Porphyrin and metalloporphyrin binding to DNA polymers: rate and equilibrium binding studies

Interactions of meso-tetrakis(4-N-methylpyridiniumyl)porphyrin [TMpyP(4)] with poly[d(G-C)].poly[d(G-C)] [poly[d(G-C)2] and poly[d(A-T)].poly[d(A-T)] [poly[d(A-T)2] were studied by equilibrium dialysis and stopped-flow dissociation kinetics as a function of [Na+]. Metalloderivatives of TMpyP(4), NiTMpyP(4), and ZnTMpyP(4) were also investigated. The apparent equilibrium binding constants (Kobs) were approximately the same for TMpyP(4) binding to either poly[d(G-C)2] or poly[d(A-T)2] and decreased with increasing [Na+]. The slopes of the plots of log Kobs vs log [Na+] were similar, with values close to -2.7. Contrary to implications in previously reported studies, these data do not indicate that TMpyP(4) prefers to bind to GC sites at low ionic strength and to AT sites at high ionic strength. In contrast, binding of ZnTMpyP(4) to these two polymers is very different. Comparisons of Kobs values at 0.065 M [Na+] indicate that ZnTMpyP(4) binding to AT sites is approximately 200 times more favorable than binding to GC sites, a finding in agreement with previous qualitative observations. Although the binding of the Zn species to the GC polymer was too weak for us to assess the salt effect, the plot of log Kobs vs log [Na+] gave a slope of -2.0 for ZnTMpyP(4) binding to poly[d(A-T)2]. Application of condensation theory for polyelectrolytes suggests similar charge interactions for ZnTMpyP(4) and for TMpyP(4) binding to poly[d(A-T)2]. Likewise, the rates of dissociation from poly[d(A-T)2] were similar for TMpyP(4) and ZnTMpyP(4) [and also NiTMpyP(4)]. However, whereas TMpyP(4) [and NiTMpyP(4)] dissociation from poly[d(G-C)2] was measurable, that for ZnTMpyP(4) was too fast to measure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Netropsin and bis-netropsin analogs as inhibitors of the catalytic activity of mammalian DNA topoisomerase II and topoisomerase cleavable complexes.

This study examined the ability of netropsin and related minor groove binders to interfere with the actions of DNA topoisomerases II and I. We evaluated a series of netropsin dimers linked with flexible aliphatic chains of different lengths. These agents are potentially able to occupy longer stretches of DNA than the parental drug as a result of bidentate binding. Both netropsin and its dimers were found: (i) to inhibit the catalytic activity of isolated topoisomerase II and (ii) to interfere with the stabilization of the cleavable complexes of topoisomerase II and I in nuclei. Dimers with linkers consisting of 0-4 and 6-9 methylene groups (n) were far more inhibitory than netropsin against isolated enzyme and in the nuclear system. The compound with n = 5 was less active than netropsin in both assays while the dimer with n = 10 inhibited only the isolated enzyme. The comparison of dimers with fixed linker length (n = 2) but varying number of N-methylpyrrole residues (from 1 to 3) revealed that the inhibitory properties were enhanced with increasing number of N-methylpyrrole units. For dimers with varying linker length, drug ability to inhibit catalytic activity of isolated topoisomerase II was positively correlated with calf thymus DNA association constants. In contrast, no such correlation existed in nuclei. However, the inhibitory effects in the nuclear system were correlated with the association constants for poly(dAdT). The results indicate that bidentate binding can significantly enhance anti-topoisomerase activity of netropsin related dimeric minor groove binders. However, other factors such as the length of the linker, the number of pyrrole moieties and the nature of the target (isolated enzyme/DNA versus chromatin in nuclei) also contribute to these activities.     

DNA sequence dependent binding modes of 4',6-diamidino-2-phenylindole (DAPI)

The interactions of DAPI with natural DNA and synthetic polymers have been investigated by hydrodynamic, DNase I footprinting, spectroscopic, binding, and kinetic methods. Footprinting results at low ratios (compound to base pair) are similar for DAPI and distamycin. At high ratios, however, GC regions are blocked from enzyme cleavage by DAPI but not by distamycin. Both poly[d(G-C)]2 and poly[d(A-T)]2 induce hypochromism and shifts of the DAPI absorption band to longer wavelengths, but the effects are larger with the GC polymer. NMR shifts of DAPI protons in the presence of excess AT and GC polymers are significantly different, upfield for GC and mixed small shifts for AT. The dissociation rate constants and effects of salt concentration on the rate constants are also quite different for the AT and the GC polymer complexes. The DAPI dissociation rate constant is larger with the GC polymer but is less sensitive to changes in salt concentration than with the AT complex. Binding of DAPI to the GC polymer and to poly[d(A-C)].poly[d(G-T)] exhibits slight negative cooperativity, characteristic of a neighbor-exclusion binding mode. DAPI binding to the AT polymer is unusually strong and exhibits significant positive cooperativity. DAPI has very different effects on the bleomycin-catalyzed cleavage of the AT and GC polymers, a strong inhibition with the AT polymer but enhanced cleavage with the GC polymer. All of these results are consistent with two totally different DNA binding modes for DAPI in regions containing consecutive AT base pairs versus regions containing GC or mixed GC and AT base pair sequences. The binding mode at AT sites has characteristics which are similar to those of the distamycin-AT complex, and all results are consistent with a cooperative, very strong minor groove binding mode. In GC and mixed-sequence regions the results are very similar to those observed with classical intercalators such as ethidium and indicate that DAPI intercalates in DNA sequences which do not contain at least three consecutive AT base pairs.     

Acridine dimers: influence of the intercalating ring and of the linking-chain nature on the equilibrium and kinetic DNA-binding parameters

The rigidity of the linking chain of bifunctional intercalators in the ditercalinium series was shown to be critical for antitumor activity. In order to study the influence of the rigidity of the linking chain on the DNA-binding properties of DNA bifunctional intercalators, fluorescent 9-aminoacridine and 2-methoxy-6-chloro-9-aminoacridine analogues with chains of variable rigidity were synthesized. 1H-NMR studies show that the conformation of 9-aminoacridine dimers is almost independent of the nature of the linking chain. A strong self-stacking of the aromatic rings of the 2-methoxy-6-chloro-9-aminoacridine is observed for dimers with flexible chains but not for those with rigid chains. All the dimers having a linking chain long enough to bisintercalate in DNA according to the excluded site model are indeed bisintercalators. The kinetic association constant of all monomers and dimers for poly[d(A-T)].poly[d(A-T)] are in the same range (2-4 x 10(7) M-1 s-1). The large increase of DNA binding affinity observed for the dimers is always associated with the expected decrease of the dissociation rate constant. The effect of chain rigidity and pH on the calf thymus DNA binding of 9-aminoacridine and 2-methoxy-6-chloro-9-aminoacridine dimers is quite different. In the series of 9-aminoacridine the pKa of the dimers remains high and therefore no difference of DNA-binding affinity is observed between pH 5 and 7.4. The rigidity of the linking chain does not significantly alter the DNA-binding affinity. In the 2-methoxy-6-chloro-9-aminoacridine series, the pKa of all dimers became smaller than the physiological pH and a dramatic decrease of DNA-binding affinity is observed when the pH is increased from pH 5 to 7.4. This decrease appears significantly smaller for dimers with rigid chains. A similar dramatic decrease of binding affinity at pH 7.4 is not observed for poly[d(A-T)].poly[d(A-T)]. This factor makes these dimers strongly specific for the alternating polymer at pH 7.4.     

Stopped-flow kinetic analysis of the interaction of anthraquinone anticancer drugs with calf thymus DNA, poly[d(G-C)].poly[d(G-C)], and poly[d(A-T)].poly[d(A-T)]

The sodium dodecyl sulfate driven dissociation reactions of daunorubicin (1), mitoxantrone (2), ametantrone (3), and a related anthraquinone without hydroxyl groups on the ring or side chain (4) from calf thymus DNA, poly[d(G-C)]2, and poly[d(A-T)]2 have been investigated by stopped-flow kinetic methods. All four compounds exhibit biphasic dissociation reactions from their DNA complexes. Daunorubicin and mitoxantrone have similar dissociation rate constants that are lower than those for ametantrone and 4. The effect of temperature and ionic strength on both rate constants for each compound is similar. An analysis of the effects of salt on the two rate constants for daunorubicin and mitoxantrone suggests that both of these compounds bind to DNA through a mechanism that involves formation of an initial outside complex followed by intercalation. The daunorubicin dissociation results from both poly[d(G-C)]2 and poly[d(A-T)]2 can be fitted with a single exponential function, and the rate constants are quite close. The ametantrone and 4 polymer dissociation results can also be fitted with single exponential curves, but with these compounds the dissociation rate constants for the poly[d(G-C)]2 complexes are approximately 10 times lower than for the poly[d(A-T)]2 complexes. Mitoxantrone also has a much slower dissociation rate from poly[d(G-C)]2 than from poly[d(A-T)]2, but its dissociation from both polymers exhibits biphasic kinetics. Possible reasons for the biphasic behavior with the polymers, which is unique to mitoxantrone, are selective binding and dissociation from the alternating polymer intercalation sites and/or dual binding modes of the intercalator with both side chains in the same groove or with one side chain in each groove.     

Poly(dA).poly(dT) exists in an unusual conformation under physiological conditions: propidium binding to poly(dA).poly(dT) and poly[d(A-T)].poly[d(A-T)]

The binding of propidium to poly(dA).poly(dT) [poly(dA.dT)] and to poly[d(A-T)].poly[d(A-T)] [poly[d(A-T)2]] has been compared under a variety of solution conditions by viscometric titrations, binding studies, and kinetic experiments. The binding of propidium to poly[d(A-T)2] is quite similar to its binding to calf thymus deoxyribonucleic acid (DNA). The interaction with poly(dA.dT), however, is quite unusual. The viscosity of a poly(dA.dT) solution first decreases and then increases in a titration with propidium at 18 degrees C. The viscosity of poly[d(A-T)2] shows no decrease in a similar titration. Scatchard plots for the interaction of propidium with poly(dA.dT) show the classical upward curvature for positive cooperativity. The curvature decreases as the temperature is increased in binding experiments. A van't Hoff plot of the observed binding constants yields an apparent positive enthalpy of approximately +6 kcal/mol for the propidium-poly(dA.dT) interaction. Propidium binding to poly[d(A-T)2] shows no evidence for positive cooperativity, and the enthalpy change for the reaction is approximately -9 kcal/mol. Both the magnitude of the dissociation constants and the effects of ionic strength are quite similar for the dissociation of propidium from poly(dA-T)2] and from poly[d(A-T)2], suggesting that the intercalated states are similar for the two complexes. The observed association reactions, under pseudo-first-order conditions, are quite different. Plots of the observed pseudo-first-order association rate constant vs. polymer concentration have much larger slopes for propidium binding to poly[d(A-T)2] than to poly(dA.dT).(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of Hoechst 33258 and its Derivatives to DNA

Abstract

In the present work, we employed UV-VIS spectroscopy, fluorescence methods, and circular dichroism spectroscopy (CD) to study the interaction of dye Hoechst 33258, Hoechst 33342, and their derivatives to poly[d(AT)]·poly[d(AT)], poly(dA)·poly(dT), and DNA dodecamer with the sequence 5′-CGTATATATACG-3′. We identified three types of complexes formed by Hoechst 33258, Hoechst 33342, and methylproamine with DNA, corresponding to the binding of each drug in monomer, dimer, and tetramer forms. In a dimer complex, two dye molecules are sandwiched in the same place of the minor DNA groove. Our data show that Hoechst 33258, Hoechst 33342, and methylproamine also form complexes of the third type that reflects binding of dye associates (probably tetramers) to DNA. Substitution of a hydrogen atom in the ortho position of the phenyl ring by a methyl group has a little effect on binding of monomers to DNA. However it reduces strength of binding of tetramers to DNA. In contrast, a Hoechst derivative containing the ortho-isopropyl group in the phenyl ring exhibits a low affinity to poly(dA)·poly(dT) and poly[d(AT)]·poly[d(AT)] and binds to DNA only in the monomer form. This can be attributed to a sterical hindrance caused by the ortho-isopropyl group for side-by-side accommodation of two dye molecules in the minor groove. Our experiments show that mode of binding of Hoechst 33258 derivatives and their affinity for DNA depend on substituents in the ortho position of the phenyl ring of the dye molecule. A statistical mechanical treatment of binding of Hoechst 33258 and its derivatives to a polynucleotide lattice is described and used for determination of binding parameters of Hoechst 33258 and its derivatives to poly[d(AT)]·poly[d(AT)] and poly(dA)·poly(dT).     

Intercalative and nonintercalative binding of large cationic porphyrin ligands to polynucleotides

The interactions of two positional isomers and one analogue of meso-tetra (4-N-methylpyridyl) porphine, with the synthetic polynucleotides poly[d(A-T)] . poly[d(A-T)] and poly[d(G-C)] . poly[d(G-C)] have been investigated by circular dichroism. All four porphyrins were found to bind to the polynucleotides as shown by the induction of circular dichroism in their Soret bands. Furthermore, the sign of the induced ellipticity reflects selective occupation of binding sites by the porphyrin ligands. The conformational lability of poly[d(A-T)] X poly[d(A-T)] was found to be appreciable as micromolar amounts of meso-substituted 4-N-methylpyridyl, 3-N-methylpyridyl, and p-N-trimethylanilinium porphines induced a CD spectrum similar but not identical to that of DNA in the Z-form, i.e. a negative band at 280 nm and a positive band at 259 nm. The effect of porphyrin binding to poly[d(G-C)] X poly[d(G-C)] was less pronounced and dissimilar to that seen in the AT polymer.     

DNA sequence recognition of thiazole-containing cross-linked polyamides can be favored

The binding ability of cross-linked thiazolated polyamides (containing the base sequence-reading elements thiazole(Th)-pyrrole(Py)-pyr-role(Py) and thiazole(Th)-imidazole(Im)-pyrrol(Py) to various DNA dodecamers has been investigated. CD titration experiments at high salt concentration demonstrate that the dimers with a heptanediyl linker (C7 dimer) show a significantly higher sequence specificity than their corresponding monomers. The dimer of Th-Py-Py primarily prefers binding to pure AT sequences and that of Th-Im-Py to the dodecamer sequences containing a GC pair within the central sequence (e.g. AACGTT). Surprisingly, the sequence binding ability is strongly influenced by the presence of a T-A step: e.g. Th-Py-Py has a similar affinity to the sequences TTTAAA and ATCGTA; likewise Th-Im-Py shows a preference for these sequences. The CD results correlate with footprinting data. Related biochemical studies on the effect of polyamides on DNA gyrase activity in vitro show that the C7 dimers most effectively inhibit the enzyme activity compared with the monomers and the natural reference minor groove binder distamycin. The highest inhibitory potency is observed for the Th-Py-Py-dimer. The role of the T-A step in binding of the cross-linked dimer to the minor groove is discussed in light of the sequence recognition of the TATA box binding protein.     

The interaction of substituted and rigidly linked diquinolines with DNA

Viscometric measurements with circular and sonicated rodlike DNA fragments were used to explore whether ring substituents or conformationally restricted linkers promote bifunctional intercalation amongst a series of binuclear 4-aminoquinolines bridged via their 4-amino group. We find that ligands comprising unsubstituted quinolines and piperazine or pyrazole linkages bisintercalate. Quinoline-substituted alkyl-linked dimers intercalate in either a mixed monofunctional-bifunctional mode or bind with only one of their chromophores intercalated depending on the nature of the substituents. Equilibrium dialysis measurements show that the binding affinity for calf thymus DNA of the compounds studied ranges from (1.2-12) . 10(4) M-1 in buffer of ionic strength 0.1. Both co-operative and antico-operative binding isotherms were obtained and there is evidence for a second binding mode for the piperazine-linked diquinoline at saturating binding levels. For this compound the high-affinity association constant decreases with increasing ionic strength, 3.4 cations being released per bound ligand molecule. Partition dialysis measurements with DNAs of differing base composition indicate that the compounds studied are either AT selective or sequence neutral depending on ligand structure. For example, the pyrazole linker imparts a marked specificity for binding to AT-rich DNA, whereas the piperazine linker does not. Kinetic measurements using the surfactant-sequestration method reveal that DNA-diquinoline complexes dissociate very rapidly by complex mechanisms with rate constants greater than 100 s-1 in buffer of ionic strength 0.1.     

Sequence-dependent variation in the conformation of DNA

The specificity of action of the enzyme DNAase I on double-stranded DNA polymers of defined sequence has been investigated. The results obtained with the alternating copolymers poly[d(A-T)] · poly[d(A-T)] and poly[d(G-C)] · poly[d(G-C)] support the suggestion of Klug et al. (1979) that regions of double-stranded DNA containing alternating purine-pyrimidine sequences may exist as structural variants of the classical B-form under physiological salt conditions. Digestion of defined oligomers containing alternating dG-dC sequences indicate that these too exist in some “alternating-B” structure in solution under similar conditions. The results obtained with the oligomers also provide a number of insights into the mode of action of DNAase I.In the case of the B-DNA dodecamer d(C-G-C-G-A-A-T-T-C-G-C-G), for which the crystal structure has been solved (Dickerson &; Drew, 1981), there is a very good correlation between the sites of rapid DNAase I cutting and positions of high local helical twist.     

Conformational changes of nucleic acids and poly (d(A-T)-d(A-T)) caused by photoaddition of furocoumarins.

DNA's of various AT content, poly[d(A-T)-d(A-T)], and double-stranded RNA were irradiated with UV light at 365 nm in the presence of linear (xanthotoxin) or angular (angelicin) furocoumarins. The covalent photobinding is strongly dependent on the spatial arrangement of furocoumarin molecules at the polymer conformation. CD measurements demonstrate that the bifunctional photochemical binding of xanthotoxin with double-stranded DNA's and poly[d(A-T)-d(A-T)] is accompanied by conformational changes which involve probably decreasing helical twisting of the double helix. This effect is greatly enhanced with increasing AT content. The formation of A-like structures is very unlikely since the B leads to A transition induced by ethanol addition was found to be strongly suppressed in xanthotoxin photoreacted DNA. The B-type helix appears to be the most sensitive conformation with minor restriction to produce photochemically induced cross-links.     

The binding of fd gene 5 protein to polydeoxynucleotides: evidence from CD measurements for two binding modes

Circular dichroism measurements were used to study the binding of fd gene 5 protein to fd DNA, to six polydeoxynucleotides (poly[d(A)], poly[d(T)], poly[d(I)], poly[d(C)], poly[d(A-T)], and the random copolymer poly[d(A,T)]), and to three oligodeoxynucleotides (d(pA)20, d(pA)7, and d(pT)7). Titrations of these DNAs with fd gene 5 protein were generally done in a low ionic strength buffer (5 mM Tris-HCl, pH 7.0 or 7.8) to insure tight binding, needed to obtain stoichiometric endpoints. By monitoring the CD of the nucleic acids above 250 nm, where the protein has no significant intrinsic optical activity, we found that there were two modes of binding, with the number of nucleotides covered by a gene 5 protein monomer (n) being close to either 4 or 3. These stoichiometries depended upon which polymer was titrated as well as upon the protein concentration. Single endpoints at nucleotide/protein molar ratios close to 3 were found during titrations of poly[d(T)] and fd DNA (giving n = 3.1 and 2.8 +/- 0.2, respectively), while CD changes with two apparent endpoints at nucleotide/protein molar ratios close to 4 and approximately 3 were found during titrations of poly[d(A)], poly[d(I)], poly[d(A-T)], and poly[d(A,T)] (with the first endpoints giving n = 4.1 4.0, 4.0, and 4.1 +/- 0.3, respectively). Calculations showed that the CD changes we observed during these latter titrations were consistent with a switch between two non-interacting binding modes of n = 4 and n = 3. We found no evidence for an n = 5 binding mode. One implication of our results is that the Brayer and McPherson model for the helical gene 5 protein-DNA complex, which has 5 nucleotides bound per protein monomer (G. Brayer and A. McPherson, J. Biomol. Struct. and Dyn. 2, 495-510, 1984), cannot be correct for the detailed solution structure of the complex. We interpreted the CD changes above 250 nm upon binding of the gene 5 protein to single-stranded DNAs to be the result of a slight unstacking of the bases, along with a significant alteration of the CD contributions of the individual nucleotides in the case of A-and/or T-containing DNAs. Interestingly, CD contributions attributed to nearest-neighbor interactions in free poly[d(A-T)], poly[d(A,T)], poly[d(A)], and poly[d(T)] were partially maintained in the CD spectra of the protein-saturated polymers, so that neighboring nucleotides, when bound to the protein at 20 degrees C, appeared to interact with one another in much the same manner as in the free polymers at 50 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Parallel double stranded helices and the tertiary structure of nucleic acids

Thermal denaturation of four oligonucleotides, viz. 3'-d(AT)5pO(CH2)6Opd(AT)5-3'(par(AT], 3'-d(AT)5pO(CH2)6Opd(AT)5-5'(anti(AT],3'-d(A)10pO(CH2) 6Op(T)10-3'(par(A-T], and 3'-d(A)10pO(CH2)6Opd(T)10-5' (anti(A-T], was studied in 0.01 M phosphate buffer, pH 7, in the presence of 0.1, 0.25, 0.5 and 1.0 M NaCl. All the oligomers were found to exist at a lower temperature (0 to 20 degrees C) as complexes composed either of two oligomer molecules (a canonical duplex) or of more oligomer molecules whereas, at a higher temperature (30 to 70 degrees C), they formed hairpins with a parallel (par(AT) and par(A-T] or antiparallel (anti(AT) and anti(A-T) orientation of the chains. Melting curves (A260(T] were used to calculate thermodynamic parameters for the formation of hairpins and "low-temperature" duplexes. Experiments on ethidium bromide binding to the oligonucleotides have shown that the oligomer anti(A-T) exists, at a low ionic strength, as a four stranded complex ("quadruplex") contains two antiparallel helices, d(A).d(T), which have a parallel orientation and are bound to one another owing to the formation of additional hydrogen bonds between nucleic acid bases. The possible biological function of quadruplexes is discussed.     

A study of the interactions of some polypyridylruthenium (II) complexes with DNA using fluorescence spectroscopy, topoisomerisation and thermal denaturation.

The nature of binding of Ru(phen) 2+ (I), Ru(bipy) 2+ (II), Ru(terpy) 2+ (III) (phen = 1,10-phenanthroline, bipy 3 = 2,2'-bipyridyl, 3 terpy = 2,2'2," - 2 terpyridyl) to DNA, poly[d(G-C)] and poly[d(A-T)] has been compared by absorption, fluorescence, DNA melting and DNA unwinding techniques. I binds intercalatively to DNA in low ionic strength solutions. Topoisomerisation shows that it unwinds DNA by 22 degrees +/- 1 per residue and that it thermally stabilizes poly[d(A-T)] in a manner closely resembling ethidium. Poly[d(A-T)] induces greater spectral changes on I than poly[d(G-C)] and a preference for A-T rich regions is indicated. I binding is very sensitive to Mg2+ concentration. In contrast to I the binding of II and III appears to be mainly electrostatic in nature, and causes no unwinding. There is no evidence for the binding of the neutral Ru(phen)2 (CN)2 or Ru(bipy)2 (CN)2 complexes. DNA is cleaved, upon visible irradiation of aerated solutions, in the presence of either I or II.     

Characterization of ciprofloxacin binding to the linear single- and double-stranded DNA

The binding of ciprofloxacin to natural and synthetic polymeric DNAs was investigated at different solvent conditions using a combination of spectroscopic and hydrodynamic techniques. In 10 mM cacodylate buffer (pH 7.0) containing 108.6 mM Na(+), no sequence preferences in the interaction of ciprofloxacin with DNA was detected, while in 2 mM cacodylate buffer (pH 7.0) containing only 1.7 mM Na(+), a significant binding of ciprofloxacin to natural and synthetic linear double-stranded DNA was observed. At low ionic strength of solution, ciprofloxacin binding to DNA duplex containing alternating AT base pairs is accompanied by the largest enhancement in thermal stability (e.g. DeltaT(m) approximately 10 degrees C for poly[d(AT)].poly[d(AT)]), and the most pronounced red shift in the position of the maximum of the fluorescence emission spectrum (lambda(max)). Similar red shift in the position of lambda(max) is also observed for ciprofloxacin binding to dodecameric duplex containing five successive alternating AT base pairs in the row. On the other hand, ciprofloxacin binding to poly[d(GC)].poly[d(GC)], calf thymus DNA and dodecameric duplex containing a mixed sequence is accompanied by the largest fluorescence intensity quenching. Addition of NaCl does not completely displace ciprofloxacin bound to DNA, indicating the binding is not entirely electrostatic in origin. The intrinsic viscosity data suggest some degree of ciprofloxacin intercalation into duplex.     

Effects of excluded volume upon protein stability in covalently cross-linked proteins with variable linker lengths

To explore the effects of molecular crowding and excluded volume upon protein stability, we used a series of cross-linking reagents with nine different single-cysteine mutants of staphylococcal nuclease to make covalently linked dimers. These cross-linkers ranged in length from 10.5 to 21.3 A, compelling separations which would normally be found only in the most concentrated protein solutions. The stabilities of the dimeric proteins and monomeric controls were determined by guanidine hydrochloride and thermal denaturation. Dimers with short linkers tend to exhibit pronounced three-state denaturation behavior, as opposed to the two-state behavior of the monomeric controls. Increasing linker length leads to less pronounced three-state behavior. The three-state behavior is interpreted in a three-state model where cross-linked native protein dimer, N-N, interconverts in a two-state transition with a dimer where one protein subunit is denatured, N-D. The remaining native protein in turn can denature in another two-state transition to a state, D-D, in which both tethered proteins are denatured. Three-state behavior is best explained by excluded volume effects in the denatured state. For many dimers, linkers longer than 17 A removed most three-state character. This sets a limit on the flexibility and size of the denatured state. Notably, in contradiction to theoretical predictions, these cross-linked dimers were not stabilized. The failure of these predictions is possibly due to neglect of the alteration in hydrophobic exposure that accompanies any significant reduction in the conformational space of the denatured state.     

Non-histone chromosomal protein HMG1 reduces the histone H5-induced changes in c.d. spectra of DNA: the acidic C-terminus of HMG1 is necessary for binding to H5

Chemical cross-linking was used to study the interaction between non-histone high-mobility-group (HMG)1 and histone H5 in free solution. The presence of acidic C-terminal domain in HMG1 was shown to be a prerequisite for HMG1 binding to histone H5. The objective of this communication is to ascertain whether HMG1 could affect the conformation of DNA associated with a linker histone H5. Complexes of histone H5 with chicken erythrocyte DNA or an alternating purine-pyrimidine polynucleotide poly[d(A-T)] were prepared at different molar ratios H5/DNA. Changes in DNA conformation in the complexes with histone H5 or H5/HMG1 were monitored by circular dichroism (c.d.). Depending on the molar ratio H5/poly[d(A-T)], under conditions limiting the complex aggregation, three distinct types of c.d. spectra were observed. The addition of HMG1 to H5-DNA complexes reduced in all cases the histone H5-induced conformational changes in poly[d(A-T)]. The sensitivity of H5-poly[d(A-T)] complexes to HMG1 was inversely proportional to the amount of H5 in the complex. The effect of HMG1 was not observed upon removal of the acidic C-terminal domain of HMG1.     

Temperature dependence of the volumetric parameters of drug binding to poly[d(A-T)].Poly[d(A-T)] and Poly(dA).Poly(dT)

We report the temperature and salt dependence of the volume change (DeltaVb) associated with the binding of ethidium bromide and netropsin with poly(dA).poly(dT) and poly[d(A-T)].poly[d(A-T)]. The DeltaV(b) of binding of ethidium with poly(dA).poly(dT) was much more negative at temperatures approximately 70 degrees C than at 25 degrees C, whereas the difference is much smaller in the case of binding with poly[d(A-T)].poly[d(A-T)]. We also determined the volume change of DNA-drug interaction by comparing the volume change of melting of DNA duplex and DNA-drug complex. The DNA-drug complexes display helix-coil transition temperatures (Tm several degrees above those of the unbound polymers, e.g., the Tm of the netropsin complex with poly(dA)poly(dT) is 106 degrees C. The results for the binding of ethidium with poly[d(A-T)].poly[d(A-T)] were accurately described by scaled particle theory. However, this analysis did not yield results consistent with our data for ethidium binding with poly(dA).poly(dT). We hypothesize that heat-induced changes in conformation and hydration of this polymer are responsible for this behavior. The volumetric properties of poly(dA).poly(dT) become similar to those of poly[d(A-T)].poly[d(A-T)] at higher temperatures.