Interaction of DNA with lysine-rich polypeptides and proteins. The influence of polypeptide composition and secondary structure

Using X-ray diffraction we have studied fibres obtained from complexes of DNA with lysine-rich polypeptides and with proteins that have different conformations, to ascertain whether the conformations of the polypeptides and the DNA are maintained upon interaction. Substances investigated include N-acetyl-Lys-Ala-Tyr-Ala-Lys-ethylamide, random poly(Leu50, Lys50), sequential poly(Leu-Lys), poly(Val-Lys), poly(Ala-Lys), poly(Lys-Ala-Ala-Lys), poly(Lys-Ala-Ala), poly(Lys-Leu-Ala), poly(Lys-Ala-Gly), protein phi 0 from sea cucumber spermatozoa, histone H1 and two fragments of this protein obtained by chemical cleavage. In general, the B form of DNA with ten base-pairs per helical turn is maintained upon interaction at high levels of humidity. The A form is never observed; it appears to be forbidden in a protein environment. No evidence for transition into any novel DNA conformation has been observed, although the B form is altered in some cases, in particular upon dehydration. Such alteration occurs always in the sense of tightening the double helix, so that the number of base-pairs per helical turn diminishes. The polypeptides may interact with DNA in both the alpha and beta conformations. We have found different types of complexes in which either a monolayer or a double layer of beta-pleated sheets is intercalated between layers of DNA molecules. Alternatively, the polypeptide chain may be wrapped around the DNA, following one of the grooves. The polypeptide conformation may be either maintained or changed upon interaction. The charge density of the polypeptide is an important parameter of the interaction. When it matches the charge density of the DNA, the polypeptide conformation is maintained in most cases; otherwise it is modified. The globular part of histone H1 gives a unique X-ray pattern upon interaction, indicative of a loss of order of DNA in the complex. On the other hand, the C-terminal part of histone H1 gives a very well-ordered complex, similar to a nucleoprotamine, in spite of its lower charge density.     

Counterions which favour the C form of DNA.

Recently the relationship between the B and C forms of DNA has been questioned. We have found some amino acids and related amines which form complexes with DNA and favour the C form. We have studied such complexes by X-ray diffraction. With some of these counterions the transition between the B and C forms occurs smoothly, whereas in other cases there is a discontinuous transition. We conclude that the C form is a well-defined variant of the B form favoured by some counterions under low humidity conditions.     

DNA conformation and thermostability of aqueous solutions of beta-alanine and gamma-aminobutyric acid

It has been shown that at low concentrations of rare amino acids (from 10(-3) M to 10(-1) M of the substance) stechiometric complexes amino acid -- DNA are formed, which bring about partial substitution of counterions screening phosphate groups and to a change of spatial structure of DNA water molecules. The DNA-solvent molecular interactions are changed, accompanied by an abrupt decrease of helix-coil enthalpy transition which leads to the unwinding of DNA double helix. In the region of amino acid high concentrations (greater than 1-1,5 M) a rise of thermostability and winding of DNA double helix is observed. It has been established that B----C-like conformational transition stimulated by the rise of DNA thermostability is a result of counterions dehydration and the increase of effective ionic strength of the solution which is due to the rise of amino acid-zwitterions content in it.     

Extent of duplex DNA underwinding induced by RecA protein binding in the presence of ATP

A method is described for the accurate determination of the superhelical density (omega) of highly underwound circular DNA molecules. Using this method, duplex DNA bound by RecA protein in the presence of ATP at pH 7.5 is found to be underwound by 39.6% (omega = -0.396), corresponding to a helical periodicity of 17.4 base-pairs per turn. The underwinding is increased to 41% (17.9 base-pairs per turn) in the presence of low levels of ATP gamma S, in good agreement with the 18.6 base-pairs per turn reported previously. In spite of the extensive underwinding, the distribution of DNA topoisomers produced by RecA protein binding is small. This indicates a high degree of structural uniformity among RecA-double-stranded DNA complexes in the presence of ATP.     

Interaction of the Escherichia coli HU protein with DNA. Evidence for formation of nucleosome-like structures with altered DNA helical pitch

Nuclease digestion studies of DNA bound to the histone-like protein HU show that cuts in each strand of the DNA double helix are made with a periodicity of 8.5 base-pairs. By contrast, similar digestions of DNA in eukaryotic nucleosomes show a repeat of 10.4 base-pairs. This and other results (including circular dichroism studies) are consistent with the proposal that the pitch of the DNA double helix in the HU complex is reduced from a repeat length of 10.5 to 8.5 base-pairs per helical turn. Simultaneously, the DNA in the HU-DNA complex containing two dimers of HU per 60 base-pairs has its linking number decreased by 1.0 turn per 290 base-pairs. From these changes it is calculated that HU imposes a DNA writhe of 1.0 per three to four monomers of HU. The results suggest a model in which DNA is coiled in left-handed toroidal supercoils on the HU complex, having a stoichiometry resembling that of the half-nucleosome of eukaryotic chromatin. An important distinction is that HU complexes can restrain the same number of DNA superhelical turns as eukaryotic nucleosomes, yet the DNA retains more negative torsional tension, just as is observed in prokaryotic chromosomes in vivo. Another distinction is that HU-DNA complexes are less stable, having a dissociation half-life of 0.6 min in 50 mM-NaCl. This last property may explain prior difficulties in detecting prokaryotic nucleosome-like structures.     

Theoretical studies on the interaction of proteins and nucleic acid: II. The binding of α-helix to B-DNA

Interactions between B-DNA and homopolymeric α-helices of glycine, alanine, serine, asparagine and aspartic acid have been studied theoretically. The complexation energy has been minimised taking into account the interactions between DNA and the polypeptides as well as the internal energy of the α-helix and the interaction energy of counterions with the complex. The results obtained indicate the important role of strong hydrogen bonds between the peptide side chains and nucleic acid phosphate groups, these bonds being much stronger than specific interactions with the base-pairs. The formation of these structural bonds depends on the size of the α-helix, which in turn determines whether bridging across the major groove is possible. The steric role of the methyl group of thymine in orienting the peptide helix and the role of DNA screening cations in complex stabilization are also significant.     

Structural data suggest that the active and inactive forms of the RecA filament are not simply interconvertible.

We have used electron microscopy to examine the two major conformational states of the helical filament formed by the RecA protein of Escherichia coli. The compressed filament, formed in the absence of a nucleotide cofactor either as a self-polymer or on a single-stranded DNA molecule, is characterized in solution by about 6.1 subunits per turn of a 76 A pitch helix, and appears to be inactive with respect to all RecA activity. The active state of the filament, formed with ATP or an ATP analog on either a single or double-stranded DNA substrate, has about 6.2 subunits per turn of a 94 A pitch helix. Measurements of the contour length of RecA-covered single-stranded DNA circles in ice, formed in the absence of nucleotide cofactor, indicate that each RecA subunit binds five bases, in contrast to the three bases or base-pairs per subunit in the active state. The different stoichiometries of DNA binding suggests that the two polymeric forms are not interconvertible, as has been suggested on biochemical grounds. A three-dimensional reconstruction of the inactive state shows the same general features as the 83 A pitch filament present in the RecA crystal. This structural similarity and the fact that the crystal does not contain ATP or DNA suggests that the crystal structure is more similar to the compressed filament than the active, extended filament.     

Computer modeling demonstrates that electrostatic attraction of nucleosomal DNA is mediated by histone tails

We conducted molecular dynamics computer simulations of charged histone tail-DNA interactions in systems mimicking nucleosome core particles (NCP) . In a coarse-grained model, the NCP is modeled as a negatively charged spherical particle with flexible polycationic histone tails attached to it in a dielectric continuum with explicit mobile counterions and added salt. The size, charge, and distribution of the tails relative to the core were built to mimick real NCP. In this way, we incorporate attractive ion-ion correlation effects due to fluctuations in the ion cloud and the attractive entropic and energetic tail-bridging effects. In agreement with experimental data, increase of monovalent salt content from salt-free to physiological concentration leads to the formation of NCP aggregates; likewise, in the presence of MgCl₂, the NCPs form condensed systems via histone-tail bridging and accumulation of counterions. More detailed mechanisms of the histone tail-DNA interactions and dynamics have been obtained from all-atom molecular dynamics simulations (including water), comprising three DNA 22-mers and 14 short fragments of the H4 histone tail (amino acids 5–12) carrying three positive charges on lysine⁺ interacting with DNA. We found correlation of the DNA-DNA distance with the presence and association of the histone tail between the DNA molecules.     

Solvent-accessible surfaces of nucleic acids

Static solvent-accessible surface areas were calculated for DNA and RNA double helices of varied conformation, composition and sequence, for the single helix of poly(rC), and for a transfer RNA. The results show that for DNA and RNA double helices, two thirds of the water-accessible surface area become buried on double helix formation; phosphate oxygens retain near maximal exposure while the bases are 80% buried. Transfer RNA exposes slightly less surface per residue than does double-helical RNA, despite the presence of several additional “modified” groups, all of which are exposed significantly.When a probe corresponding to a single water molecule is used, both the total and atom type exposures are very similar for A-DNA and B-DNA, although marked differences appear in the major and minor groove exposures between the two conformations. For a given base-pair, the accessible surface area buried upon double-helical stacking is nearly constant (within 5%) for different sequences of neighboring base-pairs.For probes larger than single water molecules, there exist considerable differences in the total and atom type exposures of A-DNA and B-DNA. Conformational transitions between the A-DNA and B-DNA helical forms can thus be related to differences in the accessible areas for “structured” water, or a secondary hydration shell, rather than to interactions with individual water molecules of the primary hydration shell. The base-composition dependence of DNA helical conformation can be explained in terms of the opposing effects of thymine methyl groups of A · T base-pairs and the amino groups of G · C base-pairs upon the solvent within the grooves.The area calculations show that primarily the major groove of B-DNA and the minor groove of A-DNA have sufficient accessible surface area to be recognized by a probe size corresponding to the side-chains of amino acids.     

A heterodimerizing leucine zipper coiled coil system for examining the specificity of a position interactions: amino acids I,V, L,N, A,and K

We use a heterodimerizing leucine zipper system to examine the contribution of the interhelical a-a' interaction to dimer stability for six amino acids (A, V, L, I, K, and N). Circular dichroism (CD) spectroscopy monitored the thermal denaturation of 36 heterodimers that generate six homotypic and 30 heterotypic a-a' interactions. Isoleucine (I-I) is the most stable homotypic a-a' interaction, being 9.2 kcal/mol per dimer more stable than the A-A interaction and 4.0 kcal/mol per dimer more stable than either the L-L or V-V interaction, and 7.0 kcal/mol per dimer more stable than the N-N interaction. Only lysine was less stable than alanine. An alanine-based double-mutant thermodynamic cycle calculated coupling energies between the a and a' positions in the heterodimer. The aliphatic amino acids L, V, and I prefer to form homotypic interactions with coupling energies of -0.6 to -0.9 kcal/mol per dimer, but the heterotypic aliphatic interactions have positive coupling energies of <1.0 kcal/mol per dimer. The asparagine homotypic interaction has a coupling energy of -0.5 kcal/mol per dimer, while heterotypic interactions with the aliphatic amino acids produce coupling energies ranging from 2.6 to 4.9 kcal/mol per dimer. The homotypic K-K interaction is 2.9 kcal/mol per dimer less stable than the A-A interaction, but the coupling energy is only 0.3 kcal/mol per dimer. Heterotypic interactions with lysine and either asparagine or aliphatic amino acids produce similar coupling energies ranging from -0.2 to -0.7 kcal/mol per dimer. Thus, of the amino acids that were examined, asparagine contributes the most to dimerization specificity because of the large positive coupling energies in heterotypic interactions with the aliphatic amino acids which results in the N-N homotypic interaction.     

Mechanistic studies on the DNA linking activity of Epstein-Barr nuclear antigen 1.

The DNA replication, plasmid segregation and transactivation functions of Epstein-Barr nuclear antigen 1 (EBNA1) require the binding of EBNA1 to specific DNA recognition sites in the two non-contiguous functional elements of the Epstein-Barr virus latent origin of replication, oriP . EBNA1 molecules bound to these elements interact with each other resulting in the formation of looped individual DNA molecules and multiply linked DNA molecules. We have developed a glycerol gradient sedimentation assay suitable for quantitative analysis of the DNA linking activity of EBNA1 and used it to investigate the contribution of EBNA1 residues to the linking interaction and the mechanism of the interaction. Using overlapping internal deletion mutants, we found that two regions of EBNA1 can cause DNA linking, amino acids 40-100 and 327-377, but that the stabilities of the linked complexes formed by the two regions differ dramatically; only complexes formed through the latter region are stable to glycerol gradient sedimentation analysis. Mechanistic studies using EBNA1 in combination with GAL4-EBNA1 fusion proteins showed that linking interactions mediated by residues 327-377 are homotypic. Our results also suggest that only the DNA-bound form of EBNA1 participates in the protein-protein interactions seen in DNA linking.     

Recognition of nucleic acid bases and base-pairs by hydrogen bonding to amino acid side-chains

Sequence-specific protein-nucleic acid recognition is determined, in part, by hydrogen bonding interactions between amino acid side-chains and nucleotide bases. To examine the repertoire of possible interactions, we have calculated geometrically plausible arrangements in which amino acids hydrogen bond to unpaired bases, such as those found in RNA bulges and loops, or to the 53 possible RNA base-pairs. We find 32 possible interactions that involve two or more hydrogen bonds to the six unpaired bases (including protonated A and C), 17 of which have been observed. We find 186 "spanning" interactions to base-pairs in which the amino acid hydrogen bonds to both bases, in principle allowing particular base-pairs to be selectively targeted, and nine of these have been observed. Four calculated interactions span the Watson-Crick pairs and 15 span the G:U wobble pair, including two interesting arrangements with three hydrogen bonds to the Arg guanidinum group that have not yet been observed. The inherent donor-acceptor arrangements of the bases support many possible interactions to Asn (or Gln) and Ser (or Thr or Tyr), few interactions to Asp (or Glu) even though several already have been observed, and interactions to U (or T) only if the base is in an unpaired context, as also observed in several cases. This study highlights how complementary arrangements of donors and acceptors can contribute to base-specific recognition of RNA, predicts interactions not yet observed, and provides tools to analyze proposed contacts or design novel interactions.     

Modeling high-resolution hydration patterns in correlation with DNA sequence and conformation

Hydration around the DNA fragment d(C5T5).(A5G5) is presented from two molecular dynamics simulations of 10 and 12 ns total simulation time. The DNA has been simulated as a flexible molecule with both the CHARMM and AMBER force fields in explicit solvent including counterions and 0.8 M additional NaCl salt. From the previous analysis of the DNA structure B-DNA conformations were found with the AMBER force-field and A-DNA conformations with CHARMM parameters. High-resolution hydration patterns are compared between the two conformations and between C.G and T.A base-pairs from the homopolymeric parts of the simulated sequence. Crystallographic results from a statistical analysis of hydration sites around DNA crystal structures compare very well with the simulation results. Differences between the crystal sites and our data are explained by variations in conformation, sequence, and limitations in the resolution of water sites by crystal diffraction. Hydration layers are defined from radial distribution functions and compared with experimental results. Excellent agreement is found when the measured experimental quantities are compared with the equivalent distribution of water molecules in the first hydration shell. The number of water molecules bound to DNA was found smaller around T.A base-pairs and around A-DNA as compared to B-DNA. This is partially offset by a larger number of water molecules in hydrophobic contact with DNA around T.A base-pairs and around A-DNA. The numbers of water molecules in minor and major grooves have been correlated with helical roll, twist, and inclination angles. The data more fully explain the observed B-->A transition at low humidity.     

Visualization of drug-nucleic acid interactions at atomic resolution. III. Unifying structural concepts in understanding drug-DNA interactions and their broader implications in understanding protein-DNA interactions.

Structural information afforded by the X-ray crystallographic studies of ethidium-dinucleoside monophosphate crystalline complexes described in the preceding two papers has led to a detailed model for ethidium-DNA binding. Features of ethidium-DNA binding, in turn, have led to unifying structural concepts in understanding a wide range of drug-DNA interactions. It is possible that these concepts have still broader implications in understanding the nature of protein-DNA interactions.This paper begins by summarizing the stereochemical aspects of ethidium-DNA, actinomycin-DNA and irehdiamine-DNA binding, molecules that use intercalative and kinked-type geometries in binding to DNA. It then describes superhelical DNA structures formed by kinking DNA periodically varying numbers of base-pairs apart. κ-kinked B DNA, a structure formed by kinking DNA every ten base-pairs, is a left-handed superhelical structure that may be utilized in the organization of DNA within the nucleosome in chromatin. β-kinked B DNA is a right-handed superhelical structure formed by kinking DNA every two base-pairs. It is possible that premelting conformational changes occur in DNA which utilize elements of this structure. This would expose base-pairs to solvent denaturation, and could lower the activation energy necessary for strand separation during DNA denaturation. RNA polymerase and other DNA melting proteins could capitalize on this type of premelting conformational change when binding to DNA.The concept that conformational flexibility exists in DNA structure (and that drug intercalation is a phenomenon that reflects this flexibility) can, in addition, explain a wide variety of physicochemical data about DNA. In this paper we discuss the nature of these data in detail.     

Effects of base mismatches on the structure of the four-way DNA junction

Heteroduplex formation between imperfectly homologous DNA sequences may result in the formation of a four-way junction at which non-Watson-Crick base mismatches are present at the point of strand exchange. This raises the question of the effect of such mismatches on the structure and stability of these potential recombination intermediates. We have constructed a series of four-way DNA junctions containing single-base mismatches, and have studied the structure of the junctions by means of gel electrophoresis and chemical modification. We observed a range of effects on the structure of the junction, ranging from almost total abolition of folding through to normal accommodation into the folded structure. In some cases we observed gel electrophoretic data consistent with a dynamic equilibrium between folded and unfolded conformations, and in general the folded form was favoured at higher concentrations of cation. The effects of single mismatches on the structure of the four-way junction may be summarized in terms of: (1) the nature of the mismatch, where we note a correlation between the thermal stability of a given mismatch and its ability to be accommodated into a folded junction; or (2) the sequence context, where the effect of a given mismatch on the structure of a junction depends on the neighbouring base-pairs. These factors are illustrated by a junction, containing a C.A mismatch, that adopted alternate isomeric conformations dependent upon pH; as the state of protonation of the mispair changed, the structure was altered along with the interaction with neighbouring base-pairs. Most base mismatches may be accommodated into the folded stacked X-conformation of the four-way junction, but many require elevated cation concentration to permit the folding process to proceed. Some mismatches were found to be extremely destabilizing.     

Crystal structure and stability of a DNA duplex containing A(anti).G(syn) base-pairs

The synthetic dodecanucleotide d(CGCAAATTGGCG) has been analysed by single-crystal X-ray diffraction techniques and the structure refined to R = 0.16 and 2.25 A resolution, with the location of 94 solvent molecules. The sequence crystallizes as a full turn of a B-DNA helix with ten Watson-Crick base-pairs and two adenine-guanine mispairs. The analysis clearly shows that the mismatches are of the form A(anti).G(syn). Thermal denaturation studies indicate that the stability of the duplex is strongly pH dependent, with a maximum at pH 5.0, suggesting that the base-pair is stabilized by protonation. Three different arrangements have been observed for base-pairs between guanine and adenine and it is likely that A.G mismatch conformation is strongly influenced by dipole-dipole interactions with adjacent base-pairs.     

DNA-drug interactions. The crystal structure of d(CGATCG) complexed with daunomycin

The structure of a d(CGATCG)-daunomycin complex has been determined by single crystal X-ray diffraction techniques. Refinement, with the location of 40 solvent molecules, using data up to 1.5 A, converged with a final crystallographic residual, R = 0.25 (RW = 0.22). The tetragonal crystals are in space group P4(1)2(1)2, with cell dimensions of a = 27.98 A and c = 52.87 A. The self-complementary d(CGATCG) forms a distorted right-handed helix with a daunomycin molecule intercalated at each d(CpG) step. The daunomycin aglycon chromophore is oriented at right-angles to the long axis of the DNA base-pairs. This head-on intercalation is stabilized by direct hydrogen bonds and indirectly via solvent-mediated, hydrogen-bonding interactions between the chromophore and its intercalation site base-pairs. The cyclohexene ring and amino sugar substituent lie in the minor groove. The amino sugar N-3' forms a hydrogen bond with O-2 of the next neighbouring thymine. This electrostatic interaction helps position the sugar in a way that results in extensive van der Waals contacts between the drug and the DNA. There is no interaction between daunosamine and the DNA sugar-phosphate backbone. We present full experimental details and all relevant conformational parameters, and use the comparison with a d(CGTACG)-daunomycin complex to rationalize some neighbouring sequence effects involved in daunomycin binding.     

Polyphosphate-cation interaction in the amino acid-containing vacuole of Neurospora crassa

The vacuoles of Neurospora crassa, grown in minimal medium, contain a 1:1 ratio of basic amino acids and phosphate, the latter in the form of long-chain, inorganic polyphosphate-P. Vacuoles isolated from cells depleted of polyphosphate retain basic amino acids despite the absence of over 90% of their polyphosphate. Thus, vacuolar retention of basic amino acids is not dependent upon binding to or charge neutralization by polyphosphate. Polyphosphate was found to be the only macromolecular polyanion in vacuoles of normal or phosphate-depleted cells. Gel filtration experiments revealed that about half the polyphosphate of normal vacuoles is bound strongly by vacuolar spermidine, Mg2+, and Ca2+. The polyphosphate thus occupied was not available for basic amino acid binding. We have identified about 90% of the cations of isolated vacuoles; in addition to spermidine, Mg2+, and Ca2+, the cation pool consists mainly of arginine, ornithine, histidine, lysine, and Na+, with a small amount of K+. Isolated vacuoles appear to be almost wholly impermeable to all these ions, and in vivo, vacuoles appear to be highly selective in ion uptake by an active process. The interaction of basic amino acid with the available polyphosphate was found to reduce the chemical activity of the former. In keeping with this effect, cells with abnormally high basic amino acid-polyphosphate ratios displayed greatly swollen vacuoles, indicating considerable osmotic activity of the basic amino acids and their counterions under these conditions.     

Binding stoichiometry and structure of RecA-DNA complexes studied by flow linear dichroism and fluorescence spectroscopy. Evidence for multiple heterogeneous DNA co-ordination

The interaction between RecA and DNA (in the form of unmodified single-stranded DNA, fluorescent single-stranded DNA and double-stranded DNA) is studied with linear dichroism and fluorescence spectroscopy. RecA is found to form a complex with single-stranded DNA with a binding stoichiometry of about four nucleotides per RecA monomer, in which the DNA bases appear to have a random orientation. Addition of ATP gamma S (a non-hydrolyzable analog of ATP) reduces the stoichiometry to about three nucleotides per RecA and causes the DNA bases to adopt an orientation preferentially perpendicular to the fiber axis. This complex can incorporate an additional strand of single-stranded DNA or double-stranded DNA, yielding a total stoichiometry of six nucleotides or three nucleotides and three base-pairs, respectively, per RecA. RecA, in the presence of ATP gamma S, is also found to interact with double-stranded DNA, with a stoichiometry of about three base-pairs per RecA. In all studied complexes, the tryptophan residues in the RecA protein are oriented with their planes preferentially parallel to the fiber axis, whereas in complexes involving ATP gamma S the planes of the DNA bases are oriented preferentially perpendicular to the fiber. This virtually excludes the possibility that the tryptophan residues are intercalated in the DNA helix. On the basis of these results, a model for the research of homology in the RecA-mediated, strand-exchange reaction in the genetic recombination process is proposed.