. Lowering the concentration of Mg2+ ions to less than 2 mM increases the processivity to 60. Replacing Mg2+ by 0.2 mM Mn2+ results in 90 nucleotides being incorporated per primer binding event. Neither the presence of ATP nor the addition of noncognate deoxynucleotide triphosphates affects the processivity of the DNA polymerase alpha-primase complex. Lower processivity was induced by lowering the reaction temperature, by adding spermine, spermidine, or putrescine, in the presence of the antibiotics novobiocin and ciprofloxacin, by adding Escherichia coli single-stranded DNA binding protein, or by adding calf thymus topoisomerase II and RNase H. Three single-stranded DNA binding proteins from calf thymus, including unwinding protein 1, do not affect processivity to any significant extent. Freshly prepared DNA polymerase alpha-primase complex exhibits in addition to its processivity of 20 further discrete processivities of about 55, 90, and 105. This result suggest that further subunits of the polymerase alpha-primase complex are necessary to reconstitute the holoenzyme form of the eukaryotic replicase.     

Concentration of MgATP2- and other ions in solution. Calculation of the true concentrations of species present in mixtures of associating ions.

1. A simple method is described for calculating the free concentrations of all species in a mixture of several ionic components that associate at equilibrium to any extent and with any stoicheiometry. 2. It can readily be adapted to take account of species such as protons for which the free rather than the total concentrations are controlled. 3. It was applied to mixtures of adenine nucleotides, Mg2+ and other ions relevant to the study of glucokinase (EC 2.7.1.2), but the qualitative conclusions are not peculiar to this system. 4. ATP exists in a high and nearly constant proportion (about 80%) as MgATP2- in solutions in which the total MgCl2 concentration exceeds the total ATP concentration by 1-10 mM. 5. By contrast, the proportion of ATP present as MgATP2- varies greatly if the total MgCl2 and total ATP concentrations are varied in constant proportion.     

A molecular beacon strategy for real-time monitoring of triplex DNA formation kinetics

We used a molecular beacon (MB) containing a 15-mer triplex-forming oligonucleotide (TFO) to probe in real-time the kinetics of triplex DNA formation in the left side of the TCl tract (502-516) of the c-src proto-oncogene in vitro. The metal ions Na+, K+, and Mg2+ stabilized triplex DNA at this site. The pseudo-first-order rate constant (kpsi) and the second-order association rate constant (k1) for the binding of the MB to the target duplex in 10 mM sodium phosphate buffer, pH 7.3, increased from 3.2 +/- 0.9 to 15 +/- 2.8 x 10(-3) s(-1) and 6.4 +/- 1.8 to 30 +/- 5.6 x 102 M(-1) s(-1), respectively, on increasing the MgCl2 concentration from 1 to 2.5 mM. Similar values were obtained for the triplex DNA stabilized by NaCl (100-250 mM). Surprisingly, the values were around 2 times higher in the presence of KCl. The AG of triplex formation in the presence of 1 mM MgCl2, 150 mM NaCl, and 150 mM KCl were -7.8 +/- 0.3, -8.2 +/- 0.3 and -8.7 +/- 0.7 kcal/mol respectively, despite significant differences in the values of deltaH and deltaS, suggesting enthalpy-entropy compensation in the stabilization of the triplex DNA by these metal ions. These results show the utility of MBs ih probing triplex DNA formation and in evaluating kinetic and thermodynamic parameters important for the design and development of TFOs as triplex DNA-based therapeutic agents.