H-NMR studies on d(GCTTAAGC)2 and its complex with berenil.

Two-dimensional (2D) 1H-NMR spectroscopy has been used to analyze the structure of d(GCTTAAGC)2 and its interaction with berenil in solution. Nuclear Overhauser enhancement connectivities enabled sequential assignments of nearly all proton resonances in the self-complementary octamer duplex and demonstrated that the oligonucleotide is primarily in a B-type conformation. No major conformational changes were observed by the addition of berenil, but proton resonances of the two adenosine nucleotides shifted substantially. Intermolecular nuclear Overhauser effects between berenil and the DNA duplex revealed that the drug binds via the minor groove of d(GCTTAAGC)2 in the A.T-base-pair region. At 18 degrees C the twofold symmetry of the duplex is preserved on berenil binding. However, strongly shifted proton resonances broadened significantly. A model is proposed for the berenil-d(GCTTAAGC)2 complex involving fast exchange of berenil between two equivalent symmetry-related binding sites, which span the 5'-TAA-3' region and are asymmetrically disposed with respect to the dyad axis of the duplex. These results are compared with previous studies on the berenil-d(GCAATTGC)2 complex.     

Dispersion of meso-tetrakis(N-methylpyridinium-4-yl)porphyrin on [d(A-T)n]2 oligonucleotides

The binding modes of the free-base meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (TMPyP) complexed with [d(AT)n]2 oligonucleotides (where n=3-8, corresponding to 6 to 16 AT base pairs) were studied by circular dichroism (CD). When associated with the shortest oligonucleotide, ([d(AT)3]2), a bisignate CD spectrum was produced in the Soret absorption region at the mixing ratio between 2.0 and 0.25, corresponding to one TMPyP per 0.5 to 4 oligonucleotides. Apparent bisignate CD was attributed to a stacked TMPyP along the DNA. On the other hand, when the oligonucleotide length reaches one helical turn or longer, ([d(AT)n]2, n=6,7,8), TMPyP exhibited a positive CD signal, that corresponds to the monomeric groove binding mode, at the mixing ratio below 1.0 (one TMPyP per oligonucleotide). As the mixing ratio increases, the CD signal was best accounted for by the sum of the stacked and groove-binding TMPyP. At the intermediate oligonucleotide length ([d(AT)n]2, n=4,5), the CD spectrum appeared to be the sum of the stacked and groove binding TMPyP at all mixing ratios. Therefore, it is conclusive that the full dispersion of TMPyP requires at least one helical turn of the AT sequence at a mixing ratio below 1.0. Further increase of the mixing ratio resulted in the stacking of TMPyP even at the long oligonucleotides.     

Binding, electrochemical activation, and cleavage of DNA by cobalt(II) tetrakis-N-methylpyridyl porphyrin and its beta-pyrrole brominated derivative

The binding of nucleic acids by water-soluble cobalt(II) tetrakis-N-methylpyridyl porphyrin, (TMPyP)Co, and its highly electron-deficient derivative cobalt(II) tetrakis-N-methyl pyridyl-beta-octabromoporphyrin, (Br(8)TMPyP)Co, was investigated by UV-visible absorption, circular dichroism (CD), and electrochemical and gel electrophoresis methods. The changes of the absorption spectra during the titration of these complexes with polynucleotides revealed a shift in the absorption maxima and a hypochromicity of the porphyrin Soret bands. The intrinsic binding constants were found to be in the range of 10(5)-10(6) M(-1). These values were higher for the more electron-deficient (Br(8)TMPyP)Co. Induced CD bands were noticed in the Soret region of the complexes due to the interaction of these complexes with different polynucleotides, and an analysis of the CD spectra supported a mainly external mode of binding. Electrochemical studies revealed the cleavage of polynucleotides by (TMPyP)Co and (Br(8)TMPyP)Co in the presence of oxygen preferentially at the A-T base pair region. Gel electrophoresis experiments further supported the cleavage of nucleic acids. The results indicate that the beta-pyrrole brominated porphyrin, (Br(8)TMPyP)Co, binds strongly and cleaves nucleic acids efficiently as compared with (TMPyP)Co. This electrolytic procedure offers a unique tool in biotechnology for cleaving double-stranded DNA with specificity at the A-T regions.     

Quantitative structure of a complex between a minor-groove-specific drug and a bent DNA decamer duplex: use of 2D NMR data and NOESY constrained energy minimization

Two-dimensional nuclear magnetic resonance (2D NMR) studies on d(GA4T4C)2 and d(GT4A4C)2 [Sarma, M.H., et al. (1988) Biochemistry 27, 3423-3432; Gupta G., et al. (1988) Biochemistry 27, 7909-7919] showed that A.T pairs are propeller twisted. As a result, A/T tracts form a straight rigid structural block with an array of bifurcated inter base pair H bonds in the major groove. It was demonstrated (previous paper) that replacement of methyl group by hydrogen (changing from T to U) in the major groove does not disrupt the array of bifurcated H bonds in the major groove. In this article, we summarize results of 2D NMR and molecular mechanic studies on the effect of a minor-groove-binding A.T-specific drug on the structure d(GA4T4C)2. A distamycin analogue (Dst2) was used for this study. It is shown that Dst2 binds to the minor groove of d(GA4T4C)2 mainly driven by van der Waals interaction between A.T pairs and the drug; as a consequence, an array of bifurcated H bonds can be formed in the minor groove between amide/amino protons of Dst2 and A.T pairs of DNA. NOESY data suggest that Dst2 predominantly binds at the central 5 A.T pairs. NOESY data also reveal that, upon drug binding, d(GA4T4C)2 does not undergo any significant change in conformation from the free state; i.e., propeller-twisted A.T pairs are still present in DNA and hence the array of bifurcated H bonds must be preserved in the major groove. NOESY data for the A5-T6 sequence also indicate that there is little change in junction stereochemistry upon drug binding.     

DNase I - DNA interaction alters DNA and protein conformations.

Human DNase I is an endonuclease that catalyzes the hydrolysis of double-stranded DNA predominantly by a single-stranded nicking mechanism under physiological conditions in the presence of divalent Mg and Ca cations. It binds to the minor groove and the backbone phosphate group and has no contact with the major groove of the right-handed DNA duplex. The aim of this study was to examine the effects of DNase I - DNA complexation on DNA and protein conformations.We monitored the interaction of DNA with DNase I under physiological conditions in the absence of Mg2+, with a constant DNA concentration (12.5 mmol/L; phosphate) and various protein concentrations (10-250 micromol/L). We used Fourier transfrom infrared, UV-visible, and circular dichroism spectroscopic methods to determine the protein binding mode, binding constant, and effects of polynucleotide-enzyme interactions on both DNA and protein conformations. Structural analyses showed major DNase-PO2 binding and minor groove interaction, with an overall binding constant, K, of 5.7 x 10(5) +/- 0.78 x 10(5) (mol/L)-1. We found that the DNase I - DNA interaction altered protein secondary structure, with a major reduction in alpha helix and an increase in beta sheet and random structures, and that a partial B-to-A DNA conformational change occurred. No DNA digestion was observed upon protein-DNA complexation.     

Binding of meso-tetrakis(N-methylpyridinium-4-yl)porphyrin to AT oligomers: effect of chain length and the location of the porphyrin stacking

Circular dichroism (CD) spectra of meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (TMPyP) that are associated with various duplex and triplex AT oligomers were investigated in this study. A strong positive CD was apparent for both the TMPyP complexed with duplex d[(A-T)(12)](2), d(A)(12).d(T)(12) and triplex d(A)(12).d[(T)(12)](2) at a low mixing ratio. As the mixing ratio increased, bisignate excitonic CD was produced for TMPyP complexed with duplexes, whereas the positive CD signal remained the same for the TMPyP-d(A)(12).d[(T)(12)](2) complex. This difference in the CD spectrum in the presence of duplex and triplex oligomers indicates that the moderate stacking of TMPyP occurs at the major groove of the duplex and the monomeric binding occurs in (or near) the minor groove. When TMPyP forms a complex with duplex d[(A-T)(6)](2) only excitonic CD was observed, even at a very low mixing ratio. Therefore, at least seven or more basepairs are required for TMPyP to exhibit a monomeric CD spectrum. After close analysis of the CD spectrum, the TMPyP-poly[d(A-T)(2)] complex could be explained by a combination of the CD spectrum of the monomeric, moderately stacked, and extensively stacked TMPyP.     

5,10,15,20-Tetra-(N-methyl-3-pyridyl)porphyrin destabilizes the anti-parallel telomeric quadruplex d(TTAGGG)4

We studied the parameters of binding of 5,10,15,20-tetra-(N-methyl-3-pyridyl)porphyrin (TMPyP3) to the anti-parallel human telomeric G-quadruplex d(TTAGGG)4, the oligonucleotide dTTAGGGTTAGAG(TTAGGG)2 that does not form a quadruplex structure, as well as to the double stranded d(AC)8 x d(GT) and single stranded d(AC)8 and d(GT)8 DNAs. The analysis of absorption revealed that the binding constants and the number of DNA binding sites for TMPyP3 were d(AC)8 < d(GT)8 < d(AC)8 x d(GT)8 = d(TTAGGG)4 < dTTAGGGTTAGAG(TTAGGG)2. We demonstrated for the first time that the binding constant of TMPyP3 with the non-quadruplex chain dTTAGGGTTAGAG(TTAGGG)2 (1.3 x 10(7) M(-1)) is approximately 3 times bigger than the binding constant with the quadruplex d(TTAGGG)4 (4.6 x 10(6) M(-1)). Binding of two TMPyP3 molecules to d(TTAGGG)4 led to a decrease of thermostability of the G-quadruplex (deltaT(m) = -8 degrees C). Circular dichroism spectra of TMPyP3:d(TTAGGG)4 complexes revealed a shift of DNA structure from the G-quadruplex to an irregular chain. We hypothesize that partial destabilization of the telomeric G-quadruplex by TMPyP3 might be a reason for relatively low potency of this ligand as a telomerase inhibitor, as well as its marginal cytotoxicity for cultured tumor cells.     

Inhibition of DNA binding by human estrogen-related receptor 2 and estrogen receptor alpha with minor groove binding polyamides

Human estrogen-related receptor 2 (hERR2, ESRRB, ERRbeta, NR3B2) belongs to a class of nuclear receptors that bind DNA through sequence-specific interactions with a 5'-AGGTCA-3' estrogen response element (ERE) half-site in the major groove and an upstream 5'-TNA-3' site in the minor groove. This minor groove interaction is mediated by a C-terminal extension (CTE) of the DNA binding domain and is unique to the estrogen-related receptors. We have used synthetic pyrrole-imidazole polyamides, which bind specific sequences in the minor groove, to demonstrate that DNA binding by hERR2 is sensitive to the presence of polyamides in both the upstream minor groove CTE site and the minor groove of the ERE half-site. Thus, polyamides can inhibit hERR2 by two mechanisms, by direct steric blockage of minor groove DNA contacts mediated by the CTE and by changing the helical geometry of DNA such that major groove interactions are weakened. To confirm the generality of the latter approach, we show that the dimeric human estrogen receptor alpha (hERalpha, ESR1, NR3A1), which binds in the major groove of the ERE, can be inhibited by a polyamide bound in the opposing minor groove of the ERE. These results highlight two mechanisms for inhibition of protein-DNA interactions and extend the repertoire of DNA recognition motifs that can be inhibited by polyamides. These molecules may thus be useful for controlling expression of hERR2- or hERalpha-responsive genes.     

Interaction of pyrrolobenzodiazepine (PBD) ligands with parallel intermolecular G-quadruplex complex using spectroscopy and ESI-MS

Studies on ligand interaction with quadruplex DNA, and their role in stabilizing the complex at concentration prevailing under physiological condition, has attained high interest. Electrospray ionization mass spectrometry (ESI-MS) and spectroscopic studies in solution were used to evaluate the interaction of PBD and TMPyP4 ligands, stoichiometry and selectivity to G-quadruplex DNA. Two synthetic ligands from PBD family, namely pyrene-linked pyrrolo[2,1-c][1,4]benzodiazepine hybrid (PBD1), mixed imine-amide pyrrolobenzodiazepine dimer (PBD2) and 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4) were studied. G-rich single-stranded oligonucleotide d(5'GGGGTTGGGG3') designated as d(T(2)G(8)), from the telomeric region of Tetrahymena Glaucoma, was considered for the interaction with ligands. ESI-MS and spectroscopic methods viz., circular dichroism (CD), UV-Visible, and fluorescence were employed to investigate the G-quadruplex structures formed by d(T(2)G(8)) sequence and its interaction with PBD and TMPyP4 ligands. From ESI-MS spectra, it is evident that the majority of quadruplexes exist as d(T(2)G(8))(2) and d(T(2)G(8))(4) forms possessing two to ten cations in the centre, thereby stabilizing the complex. CD band of PBD1 and PBD2 showed hypo and hyperchromicity, on interaction with quadruplex DNA, indicating unfolding and stabilization of quadruplex DNA complex, respectively. UV-Visible and fluorescence experiments suggest that PBD1 bind externally where as PBD2 intercalate moderately and bind externally to G-quadruplex DNA. Further, melting experiments using SYBR Green indicate that PBD1 unfolds and PBD2 stabilizes the G-quadruplex complex. ITC experiments using d(T(2)G(8)) quadruplex with PBD ligands reveal that PBD1 and PBD2 prefer external/loop binding and external/intercalative binding to quadruplex DNA, respectively. From experimental results it is clear that the interaction of PBD2 and TMPyP4 impart higher stability to the quadruplex complex.     

Bcl-2 Promoter Sequence G-Quadruplex Interactions with Three Planar and Non-Planar Cationic Porphyrins: TMPyP4, TMPyP3, and TMPyP2

The interactions of three related cationic porphyrins, TMPyP4, TMPyP3 and TMPyP2, with a WT 39-mer Bcl-2 promoter sequence G-quadruplex were studied using Circular Dichroism, ESI mass spectrometry, Isothermal Titration Calorimetry, and Fluorescence spectroscopy. The planar cationic porphyrin TMPyP4 (5, 10, 15, 20-meso-tetra (N-methyl-4-pyridyl) porphine) is shown to bind to a WT Bcl-2 G-quadruplex via two different binding modes, an end binding mode and a weaker mode attributed to intercalation. The related non-planar ligands, TMPyP3 and TMPyP2, are shown to bind to the Bcl-2 G-quadruplex by a single mode. ESI mass spectrometry experiments confirmed that the saturation stoichiometry is 4:1 for the TMPyP4 complex and 2:1 for the TMPyP2 and TMPyP3 complexes. ITC experiments determined that the equilibrium constant for formation of the (TMPyP4)₁/DNA complex (K₁ = 3.7 × 10⁶) is approximately two orders of magnitude greater than the equilibrium constant for the formation of the (TMPyP2)₁/DNA complex, (K₁ = 7.0 × 10⁴). Porphyrin fluorescence is consistent with intercalation in the case of the (TMPyP4)₃/DNA and (TMPyP4)₄/DNA complexes. The non-planar shape of the TMPyP2 and TMPyP3 molecules results in both a reduced affinity for the end binding interaction and the elimination of the intercalation binding mode.     

Mechanism of damage recognition by Escherichia coli DNA photolyase

Escherichia coli DNA photolyase binds to DNA containing pyrimidine dimers with high affinity and then breaks the cyclobutane ring joining the two pyrimidines of the dimer in a light- (300-500 nm) dependent reaction. In order to determine the structural features important for this level of specificity, we have constructed a 43 base pair (bp) long DNA substrate that contains a thymine dimer at a unique location and studied its interaction with photolyase. We find that the enzyme protects a 12-16-bp region around the dimer from DNase I digestion and only a 6-bp region from methidium propyl-EDTA-Fe (II) digestion. Chemical footprinting experiments reveal that photolyase contacts the phosphodiester bond immediately 5' and the 3 phosphodiester bonds immediately 3' to the dimer but not the phosphodiester bond between the two thymines that make up the dimer. Methylation protection and interference experiments indicate that the enzyme makes major groove contacts with the first base 5' and the second base 3' to the dimer. These data are consistent with photolyase binding in the major groove over a 4-6-bp region. However, major groove contacts cannot be of major significance in substrate recognition as the enzyme binds equally well to a thymine dimer in a 44-base long single strand DNA and protects a 10-nucleotide long region around the dimer from DNase I digestion. It is therefore concluded that the unique configuration of the phosphodiester backbone in the strand containing the pyrimidine dimer, as well as the cyclobutane ring of the dimer itself are the important structural determinants of the substrate for recognition by photolyase.     

Cationic porphyrins bearing diazolium rings: synthesis and their interaction with calf thymus DNA

Two novel cationic porphyrins bearing five-membered rings at the meso-positions, meso-tetrakis(1,3-dimethylimidazolium-2-yl)porphyrin (H2TDMImP) and meso-tetrakis(1,2-dimethylpyrazolium-4-yl)porphyrin (H2TDMPzP), have been synthesized. These two compounds interact with calf thymus DNA (CTDNA) in different binding modes from that of mesotetrakis(N-methylpyridinium-4-yl)porphyrin (H2TMPyP). H2TDMImP outside binds to the minor groove of CTDNA while H2TDMPzP intercalates into CTDNA. These two novel cationic porphyrins strongly bind to CTDNA even at high ionic strength and the binding constant of H2TDMPzP to CTDNA is comparable to that of H2TMPyP. The binding of H2TDMImP to CTDNA is enthalpically driven. The favorable free energy changes in binding of H2TDMPzP to CTDNA come from the large negative enthalpy changes accompanied by small positive entropy changes.     

Use of monoacetyl-4-hydroxyaminoquinoline 1-oxide to probe contacts between guanines and protein in the minor and major grooves of DNA. Interaction of Escherichia coli integration host factor with its recognition site in the early promoter and transposition enhancer of bacteriophage Mu.

Monoacetyl-4-hydroxyaminoquinoline 1-oxide (Ac-HAQO) reacts with DNA to form adducts at the C8- and N2-positions of guanine and with the N6-position of adenine. Only the N2-guanine adduct blocks the 3'-5' exonuclease action of phage T4 DNA polymerase. Piperidine treatment cleaves the DNA at sites bearing C8-guanine adducts. The N2-position of guanine lies in the minor groove of DNA, whereas the C8-position of guanine occupies the major groove. We have taken advantage of these characteristics to employ Ac-HAQO in conjunction with either T4 DNA polymerase or piperidine in a footprinting technique to probe the interaction of the Escherichia coli integration host factor (IHF) with its binding site. We show that when IHF binds to its recognition site both the N2- and C8-positions of guanines are protected from modification by AcHAQO. In addition, the binding of IHF to DNA was prevented when either an N2- or a C8-AQO adduct was present in the binding site. When dimethylsulfate was used as the footprinting reagent, IHF protected against methylation of the N3 position of adenine in the minor groove but not the N7 position of guanine in the major groove. The difference in results obtained with the two reagents is ascribed to their relative sizes. Both DMS and AcHAQO are excluded by IHF from the minor groove, but only the larger AcHAQO molecule is excluded from the major groove.     

Specific interaction between the initiator protein (Rep) and origin of plasmid ColE2-P9

The replication initiator protein (Rep) of plasmid ColE2-P9 (ColE2) is multifunctional. We are interested in how Rep binds to the origin (Ori) to perform various functions. We used the wild type and variants of Rep to study the Rep-Ori interaction by both in vitro and in vivo approaches, including biochemical analyses of protein-DNA interactions and an in vivo replication assay. We identified three regions (I, II, and III) of Rep, located in the C-terminal half, and three corresponding binding sites (I, II, and III) in Ori which are important for Rep-Ori interaction. We showed that region I, containing a putative helix-turn-helix motif, is necessary and sufficient for specific Ori recognition, interacting with site I of the origin DNA from the major groove. Region II interacts with site II of the origin DNA, from the adjacent minor groove in the left half of Ori, and region III interacts with site III, next to the template sequence for primer synthesis, which is one and one-half turn apart from site I on the opposite surface of the origin DNA. A putative linker region located between the two DNA binding domains (regions II and III) was identified, which might provide Rep an extended conformation suitable for binding to the two separate sites in Ori. Based on the results presented in this paper, we propose a model for Rep-Ori interaction in which Rep binds to Ori as a monomer.     

Vibrational and electronic circular dichroism study of the interactions of cationic porphyrins with (dG-dC)10 and (dA-dT)10

The interactions of two different porphyrins, without axial ligands-5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin-Cu(II) tetrachloride (Cu(II)TMPyP) and with bulky meso substituents-5,10,15,20-tetrakis(N,N,N-trimethylanilinium-4-yl)porphyrin tetrachloride (TMAP), with (dG-dC)10 and (dA-dT)10 were studied by combination of vibrational circular dichroism (VCD) and electronic circular dichroism (ECD) spectroscopy at different [oligonucleotide]/[porphyrin] ratios, where [oligonucleotide] and [porphyrin] are the concentrations of oligonucleotide per base-pair and porphyrin, respectively. The combination of VCD and ECD spectroscopy enables us to identify the types of interactions, and to specify the sites of interactions: The intercalative binding mode of Cu(II)TMPyP with (dG-dC)(10), which has been well described, was characterized by a new VCD "marker" and it was shown that the interaction of Cu(II)TMPyP with (dA-dT)10 via external binding to the phosphate backbone and major groove binding caused transition from the B to the non-B conformer. TMAP interacted with the major groove of (dG-dC)10, was semi-intercalated into (dA-dT)10, and caused significant variation in the structure of both oligonucleotides at the higher concentration of porphyrin. The spectroscopic techniques used in this study revealed that porphyrin binding with AT sequences caused substantial variation of the DNA structure. It was shown that VCD spectroscopy is an effective tool for the conformational studies of nucleic acid-porphyrin complexes in solution.     

Probing site specificity of DNA binding metallointercalators by NMR spectroscopy and molecular modeling

The molecular recognition of oligonucleotides by chiral ruthenium complexes has been probed by NMR spectroscopy using the template Delta-cis-alpha- and Delta-cis-beta-[Ru(RR-picchxnMe(2)) (bidentate)](2+), where the bidentate ligand is one of phen (1,10-phenanthroline), dpq (dipyrido[3,2-f:2',3'-h]quinoxaline), or phi (9,10-phenanthrenequinone diimine) and picchxnMe(2)() is N,N'-dimethyl-N,N'-di(2-picolyl)-1,2-diaminocyclohexane. By varying only the bidentate ligand in a series of complexes, it was shown that the bidentate alone can alter binding modes. DNA binding studies of the Delta-cis-alpha-[Ru(RR-picchxnMe(2))(phen)](2+) complex indicate fast exchange kinetics on the chemical shift time scale and a "partial intercalation" mode of binding. This complex binds to [d(CGCGATCGCG)](2) and [d(ATATCGATAT)](2) at AT, TA, and GA sites from the minor groove, as well as to the ends of the oligonucleotide at low temperature. Studies of the Delta-cis-beta-[Ru(RR-picchxnMe(2))(phen)](2+) complex with [d(CGCGATCGCG)](2) showed that the complex binds only weakly to the ends of the oligonucleotide. The interaction of Delta-cis-alpha-[Ru(RR-picchxnMe(2))(dpq)](2+) with [d(CGCGATCGCG)](2) showed intermediate exchange kinetics and evidence of minor groove intercalation at the GA base step. In contrast to the phen and dpq complexes, Delta-cis-alpha- and Delta-cis-beta-[Ru(RR-picchxnMe(2))(phi)](2+) showed evidence of major groove binding independent of the metal ion configuration. DNA stabilization induced by complex binding to [d(CGCGATCGCG)](2) (measured as DeltaT(m)) increases in the order phen < dpq and DNA affinity in the order phen < dpq < phi. The groove binding preferences exhibited by the different bidentate ligands is explained with the aid of molecular modeling experiments.     

DNase I-tRNA interaction

Deoxyribonuclease I (DNase I) binds right-handed DNA duplex via a minor groove and the backbone phosphate group with no contact to the major groove. It hydrolyses double-stranded DNA predominantly by a single-stranded nicking mechanism under physiological conditions, in the presence of divalent Mg and Ca cations. Even though DNase-RNA interaction was observed, less is known about the protein-RNA binding mode and the effect of such complexation on both protein and RNA conformations. The aim of this study was to examine the effects of DNase I-tRNA interaction on tRNA and protein conformations. The interaction of DNase I with tRNA is monitored under physiological conditions, in the absence of Mg2+, using constant DNA concentration of 12.5 mM (phosphate) and various protein contents (10 microM to 250 microM). FTIR, UV-visible, and CD spectroscopic methods were used to analyze the protein binding mode, the binding constant, and the effects of polynucleotide-enzyme interaction on both tRNA and protein conformations. Spectroscopic evidence showed major DNase-PO2 and minor groove interactions with overall binding constant of K = 2.1 (+/-0.7) x 10(4) M(-1). The DNase I-tRNA interaction alters protein secondary structure with major reduction of the alpha-helix, and increases the random coil, beta-anti and turn structures, while tRNA remains in the A-conformation. No digestion of tRNA by DNase I was observed in the protein-tRNA complexes.     

Histone h1(0) and its carboxyl-terminal domain bind in the major groove of DNA

The binding of histone H1(0) to T4 bacteriophage DNA was investigated using thermal denaturation of the DNA titrated with varying concentrations of protein. The H1(0) used was expressed in and purified from a strain of E. coli and is therefore homogeneous with respect to H1 subtype and posttranslational modifications. Two types of T4 DNA were used: wild-type, which contains a modification of the cytosine residues that projects into the major groove: and a mutant type, which lacks the modification of the cytosines. Data were compared to simulated thermal denaturation curves to determine estimates for binding affinity and binding site size in base pairs of the protein. Analysis of the data yielded values of 10(8) M(-1) for K, the binding affinity, and 10 base pairs for n, the number of base pairs covered by one protein, for the mutant T4 DNA. Analysis of the wild-type DNA data suggested that the glucose projecting into the major groove of this DNA decreases the number of sites to which the H1(0) protein can bind, indicating that there are interactions between the protein and the major groove of DNA. The binding site size on this DNA is 10 base pairs, the same as on the unmodified DNA. The affinity for wild-type DNA is slightly higher, 10(9) M(-1). Data were collected and analyzed for binding of two domains of the protein as well, the carboxyl-terminal domain and the central globular domain. Binding of the carboxyl-terminal domain was quantitatively and qualitatively similar to that of the full-length protein. In contrast, binding of the globular domain was quite different: it binds much more weakly, with a K of 6 x 10(4) M(-1), and covers fewer base pairs, with an n of 3. Also, there was no evidence that the globular domain interacts with the major groove of DNA.     

Structure-specific binding of [Co(phen)2(HPIP)] to a DNA duplex containing sheared G:A mismatch base pairs

The binding of a Co(III) complex to the decanucleotide d(CCGAATGAGG)₂ containing two pairs of G:A mismatches was studied by 2D-NMR, UV absorption, and molecular modeling. NMR investigations indicate that racemic [Co(phen)₂(HPIP)]Cl₃ [HPIP = 2-(2-hydroxyphenyl) imidazo [4,5-f][1,10] phenanthroline] binds the decanucleotide by intercalation: the HPIP ligand selectively inserts between the stacked bases from the minor groove at the terminal regions and from the major groove at the sheared region. Further, molecular modeling revealed that the recognition shows strong enantioselectivity: the Λ-isomer preferentially intercalates into the T₆G₇:A₅A₄ region from the DNA major groove, while Δ-isomer favors the terminal C₁C₂:G₁₀G₉ region and intercalates from the minor groove. Detailed energy analysis suggests that the steric interaction, especially the electrostatic effect, is the primary determinants of the recognition event. Melting experiments indicate that binding stabilizes the DNA duplex and increases the melting temperature by 9.5 °C. The intrinsic binding constant of the complex to the mismatched duplex was determined to be 3.5 × 105 M⁻¹.     

Quantitative footprinting analysis of the chromomycin A3--DNA interaction.

Chromomycin A3 (CHR) binding to the duplex d(CAAGTCTGGCCATCAGTC).d(GACTGATGGCCAGACTTG) has been studied using quantitative footprinting methods. Previous NMR studies indicated CHR binds as a dimer in the minor groove. Analysis of autoradiographic spot intensities derived from DNase I cleavage of the 18-mer in the presence of various amounts of CHR revealed that the drug binds as a dimer to the sequence 5'-TGGCCA-3',3'-ACCGGT-5' in the 18-mer with a binding constant of (2.7 +/- 1.4) x 10(7) M-1. Footprinting and fluorescence data indicate that the dimerization constant for the drug in solution is approximately 10(5) M-1. Since it has been suggested that CHR binding alters DNA to the A configuration, quantitative footprinting studies using dimethyl sulfate, which alkylates at N-7 of guanine in the major groove, were also carried out. Apparently, any drug-induced alteration in DNA structure does not affect cleavage by DMS enough to be observed by these experiments.