Salt effects on internal motions of superhelical and linear pUC8 DNA. Dynamic light scattering studies

The plasmid pUC8 (2717 bp) has been studied in its native superhelical and Eco RI-linearized forms by dynamic light scattering at NaCl concentrations from 1.1 mM to 1 M. The data were analyzed using the biexponential model for the dynamic structure factor described by us in a previous paper (J. Langowski, U. Giesen and C. Lehmann, Biophys. Chem. 25 (1986) 191). As before, we could identify two decay components corresponding to the center-of-mass diffusion and to internal motions of the DNA, where the fast component could be identified as a rotational diffusion contribution in the case for superhelical, but not for linear DNA. We found that the conformation of superhelical pUC8 is not affected by changing the ionic strength, while the amplitude of the internal relaxation increases approx. 2-fold when [NaCl] is raised from 1.1 mM to 1 M. The linearized DNA shows an increase of the diffusion coefficient with ionic strength which is, however, not quite as pronounced as that found by others (Z. Kam, N. Borochov and H. Eisenberg, Biopolymers 20 (1981) 2671), and, together with the unchanged conformation of the superhelical DNA, suggests a persistence length which is not strongly dependent on ionic strength. In contrast to the increasing amplitude of internal relaxation for the superhelical DNA, this amplitude remains constant or decreases slightly for linear DNA on going from 1.1 mM to 1 M salt. Our findings are further discussed with respect to possible models of the interwound form of superhelical DNA.     

Modulation of the DNA scanning activity of the Micrococcus luteus UV endonuclease

Micrococcus luteus UV endonuclease incises DNA at the sites of ultraviolet (UV) light-induced pyrimidine dimers. The mechanism of incision has been previously shown to be a glycosylic bond cleavage at the 5'-pyrimidine of the dimer followed by an apyrimidine endonuclease activity which cleaves the phosphodiester backbone between the pyrimidines. The process by which M. luteus UV endonuclease locates pyrimidine dimers within a population of UV-irradiated plasmids was shown to occur, in vitro, by a processive or "sliding" mechanism on non-target DNA as opposed to a distributive or "random hit" mechanism. Form I plasmid DNA containing 25 dimers per molecule was incubated with M. luteus UV endonuclease in time course reactions. The three topological forms of plasmid DNA generated were analyzed by agarose gel electrophoresis. When the enzyme encounters a pyrimidine dimer, it is significantly more likely to make only the glycosylase cleavage as opposed to making both the glycosylic and phosphodiester bond cleavages. Thus, plasmids are accumulated with many alkaline-labile sites relative to single-stranded breaks. In addition, reactions were performed at both pH 8.0 and pH 6.0, in the absence of NaCl, as well as 25,100, and 250 mM NaCl. The efficiency of the DNA scanning reaction was shown to be dependent on both the ionic strength and pH of the reaction. At low ionic strengths, the reaction was shown to proceed by a processive mechanism and shifted to a distributive mechanism as the ionic strength of the reaction increased. Processivity at pH 8.0 is shown to be more sensitive to increases in ionic strength than reactions performed at pH 6.0.     

Escherichia coli DNA helicase I catalyzes a site- and strand-specific nicking reaction at the F plasmid oriT.

A site- and strand-specific nick, introduced in the F plasmid origin of transfer, initiates conjugal DNA transfer during bacterial conjugation. Recently, molecular genetic studies have suggested that DNA helicase I, which is known to be encoded on the F plasmid, may be involved in this nicking reaction (Traxler, B. A., and Minkley, E. G., Jr. (1988) J. Mol. Biol. 204, 205-209). We have demonstrated this site- and strand-specific nicking event using purified helicase I in an in vitro reaction. The nicking reaction requires a superhelical DNA substrate containing the F plasmid origin of transfer, Mg2+ and helicase I. The reaction is protein concentration-dependent but, under the conditions used, only 50-70% of the input DNA substrate is converted to the nicked species. Genetic data (Everett, R., and Willetts, N. (1980) J. Mol. Biol. 136, 129-150) have also suggested the involvement of a second F-encoded protein, the TraY protein, in the oriT nicking reaction. Unexpectedly, the in vitro nicking reaction does not require the product of the F plasmid traY gene. The implications of this result are discussed. The phosphodiester bond interrupted by helicase I has been shown to correspond exactly to the site nicked in vivo suggesting that helicase I is the site- and strand-specific nicking enzyme that initiates conjugal DNA transfer. Thus, helicase I is a bifunctional protein which catalyzes site- and strand-strand specific nicking of the F plasmid in addition to the previously characterized duplex DNA unwinding (helicase) reaction.     

Binding of NR1 plasmid DNA to membrane during replication in Escherichia coli mini-cells

Studying the replication of NR1 plasmid in E. coli mini-cells it was shown that the character of bond between plasmid DNA and membrane depends on the stage of replication cycle of the plasmid. On initiation the DNA-membrane complex is sensitive to the action of ionic force. In the process of elongation the bond of DNA molecules with the membrane is unstable if exists at all, and can be broken even by the nonionic detergent. At the final stage of replication the newly synthesized molecules form a complex with the membrane structures which is unstable in the presence of 0,5 M NaCl. The destruction of the complex followed by the open cycle of plasmid DNA coming out of it takes place under the action of ionic detergent.     

Homologous pairing and topological linkage of DNA molecules by combined action of E. coli recA protein and topoisomerase I

E. coil RecA protein and topolsomerase I, acting on superhelical DNA and circular single strands in the presence of ATP and Mg²⁺, topologically link single-stranded molecules to one another, and single-stranded molecules to duplex DNA. When super-helical DNA is relaxed by prior incubation with topoisomerase, it is a poor substrate for catenation. Extensive homology stimulates the catenation of circular single-stranded DNA and superhelical DNA, whereas little reaction occurs between these forms of the closely related DNAs of phages φX174 and G4, indicating that, in conjunction with topoisomerase I, RecA protein can discriminate perfect or nearly perfect homology from a high degree of relatedness. Circular single-stranded G4 DNA reacts with superhelical DNA of a chimeric phage, M13Goril, to form catenanes, at least half of which survive heating at 80°C following restriction cleavage in the M13 region, but few of which survive following restriction cleavage in the G4 region. Electron microscopic examination of catenated molecules cleaved in the M13 region reveals that in most cases the single-stranded G4 DNA is joined to the linear duplex M13(G4) DNA in the homologous G4 region. The junction frequently has the appearance of a D loop, with an extent equivalent to 100 or more bp. We conclude that a significant fraction of catenanes were hemicatenanes, in which the single-stranded circle was topologically linked, probably by multiple turns, to its complementary strand in the duplex DNA. These observations support the previous conclusion that RecA protein can pair a single strand with its complementary strand in duplex DNA in a side-by-side fashion without a free end in any of the three strands.     

The specific cleavage of DNA with ultrasound

Cleavages of double-stranded DNA fragments of known base pair sequence upon ultrasound irradiation at 22 and 44 kHz were studied by gel electrophoresis. The cleavage rate is found to be strongly dependent on the DNA fragment length, pH, temperature and ionic strength of the solution under study. The cleavage of double-stranded DNA occurs predominantly at sites containing alternating 5'-CpG-3' sequences. The breakage of phosphatediester bond takes place between C and G in such a way that phosphate group at the 5'-end of the guanine residue remain intact. The cleavage rate at a given DNA site is found to depend on base pair sequences at adjacent sites. Distinctly different cleavage patterns are observed when free DNA and DNA complexes with cys-diammine-Pt-bridged bis-netropsin were irradiated by ultrasound. The observed effect can be attributed to local DNA conformation changes induced upon complex formation between ligand and DNA.     

The time-resolved kinetics of superhelical DNA cleavage by BamHI restriction endonuclease

The kinetics of the cleavage of superhelical plasmid DNA (pBR322) by the restriction endonuclease, BamHI, have been analyzed in terms a compartmental model consistent with the chemistry first proposed by Rubin and Modrich (Rubin, R. A., and Modrich, P. (1978) Nucleic Acids Res. 5, 2991-2997) for analysis of the kinetics of the restriction endonuclease, EcoRI. The model was defined in terms of two compartments representing DNA substrate (bound and free), two compartments representing nicked intermediate (bound and free), one compartment representing linear product, and one compartment for free enzyme. A simultaneous analysis of concentration changes over time of the three DNA forms (superhelical, nicked, and linear) at six different enzyme concentrations was undertaken employing this compartmental model using SAAM (Simulation Analysis And Modeling) software. Results showed that rate constants characterizing the association of enzyme with superhelical DNA (6.0 x 10(5) M-1 s-1) and nicked DNA (2.8 x 10(5) M-1 s-1) were similar in magnitude and rate constants characterizing cleavage of the first (1.2 x 10(-2) s-1) and second phosphodiester bonds (3.1 x 10(-2) s-1) were also similar. The analysis yields a kinetically determined equilibrium constant of 12.9 nM for the dissociation of nicked intermediate from the enzyme. The rate constant describing the release of the nicked intermediate from the enzyme has a value of 3.7 x 10(-3) s-1. By comparing the value of this release rate constant to the value of the constant describing the second cleavage event, it can be determined that only 10% of the nicked intermediate bound to the enzyme is released as free nicked DNA and that 90% of the nicked intermediate is processed to the linear form without being released. Hence, most of the DNA is cleaved as the result of a single enzyme-DNA recognition event. No steady state assumptions were made in the analysis. The approach was to directly solve the differential equations which described the kinetic processes using an interactive method. This study demonstrates the usefulness of this approach for the analysis of kinetics of protein-DNA interactions for the restriction endonucleases.     

The number of superhelical turns in native virion SV40 DNA and minicol DNA determined by the band counting method.

By a method of overlapping the results obtained after agarose gel electrophoresis under two different sets of conditions, it has become possible to determine the number of superhelical turns in a given DNA by counting the bands present after partially relaxing the DNA (Keller and Wendel, 1974) with highly purified nicking-closing (N-C) enzyme from LA9 mouse cell nuclei. Because native supercoiled DNA is heterogeneous with respect to superhelix density, an average number of superhelical turns was determined. Virion SV40 DNA contains 26 +/- 0.5 superhelical turns, and native Minicol DNA contains 19 +/- 0.5 superhelical turns. The above are values at 0.2 M NaCl and at 37 degrees C, the condition under which the enzymatic relaxations were performed. The superhelix densities determined by the band counting method have been compared with superhelix densities determined by buoyant equilibrium in PDl-CsCl gradients. The Gray, Upholt, and Vinograd (1971) calculation procedure has been used for evaluating the superhelix densities by the latter method with the new statement, however, that relaxed DNA has zero superhelical turns. Comparison of the superhelix densities obtained by both methods permits a calculation of an unwinding angle for ethidium. The mean value from experiments with SV40 DNA is 23 +/- 3 degree. The average number of superhelical turns in SV40, 26, combined with the value, 21, obtained by both Griffith (1975) and Germond et al. (1975) for the average number of nucleosomes per SV40 genome, yields an average of 1.25 superhelical turns per 1/21 of the SV40 genome. If the regions of internucleosomal DNA are fully relaxed, 1.25 correesponds to the average number of superhelical turns with a nucleosome. When analyzed under identical conditions, the limit product generated by ligating a nicked circular substrate in the presence of 0.001 M Mg2+ at 37 degrees C (ligation conditions) is slightly more positively supercoiled than the limit product obtained when the N-C reaction is performed in 0.2 M NaCl at 37 degrees C. The difference in superhelix density as measured in gels between the two sets of limit products for both Minicol and SV40 DNAs is 0.0059 +/- 0.0005. This result indicates that the DNA duplex is overwound in the ligation solvent relative to its state in 0.2 M NaCl.     

Studies on the mechanism of DNA cleavage by ethidium.

Ethidium causes the cleavage of DNA via a light and oxygen dependent process. Using covalently closed circular DNA as a substrate, the saturation kinetics and the dependence on superhelical density of the cleavage indicate that intercalated ethidium is mainly responsible for nicking DNA. Superoxide dismutase has little effect on the reaction and catalase none. Lowering the pH inhibited the reaction. The reaction mechanism and its use in determining superhelical densities of covalently closed circular DNA's are discussed.     

DNA site recognition and reduced specificity of the Eco RI endonuclease

It has been shown previously (Polisky, B., Green, P., Garfin, D. E., McCarthy, B. J., Goodman, H. M., and Boyer, H. W. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 3310-3314; Hsu, M., and Berg, P. (1978) Biochemistry 17, 131-138) that the cleavage sequence specificity of Eco RI endonuclease can be "relaxed" by various means. In this paper this phenomenon is explored in detail, in order to obtain further insight into the nature and selectivity of sequence recognition patterns between proteins and double-stranded nucleic acids. Using conditions of low ionic strength and alkaline pH, we have mapped the positions of potentially cleavable sites in the (completely sequenced) replicative form of the bacteriophage phi X174 genome, and have deduced their sequence. The time course of digestion of phi X174 DNA suggests that double-stranded sequences reading GGATTT, AAATTT, GAATTT, and GAATTA (only "top" strands, written 5' leads to 3', are shown) are cleaved readily under these conditions, while sequences reading CAATTN (N = A, T, G) resist attack. Cleavages at (at least) the more labile sites result in cohesive ends that are religatable. End group analysis of cleaved phi X174 DNA fragments indicates the presence of a 5'-terminal adenine residue on most of the fragments; some fragments may carry a 5'-terminal guanine residue, consistent with the cleavage site sequences suggested above. Addition of Mn2+ to cleavage reactions carried out at moderate salt concentrations and near-neutral pH induces the same pattern of cleavage seen at low ionic strength and alkaline pH. These results are combined with those from other studies, and are interpreted in terms of a model for the site-specific interaction of the Eco RI endonuclease with its substrate, considering both the effects of changes in DNA sequence and of environmental alterations. The resulting model is compared with data developed on similar grounds for Eco RI methylase (see Woodbury, C. P., Downey, R. L., and von Hippel, P. H. (1980) J. Biol. Chem. 255, 11526-11533), and attempts are made to define both common and differing molecular facets of the DNA recognition specificity of these companion (but genetically distinct) enzymes.     

Bleomycin cleaves DNA depending on DNA primary, secondary, and tertiary structures

The cleavage by bleomycin-Fe(II) complex in the presence of dithiothreitol was investigated by using 3'- or 5'-end-labeled DNA containing the region of the bacteriophage G4 origin of complementary strand synthesis as substrates. Bleomycin cleaved single-stranded DNA substrates preferentially at inverted repeat sequences, which potentially form stem-and-loop structures, in addition to the primary sequence specificity previously reported. DNA sequences preferentially cleaved in the double-stranded substrate were resistant when they lay outside the stem regions. These results suggest the formation of three predicted stem-and-loop structures and other possible secondary structures near the replication origin. Changes of the degree of bleomycin-induced DNA cleavage in a NaCl concentration between 0 and 50 mM suggest that a subtle change of ionic conditions within the double helix, or of DNA conformation, or of both, may occur at 0-50 mM NaCl. Bleomycin appears to be a useful reagent for analyzing secondary and tertiary structures of DNA.     

Structures of homopurine-homopyrimidine tract in superhelical DNA

For homopurine-homopyrimidine tracts in superhelical DNA, we propose a structure involving Watson-Crick and Hoogsteen paired triple helixes, hairpin loops and unstructured domains. Topologically, the whole structure is equivalent to an open region. The proposed structure is consistent with available S1 cleavage, pH and alkylation data and energetics under superhelical stress; this new structure is a much more probable candidate than the one proposed by us recently (V.I. Lyamichev, S.M. Mirkin & M.D. Frank-Kamenetskii, J. Biomole. Str. Dyns 3, 327-338, 1985).     

Copper-bleomycin has no significant DNA cleavage activity

In contrast to a very recent report [Ehrenfeld, G. M., Rodriguez, L. O., Hecht, S. M., Chang, C., Basus, V. J., & Oppenheimer, N. J. (1985) Biochemistry 24, 81-92], the present careful reexamination demonstrated that copper-bleomycin systems have no significant DNA cleavage activity. In the presence of dithiothreitol, the bleomycin-Cu(II) complex showed little activity for DNA degradation. The DNA strand scission by the Cu(I)-bleomycin-dithiothreitol system was remarkably depressed by deferoxamine rather than by bathocuproine, suggesting the effect of trace amounts of contaminating iron in the experiments. It seems highly unlikely that the DNA breakage activity due specifically to the Cu(I)-bleomycin complex system is substantially strong. Our results indicate that the metal really relevant to the DNA cleavage by bleomycin is iron not copper.     

Structure and function of a family 10 beta-xylanase chimera of Streptomyces olivaceoviridis E-86 FXYN and Cellulomonas fimi Cex

The catalytic domain of xylanases belonging to glycoside hydrolase family 10 (GH10) can be divided into 22 modules (M1 to M22; Sato, Y., Niimura, Y., Yura, K., and Go, M. (1999) Gene (Amst.) 238, 93-101). Inspection of the crystal structure of a GH10 xylanase from Streptomyces olivaceoviridis E-86 (SoXyn10A) revealed that the catalytic domain of GH10 xylanases can be dissected into two parts, an N-terminal larger region and C-terminal smaller region, by the substrate binding cleft, corresponding to the module border between M14 and M15. It has been suggested that the topology of the substrate binding clefts of GH10 xylanases are not conserved (Charnock, S. J., Spurway, T. D., Xie, H., Beylot, M. H., Virden, R., Warren, R. A. J., Hazlewood, G. P., and Gilbert, H. J. (1998) J. Biol. Chem. 273, 32187-32199). To facilitate a greater understanding of the structure-function relationship of the substrate binding cleft of GH10 xylanases, a chimeric xylanase between SoXyn10A and Xyn10A from Cellulomonas fimi (CfXyn10A) was constructed, and the topology of the hybrid substrate binding cleft established. At the three-dimensional level, SoXyn10A and CfXyn10A appear to possess 5 subsites, with the amino acid residues comprising subsites -3 to +1 being well conserved, although the +2 subsites are quite different. Biochemical analyses of the chimeric enzyme along with SoXyn10A and CfXyn10A indicated that differences in the structure of subsite +2 influence bond cleavage frequencies and the catalytic efficiency of xylooligosaccharide hydrolysis. The hybrid enzyme constructed in this study displays fascinating biochemistry, with an interesting combination of properties from the parent enzymes, resulting in a low production of xylose.     

Thermodynamic parameters governing interaction of EcoRI endonuclease with specific and nonspecific DNA sequences

Equilibrium binding of EcoRI endonuclease to DNA has been analyzed by nitrocellulose filter and preferential DNA cleavage methods. Association constants for pBR322 and a 34-base pair molecule containing the EcoRI site of this plasmid in a central position were determined to be 1.9 X 10(11) M-1 and 1.0 X 10(11) M-1 at 37 degrees C, respectively, with the stoichiometry of binding being 0.8 +/- 0.1 mol of endonuclease dimer per mol of DNA. In contrast, the affinity of the enzyme for a pBR322 derivative from which the EcoRI site has been deleted is 3.2 X 10(9) M-1 as judged by competitive binding experiments. If it is assumed that each base pair can define the beginning of a nonspecific binding site, this value corresponds to an affinity for nonspecific sites of 7.4 X 10(5) M-1. Furthermore, the affinity of the endonuclease for the EcoRI-methylated sequence is at least three orders of magnitude less than that for the unmodified recognition site. The dependence on temperature and ionic strength of the equilibrium constant governing specific interactions has also been examined. The temperature dependence of the reaction indicates that entropy increase accounts for 70% of the free energy of specific binding at 37 degrees C. Affinity of the endonuclease for the EcoRI site is highly dependent on NaCl concentration. Analysis of this dependence according to the theory of Record and colleagues (Record, T. M., Jr., Lohman, T. M., and deHaseth, P. (1976) J. Mol. Biol. 107, 145-158) has implicated 8 ion pairs in the stability of specific complexes, a value identical with the number of phosphate contacts determined by ethylation interference analysis (Lu, A. L., Jack, W. E., and Modrich, P. (1981) J. Biol. Chem. 256, 13200-13206). Extrapolation to 1 M NaCl suggests that nonelectrostatic interactions account for 40% of the free energy change associated with specific complex formation.     

Site-directed mutagenesis of residues involved in G Strand DNA binding by Escherichia coli DNA topoisomerase I

Crystal structures of complexes between type IA DNA topoisomerases and single-stranded DNA suggest that the residues Ser-192, Arg-195, and Gln-197 in a conserved region of Escherichia coli topoisomerase I may be important for direct interactions with phosphates on the G strand of DNA, which is the substrate for DNA cleavage and religation (Changela A., DiGate, R. J., and Mondragón, A. (2001) Nature 411, 1077-1081; Perry, K., and Mondragón, A. (2003) Structure 11, 1349-1358). Site-directed mutagenesis experiments altering these residues to alanines and other amino acids were carried out to probe the relevance of these interactions in the catalytic activities of the enzyme. The results show that the side chains of Arg-195 and Gln-197 are required for DNA cleavage by the enzyme and are likely to be important for positioning of the G strand of DNA at the active site prior to DNA cleavage. Mutation of Ser-192 did not affect DNA binding and cleavage but nevertheless decreased the overall rate of relaxation of supercoiled DNA probably because of its participation in a later step of the reaction pathway.     

Circular dichroism of superhelical DNA

The circular dichroism (CD) spectra of a number of superhelical DNA's have been measured. The introduction of negative superhelical turns causes an increase in magnitude of the positive band around 280 mμ, while the trough around 250mμ is little affected. For two samples of λb2b5c DNA (20 Mdalton) containing different number of negative superhelical turns, the magnitude of the positive band relative to that of the nicked control increases with increasing number of superhelical turns. In 2M NaCl, the small (1.45 Mdalton) superhelical DNA from E. coli 15 shows an unusually large difference in CD compared with that of the same DNA with a few single-chain scissions per molecule. This large difference is not observed in a medium containing p. 0.11M NaCl. These results indicate that the double helix in a superhelical DNA is perturbed somewhat due to the bending and torsional forces in such a molecule. The magnitude of such structural alteration seems to depend on the number of superhelical turns per unit length, the size of the DNA molecule, as well as the ionic medium.     

Control of bacterial DNA supercoiling

Two DNA topoisomerases control the level of negative supercoiling in bacterial cells. DNA gyrase introduces supercoils, and DNA topoisomerase I prevents supercoiling from reaching unacceptably high levels. Perturbations of supercoiling are corrected by the substrate preferences of these topoisomerases with respect to DNA topology and by changes in expression of the genes encoding the enzymes. However, supercoiling changes when the growth environment is altered in ways that also affect cellular energetics. The ratio of [ATP] to [ADP], to which gyrase is sensitive, may be involved in the response of supercoiling to growth conditions. Inside cells, supercoiling is partitioned into two components, superhelical tension and restrained supercoils. Shifts in superhelical tension elicited by nicking or by salt shock do not rapidly change the level of restrained supercoiling. However, a steady-state change in supercoiling caused by mutation of topA does alter both tension and restrained supercoils. This communication between the two compartments may play a role in the control of supercoiling.     

Coordinate ion pair formation between EcoRI endonuclease and DNA

The free energy of the binding reaction between EcoRI restriction endonuclease and a specific cognate dodecadeoxynucleotide (d(CGCGAATTCGCG)) has contributions from both electrostatic and nonelectrostatic components. These contributions were dissected by measuring the effects of varying salt concentration on the equilibrium binding constant and applying the thermodynamic analyses of Record et al. (Record, M. T., Jr., Lohman, T. M., and deHaseth, P. L. (1976) J. Mol. Biol. 107, 145-158). Endonuclease mutation S187 (Arg 187 to Ser) (Greene, P. J., Gupta, M., Boyer, H. W., Brown, W. E., and Rosenberg, J. M. (1981) J. Biol. Chem. 256, 2143-2153) did not significantly affect the nonelectrostatic component but did perturb the electrostatic contribution to the binding energy (we are numbering the amino acid residues according to the DNA sequence). The former was determined by extrapolating the linear portion of the salt dependence curve (0.125 to 0.25 M KCl) to 1 M ionic strength, with the same result for both wild type and S187 endonucleases at both pH 6.0 and 7.4 (-8.5 +/- 1.5 kcal/mol or greater than 50% of the total binding free energy). The slopes of these same curves yield estimates of eight ionic interactions between wild type endonuclease and the DNA at both pH values. By contrast, binding of EcoRI-S187 to dodecanucleotide involves six charge-charge interactions at pH 6.0. Only two ionic interactions are observed at pH 7.4. This was unexpected since gel permeation chromatography demonstrated that the recognition complex for both wild type and S187 proteins contains an enzyme dimer and a DNA duplex. EcoRI-S187 endonuclease retains wild type DNA sequence specificity, and the rate of the phosphodiester hydrolysis step is also unchanged. Thus, electrostatic interactions are functionally separable from sequence recognition and strand cleavage. Our results also establish that arginine 187 plays a key role in the electrostatic function and suggest that it might be located at the DNA-protein interface. The disproportionate loss of ion pairs at pH 7.4 can be rationalized by a model which suggests that six conformationally mobile ionic groups on the protein act in a coordinated manner during the interaction with DNA.