Localization of chromatin proteins within DNA grooves by methylation of chromatin with dimethyl sulphate

The use of the comparative modification with ³H-dimethyl sulphate (DMS) of free DNA and DNA in different complexes is proposed to evaluate the shielding of the minor and major grooves of the DNA double helix and to determine the presence of single-stranded DNA in the complexes.Glucosyl groups in DNA of T6 phage protect, as expected, the major groove, and actinomycin d in its complex with DNA shields the minor groove against methylation with DMS.The data obtained suggest that histones and protamine in reconstituted nucleohistone and nucleoprotamine are allocated within partly the major groove leaving the minor groove open, while polylysine does not seem to be buried within either of the grooves, and cations of cetyltrimethylammonium lie within the minor groove of DNA.     

DNA determinants important in sequence recognition by Eco RI endonuclease

Alkylation interference and protection methods (Siebenlist, U., and Gilbert, W., (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 122-126) have been utilized to deduce potential DNA contacts involved in specific complex formation between Eco RI endonuclease and its recognition sequence. The endonuclease protected the N7 position (major groove) of the dG and the N3 position (minor groove) of both dA residues within the Eco RI sequence against alkylation by dimethylsulfate, d(GpApApTpTpC), suggesting the presence of poly-peptide in both grooves in the vicinity of affected nitrogens. Results of methylation interference analysis suggest that the N7 of the Eco RI site dG and the N3 of the central dA, d(GpApApTpTpC), are utilized as contacts by the enzyme. The failure to observe interference upon methylation of the 5'-penultimate dA within the sequence implies that the endonuclease does not bond to the N3 of this residue, despite the fact that it is protected against alkylation by the protein. Ethylation interference patterns suggest four major phosphate contacts between endonuclease and each DNA strand. Two of these phosphates are 5'-external to the Eco RI sequence, d(pNpGpApApTpTpC), suggesting involvement of outside phosphates in electrostatic interactions. Moreover, alkylation protection and interference effects on the two DNA strands display perfect 2-fold symmetry. Thus, the endonuclease interacts with a minimum of 10 nucleotide pairs to yield a DNA-protein complex characterized by elements of symmetry. In contrast, specific alkylation effects were not observed in comparable experiments with the endonuclease and a DNA which had been previously methylated by the Eco RI modification enzyme.     

Molecular mechanics studies of parallel and antiparallel phosphate-methylated DNA

Methylation of phosphate groups in oligo-dT strands leads to a parallel duplex with T · T base pairs. Molecular mechanics calculations on parallel d(TTTTTT)₂ show it to be a symmetric right-handed helix with B-DNA conformational characteristics. Phosphate methylation stabilizes the duplex by ca. 41 kcal/mol, due to removal of the interstrand phosphate electrostatic repulsions. The chirality introduced with phosphate methylation is important for the molecular geometry, since R_P methylation predominantly influences the conformation around the ζ bond (P? O_3′), while S_P methylation mostly changes the α conformation (P? O_5′). This is also true in antiparallel helices with methylated phosphates, as is shown by molecular mechanics calculations on d(GCGCGC)₂. These results may be of relevance to protein–DNA interactions, where phosphate charges are also shielded. As the pro-S_P oxygen is most available in a right-handed helix, we suggest changes around the α bond to occur upon protein complexation, leading to a widening of the major groove in the d(GCGCGC)₂ duplex (from 12 to 13 Å) and reduced minor groove (from 6 to 5 Å).     

DNA recognition by DNase I

Bovine pancreatic DNase I shows a strong preference for double-stranded substrates and cleaves DNA with strongly varying cutting rates suggesting that the enzyme recognises sequence-dependent structural variations of the DNA double helix. The complicated cleavage pattern indicates that several local as well as global helix parameters influences the cutting frequency of DNase I at a given bond. The high resolution crystal structures of two DNase I-DNA complexes showed that the enzyme binds tightly in the minor groove, and to the sugar-phosphate backbones of both strands, and thereby induces a widening of the minor groove and a bending towards the major grooves. In agreement with biochemical data this suggests that flexibility and minor groove geometry are major parameters determining the cutting rate of DNase I. Experimental observations showing that the sequence environmental of a dinucleotide step strongly affects its cleavage efficiency can be rationalized by that fact that six base pair are in contact with the enzyme. Mutational analysis based on the structural results has identified critical residues for DNA binding and cleavage and has lead to a proposal for the catalytic mechanism.     

Elucidating a key intermediate in homologous DNA strand exchange: structural characterization of the RecA-triple-stranded DNA complex using fluorescence resonance energy transfer

The RecA protein of Escherichia coli plays essential roles in homologous recombination and restarting stalled DNA replication forks. In vitro, the protein mediates DNA strand exchange between single-stranded (ssDNA) and homologous double-stranded DNA (dsDNA) molecules that serves as a model system for the in vivo processes. To date, no high-resolution structure of the key intermediate, comprised of three DNA strands simultaneously bound to a RecA filament (RecA-tsDNA complex), has been reported. We present a systematic characterization of the helical geometries of the three DNA strands of the RecA-tsDNA complex using fluorescence resonance energy transfer (FRET) under physiologically relevant solution conditions. FRET donor and acceptor dyes were used to label different DNA strands, and the interfluorophore distances were inferred from energy transfer efficiencies measured as a function of the base-pair separation between the two dyes. The energy transfer efficiencies were first measured on a control RecA-dsDNA complex, and the calculated helical parameters (h approximately 5 A, Omega(h) approximately 20 degrees ) were consistent with structural conclusions derived from electron microscopy (EM) and other classic biochemical methods. Measurements of the helical parameters for the RecA-tsDNA complex revealed that all three DNA strands adopt extended and unwound conformations similar to those of RecA-bound dsDNA. The structural data are consistent with the hypothesis that this complex is a late, post-strand-exchange intermediate with the outgoing strand shifted by about three base-pairs with respect to its registry with the incoming and complementary strands. Furthermore, the bases of the incoming and complementary strands are displaced away from the helix axis toward the minor groove of the heteroduplex, and the bases of the outgoing strand lie in the major groove of the heteroduplex. We present a model for the strand exchange intermediate in which homologous contacts preceding strand exchange arise in the minor groove of the substrate dsDNA.     

Geometry of the DNA strands within the RecA nucleofilament: role in homologous recombination

Homologous recombination consists of exchanging DNA strands of identical or almost identical sequence. This process is important for both DNA repair and DNA segregation. In prokaryotes, it involves the formation of long helical filaments of the RecA protein on DNA. These filaments incorporate double-stranded DNA from the cell's genetic material, recognize sequence homology and promote strand exchange between the two DNA segments. DNA processing by these nucleofilaments is characterized by large amplitude deformations of the double helix, which is stretched by 50% and unwound by 40% with respect to B-DNA. In this article, information concerning the structure and interactions of the RecA, DNA and ATP molecules involved in DNA strand exchange is gathered and analyzed to present a view of their possible arrangement within the filament, their behavior during strand exchange and during ATP hydrolysis, the mechanism of RecA-promoted DNA deformation and the role of DNA deformation in the process of homologous recombination. In particular, the unusual characteristics of DNA within the RecA filament are compared to the DNA deformations locally induced by architectural proteins which bind in the DNA minor groove. The possible role and location of two flexible loops of RecA are discussed.     

Control of DNA minor groove width and Fis protein binding by the purine 2-amino group

The width of the DNA minor groove varies with sequence and can be a major determinant of DNA shape recognition by proteins. For example, the minor groove within the center of the Fis–DNA complex narrows to about half the mean minor groove width of canonical B-form DNA to fit onto the protein surface. G/C base pairs within this segment, which is not contacted by the Fis protein, reduce binding affinities up to 2000-fold over A/T-rich sequences. We show here through multiple X-ray structures and binding properties of Fis–DNA complexes containing base analogs that the 2-amino group on guanine is the primary molecular determinant controlling minor groove widths. Molecular dynamics simulations of free-DNA targets with canonical and modified bases further demonstrate that sequence-dependent narrowing of minor groove widths is modulated almost entirely by the presence of purine 2-amino groups. We also provide evidence that protein-mediated phosphate neutralization facilitates minor groove compression and is particularly important for binding to non-optimally shaped DNA duplexes.     

Minor groove deformability of DNA: a molecular dynamics free energy simulation study

The conformational deformability of nucleic acids can influence their function and recognition by proteins. A class of DNA binding proteins including the TATA box binding protein binds to the DNA minor groove, resulting in an opening of the minor groove and DNA bending toward the major groove. Explicit solvent molecular dynamics simulations in combination with the umbrella sampling approach have been performed to investigate the molecular mechanism of DNA minor groove deformations and the indirect energetic contribution to protein binding. As a reaction coordinate, the distance between backbone segments on opposite strands was used. The resulting deformed structures showed close agreement with experimental DNA structures in complex with minor groove-binding proteins. The calculated free energy of minor groove deformation was approximately 4-6 kcal mol(-1) in the case of a central TATATA sequence. A smaller equilibrium minor groove width and more restricted minor groove mobility was found for the central AAATTT and also a significantly ( approximately 2 times) larger free energy change for opening the minor groove. The helical parameter analysis of trajectories indicates that an easier partial unstacking of a central TA versus AT basepair step is a likely reason for the larger groove flexibility of the central TATATA case.     

Preferential protection of the minor groove of non-operator DNA by lac repressor against methylation by dimethyl sulphate.

The binding of lactose repressor to non-operator DNA was studied by the modification of several DNA's, including glycosylated DNA, with dimethyl sulphate, which affects the minor and major grooves of DNA and single stranded DNA regions. The non-specific binding of the repressor to DNA protected the minor groove but apparently not the major groove of the DNA double helix against methylation and did not increase the content of single stranded DNA regions. This suggests that the repressor on binding to non-operator DNA makes contacts mainly in the minor groove of DNA and does not uncoil the DNA double helix. This is different from the interaction of the repressor with lactose operator DNA which occurs, as shown by Gilbert et al. (1), along both the major and the minor groove.     

Sites of contact between lambda operators and lambda repressor.

DNA bearing lambda operator sequences was methylated by dimethyl sulfate (DMS) in the presence or absence of lambda repressor. Under the experimental conditions, DMS methylates only the purine residues. The presence of lambda repressor affects only the methylation of certain G residues in the operators. Repressor blocks the methylation of certain G's and enhances the methylation of other G's. Since the reactive ring-nitrogen of G lies in the major groove of double-stranded DNA, and the reactive ring-nitrogen of A lies in the minor groove, the above results imply that the repressor makes contacts in the major groove of the helix. The repressor effect on G-methylation is sharply confined to the three 17 base pair units within each lambda operator previously proposed as the repressor-binding sites.     

DNA recognition by β-sheets

The modes of DNA recognition by β-sheets are analyzed by using the known crystal and solution three-dimensional structures of DNA-protein complexes. Close fitting of the protein surface and the DNA surface determines the binding geometry. Interaction takes place so that essentially the N-to-C direction of the β-strands either follows or crosses the DNA groove. Upon following the major groove a two-stranded antiparallel β-sheet dives into the groove and contacts DNA bases with its convex side facing the DNA, while upon following the minor groove, it binds around the sugar-phosphate backbones, with its opposite concave side shielding the DNA. In order for the β-strands crossing the minor groove to interact with the DNA, the dinucleotide steps need to almost totally helically untwist and roll around major groove. The β-sheet, on the other hand, needs to adopt a concave curvature on the binding surface in the direction that follows the DNA minor groove, and a convex surface in the direction that bridges the sugar-phosphate backbones across the groove. The result is to produce a hyperbolic paraboloidal DNA-binding surface. © 1998 John Wiley & Sons, Inc. Biopoly 44: 335–359, 1997     

A study of unwinding of DNA and shielding of the DNA grooves by RNA polymerase by using methylation with dimethylsulphate.

The dimethylsulphate method has been used to study the complexes of RNA polymerase (Escherichia coli) with DNA of T7 phage, poly[d(A--T)] and fragments of calf thymus DNA protected against DNase digestion by RNA polymerase. The binding of RNA polymerase to DNA significantly increases the formation of 1-methyl-adenine produced by methylation of the single-stranded DNA region, diminishes by about 10% the formation of 3-methyl-adenine by methylation within the minor groove and does not affect the formation of 7-methyl-guanine by methylation within the major DNA groove. The presence of nascent RNA decreases the formation of 1-methyl-adenine in DNA of the complex by about 30%. The initiation of RNA synthesis or RNA synthesis itself does not influence the methylation of the major groove but shielding of the minor groove increases by about twice as much. These results suggest that RNA polymerase, upon binding, breaks Watson-Crick base-pairing in a DNA region of about 15-base-pairs long, that nascent RNA forms a duplex with DNA of about 10-base-pairs long; and that the enzyme weakly interacts with DNA along its grooves and preferentially makes contacts with the minor groove.     

The potential role of DNA methylation in the response to 2,3,7,8-tetrachlorodibenzo-p-dioxin

The Ah receptor is an intracellular protein that binds the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin. The liganded receptor interacts with a specific DNA recognition motif located within a dioxin-responsive enhancer upstream of the CYP1A1 gene. Methylation protection and methylation interference studies indicate that the liganded receptor contacts both DNA strands, at 4 guanine residues contained within the recognition motif. These findings imply that the liganded receptor interacts with its cognate enhancer within the major groove of the DNA helix. Cytosine methylation of the recognition motif at CpG dinucleotides diminishes the protein-DNA interaction, as measured by gel retardation. Furthermore, methylation at cytosine inhibits the enhancer function of the DNA. These findings imply that DNA methylation can diminish the response to dioxin by impeding the Ah receptor-enhancer interaction.     

An unexpected major groove binding of netropsin and distamycin A to tRNA(phe)

Crystalline complexes of yeast tRNA(phe) and the oligopeptide antibiotics netropsin and distamycin A were prepared by diffusing drugs into crystals of tRNA. X-ray structure analyses of these complexes reveal a single common binding site for both drugs which is located in the major or deep groove of the tRNA T-stem. The netropsin-tRNA complex is stabilized by specific hydrogen bonds between the amide groups of the drug and the tRNA bases G51 O(6), U52 O(4) and G53 N(7) on one strand, and is further stabilized by electrostatic interactions between the positively charges guanidino side chain of the drug and the tRNA phosphate P53 on the same strand and the positively charged amidino propyl side chain and the phosphates P61, P62 and P63 on the opposite strand of the double helix. These results are in contrast to the implicated minor groove binding of these drugs to non-guanine sequences in DNA. The binding to the GUG sequence in tRNA implies that major groove binding to certain DNA sequences is possible.     

The double helix is dehydrated: evidence from the hydrolysis of acridinium ester-labeled probes

A highly chemiluminescent reporter molecule, acridinium ester (AE), was tethered to single-stranded oligonucleotide probes and hybridized to complementary as well as mismatched target sequences. When tethered to single-stranded probes, AE was readily hydrolyzed by water or hydroxide ion. In contrast, when hybridized to a complementary target, hydrolysis of the AE probe was markedly inhibited. Mismatches near AE eliminated the ability of the double helix to strongly inhibit AE hydrolysis. To establish the molecular basis for these remarkable hydrolysis properties of AE-labeled probes, the binding and hydrolysis mechanisms of AE-labeled probes were examined. When tethered to single- or double-stranded nucleic acids, hydrolysis of AE was found to proceed by generalized base catalysis in which a base abstracts a proton from water and the resulting hydroxide ion then hydrolyzes AE. Analysis of the hydrolysis rates of AE bound to DNA revealed that AE binds the minor groove of DNA and that its hydrolysis is inhibited by low water activity within the minor groove of the helix. Depending upon the sequence of the DNA, the water activity of the minor groove was estimated to be at least 2-4-fold lower than bulk solution. Hydrolysis measurements of AE tethered to RNA as well as RNA/DNA hybrids argued that the grooves of these double helices are also dehydrated relative to bulk solution. Remarkably, mismatched bases, regardless of their structure or sequence context, enhanced hydrolysis of AE by inducing hydration of the double helix that spread approximately five base pairs on either side of the mismatch.     

Binding to the minor groove of the double-strand, tau protein prevents DNA from damage by peroxidation

Tau, an important microtubule associated protein, has been found to bind to DNA, and to be localized in the nuclei of both neurons and some non-neuronal cells. Here, using electrophoretic mobility shifting assay (EMSA) in the presence of DNA with different chain-lengths, we observed that tau protein favored binding to a 13 bp or a longer polynucleotide. The results from atomic force microscopy also showed that tau protein preferred a 13 bp polynucleotide to a 12 bp or shorter polynucleotide. In a competitive assay, a minor groove binder distamycin A was able to replace the bound tau from the DNA double helix, indicating that tau protein binds to the minor groove. Tau protein was able to protect the double-strand from digestion in the presence of DNase I that was bound to the minor groove. On the other hand, a major groove binder methyl green as a negative competitor exhibited little effect on the retardation of tau-DNA complex in EMSA. This further indicates the DNA minor groove as the binding site for tau protein. EMSA with truncated tau proteins showed that both the proline-rich domain (PRD) and the microtubule-binding domain (MTBD) contributed to the interaction with DNA; that is to say, both PRD and MTBD bound to the minor groove of DNA and bent the double-strand, as observed by electron microscopy. To investigate whether tau protein is able to prevent DNA from the impairment by hydroxyl free radical, the chemiluminescence emitted by the phen-Cu/H(2)O(2)/ascorbate was measured. The emission intensity of the luminescence was markedly decreased when tau protein was present, suggesting a significant protection of DNA from the damage in the presence of hydroxyl free radical.     

An Unexpected Major Groove Binding of Netropsin and Distamycin A to tRNAphe

Abstract

Crystalline complexes of yeast tRNA^phe and the oligopeptide antibiotics netropsin and distamycin A were prepared by diffusing drugs into crystals of tRNA. X-ray structure analyses of these complexes reveal a single common binding site for both drugs which is located in the major or deep groove of the tRNA T-stem. The netropsin-tRNA complex is stabilized by specific hydrogen bonds between the amide groups of the drug and the tRNA bases G51 0(6), U52 0(4) and G53 N(7) on one strand, and is further stabilized by electrostatic interactions between the positively charges guanidino side chain of the drug and the tRNA phosphate P53 on the same strand and the positively charged amidino propyl side chain and the phosphates P61, P62 and P63 on the opposite strand of the double helix. These results are in contrast to the implicated minor groove binding of these drugs to non-guanine sequences in DNA. The binding to the GUG sequence in tRNA implies that major groove binding to certain DNA sequences is possible.