DNA Phosphate Backbone Research Articles

Structure-based Analysis of the Metal-dependent Mechanism of H-N-H Endonucleases

Controversy surrounds the metal-dependent mechanism of H-N-H endonucleases, enzymes involved in a variety of biological functions, including intron homing and DNA repair. To address this issue we determined the crystal structures for complexes of the H-N-H motif containing bacterial toxin colicin E9 with Zn(2+), Zn(2+).DNA, and Mg(2+).DNA. The structures show that the rigid V-shaped architecture of the active site does not undergo any major conformational changes on binding to the minor groove of DNA and that the same interactions are made to the nucleic acid regardless of which metal ion is bound to the enzyme. The scissile phosphate contacts the single metal ion of the motif through distortion of the DNA brought about by the insertion of the Arg-96-Glu-100 salt bridge into the minor groove and a network of contacts to the DNA phosphate backbone that straddle the metal site. The Mg(2+)-bound structure reveals an unusual coordination scheme involving two H-N-H histidine residues, His-102 and His-127. The mechanism of DNA cleavage is likely related to that of other single metal ion-dependent endonucleases, such as I-PpoI and Vvn, although in these enzymes the single alkaline earth metal ion is coordinated by oxygen-bearing amino acids. The structures also provide a rationale as to why H-N-H endonucleases are inactive in the presence of Zn(2+) but active with other transition metal ions such as Ni(2+). This is because of coordination of the Zn(2+) ion through a third histidine, His-131. "Active" transition metal ions are those that bind more weakly to the H-N-H motif because of the disengagement of His-131, which we suggest allows a water molecule to complete the catalytic cycle.

DNA-Thumb Interactions and Processivity of T7 DNA Polymerase in Comparison to Yeast Polymerase η

The replicative polymerase of bacteriophage T7 is structurally and mechanistically well characterized. The crystal structure of T7 DNA polymerase or gene 5 protein complexed to its processivity factor, Escherichia coli thioredoxin, a primer-template, and a dideoxynucleotide reveals how this enzyme interacts with the 3'-end of the primer-template, but does not show how thioredoxin confers processivity to the polymerase. In the crystal structure highly conserved amino acids Asn(335) and Ser(338) of the thumb subdomain of T7 DNA polymerase are seen to interact with phosphates 7 and 8 of the DNA template strand. Results with a mutant T7 DNA polymerase in which aliphatic residues are substituted for these amino acids and experiments with different length and methylphosphonate-modified primer-templates demonstrate that these interactions are essential for processive synthesis and d(A.T)(n) tract bypass. Our data with methylphosphonate-modified DNA suggests that thioredoxin confers processivity to T7 DNA polymerase in part by causing an interaction with the phosphate backbone or minor groove of DNA. Residues Asn(335) and Ser(338) may also function with a nearby helix-loop-helix motif located at residues 339-372 to enclose the DNA during processive synthesis. Our results suggest that this structure must be held close to the DNA by ionic interactions to function. These interactions also allow for DNA sliding but physically block the passage of a 3T bulge in the template. In contrast, yeast polymerase eta, a polymerase that non-mutagenically repairs cis-syn thymidine dimers, allows the same bulge to slide past its thumb subdomain during synthesis. A relaxed thumb interaction with the DNA could account for the notably low processivity of polymerase eta.

Histone Acetylation and Cancer

Acta Oncologica Vol. 42, No. 7, p. 792, 2003Received 7 March 2003Accepted 14 April 2003To the Editor:The organization of chromatin is crucial for the regulation of geneexpression. In particular, both the positioning and properties ofnucleosomes influence promoter-specific transcription in responseto extracellular or intracellular signals (1). There is a growing bodyof evidence to suggest that the acetylation and deacetylation ofhistones play significant roles in transcriptional regulation ofeukaryotic cells. To date, several papers have been published abouttheir role in gene expression (2). They are catalysed by specificenzyme families, histone acetyltransferases (HATs) and deacety-lases (HDACs). HATs have been identified as transcriptionalcoactivators and HDACs as yeast transcriptional regulators. Thereare at least four groups of proteins with intrinsic HAT activity,while eight HDACs belonging to three distinct classes have beendescribed in mammalian cells (1).The balance between acetylation and deacetylation is animportant factor in regulating gene expression and is thus linkedto the control of cell fate. Disruption of HAT or HDAC activity ispossibly associated with cancer development (3). Genes that encodeHAT enzymes are either translocated, or amplified, or over-expressed, or mutated in different types of cancers. Nucleosomescontaining highly charged hypoacetylated histones bind tightly tothe phosphate backbone of DNA, inhibiting transcription, pre-sumably because transcription factors, regulatory complexes andRNA polymerase do not have access to DNA. Acetylationneutralizes the charge of the histones and generates a more openDNA conformation. From molecular analyses of human diseases itis suggested that changes in acetylation may play a role in theuncontrolled cell growth of cancer. Since aberrant histone acetyla-tion has been linked to malignant diseases in some cases, HDACinhibitors have potential as new drugs due to their ability tomodulate transcription and to induce differentiation and apoptosis.Today’s treatment strategies are mostly limited to general HDACinhibitors, some of which have pleiotropic and/or unknown effectsin addition to deacetylase repression. Several drugs that are able toinhibit HDACs are at present undergoing clinical trials (4). Theseinclude short chain fatty acids (such as 4-phenylbutyrate andvalporic acid), hydroxamic acids (such as suberoylanilide hydro-xamic acid or SAHA, pyroxamide, trichostatin A or TSA,oxamflatin and CHAPs), cyclic tetrapeptides (such as trapoxin,apicidin and depsipeptide*

Full-length archaeal Rad51 structure and mutants: mechanisms for RAD51 assembly and control by BRCA2.

To clarify RAD51 interactions controlling homologous recombination, we report here the crystal structure of the full-length RAD51 homolog from Pyrococcus furiosus. The structure reveals how RAD51 proteins assemble into inactive heptameric rings and active DNA-bound filaments matching three-dimensional electron microscopy reconstructions. A polymerization motif (RAD51-PM) tethers individual subunits together to form assemblies. Subunit interactions support an allosteric 'switch' promoting ATPase activity and DNA binding roles for the N-terminal domain helix-hairpin-helix (HhH) motif. Structural and mutational results characterize RAD51 interactions with the breast cancer susceptibility protein BRCA2 in higher eukaryotes. A designed P.furiosus RAD51 mutant binds BRC repeats and forms BRCA2-dependent nuclear foci in human cells in response to gamma-irradiation-induced DNA damage, similar to human RAD51. These results show that BRCA2 repeats mimic the RAD51-PM and imply analogous RAD51 interactions with RAD52 and RAD54. Both BRCA2 and RAD54 may act as antagonists and chaperones for RAD51 filament assembly by coupling RAD51 interface exchanges with DNA binding. Together, these structural and mutational results support an interface exchange hypothesis for coordinated protein interactions in homologous recombination.

DNA binding and cleavage by the periplasmic nuclease Vvn: a novel structure with a known active site.

The Vibrio vulnificus nuclease, Vvn, is a non-specific periplasmic nuclease capable of digesting DNA and RNA. The crystal structure of Vvn and that of Vvn mutant H80A in complex with DNA were resolved at 2.3 A resolution. Vvn has a novel mixed alpha/beta topology containing four disulfide bridges, suggesting that Vvn is not active under reducing conditions in the cytoplasm. The overall structure of Vvn shows no similarity to other endonucleases; however, a known 'betabetaalpha-metal' motif is identified in the central cleft region. The crystal structure of the mutant Vvn-DNA complex demonstrates that Vvn binds mainly at the minor groove of DNA, resulting in duplex bending towards the major groove by approximately 20 degrees. Only the DNA phosphate backbones make hydrogen bonds with Vvn, suggesting a structural basis for its sequence-independent recognition of DNA and RNA. Based on the enzyme-substrate and enzyme-product structures observed in the mutant Vvn-DNA crystals, a catalytic mechanism is proposed. This structural study suggests that Vvn hydrolyzes DNA by a general single-metal ion mechanism, and indicates how non-specific DNA-binding proteins may recognize DNA.

DNA-templated nanotube localization.

Carbon nanotubes are nanometer-scale materials with important properties, but their use in nanofabrication will require further development of methods for controlled positioning at well-defined locations on surfaces. We have devised an approach for specifically localizing single-walled carbon nanotubes (SWNTs) onto 1-pyrenemethylamine (PMA)-decorated lambda-DNA molecules aligned on Si surfaces. PMA is used as a bridging compound because its amine group is attracted electrostatically to the negatively charged phosphate backbone of DNA, while the pyrenyl group in PMA interacts with SWNT surfaces through pi-stacking forces. From a total of 60 atomic force microscopy images obtained on three different substrates, we determined that 63% of SWNTs observed on the surfaces were anchored along DNA, and these nanotubes covered approximately 5% of the total DNA length. DNA-templated nanopositioning offers intriguing possibilities for the bottom-up assembly of materials at the nanometer scale.

Features of a Smad3 MH1-DNA Complex: ROLES OF WATER AND ZINC IN DNA BINDING

The Smad family of proteins mediates transforming growth factor-beta signaling from cell membrane to the nucleus. In the nucleus, Smads serve as transcription factors by directly binding to specific DNA sequences and regulating the expression of ligand-response genes. A previous structural analysis, at 2.8-A resolution, revealed a novel DNA-binding mode for the Smad MH1 domain but did not allow accurate assignment of the fines features of protein-DNA interactions. The crystal structure of a Smad3 MH1 domain bound to a palindromic DNA sequence, determined at 2.4-A resolution, reveals a surprisingly important role for water molecules. The asymmetric placement of the DNA-binding motif (a conserved 11-residue beta-hairpin) in the major groove of DNA is buttressed by seven well ordered water molecules. These water molecules make specific hydrogen bonds to the DNA bases, the DNA phosphate backbones, and several critical Smad3 residues. In addition, the MH1 domain is found to contain a bound zinc atom using four invariant residues among Smad proteins, three cysteines and one histidine. Removal of the zinc atom results in compromised DNA binding activity. These results define the Smad MH1 domain as a zinc-coordinating module that exhibits unique DNA binding properties.

The Role of the Minor Groove Substituents in Indirect Readout of DNA Sequence by 434 Repressor

The sequence of non-contacted bases at the center of the 434 repressor binding site affects the strength of the repressor-DNA complex by influencing the structure and flexibility of DNA (Koudelka, G. B., and Carlson, P. (1992) Nature 355, 89-91). We synthesized 434 repressor binding sites that differ in their central sequence base composition to test the importance of minor groove substituents and/or the number of base pair hydrogen bonds between these base pairs on DNA structure and strength of the repressor-DNA complex. We show here that the number of base pair H-bonds between the central bases apparently has no role in determining the relative affinity of a DNA site for repressor. Instead we find that the affinity of DNA for repressor depends on the absence or presence the N2-NH(2) group on the purine bases at the binding site center. The N2-NH(2) group on bases at the center of the 434 binding site appears to destabilize 434 repressor-DNA complexes by decreasing the intimacy of the specific repressor-DNA contacts, while increasing the reliance on protein contacts to the DNA phosphate backbone. Thus, the presence of an N2-NH(2) group on the purines at the center of a binding site globally alters the precise conformation of the protein-DNA interface.

Phosphorylation-dependent Down-regulation of c-Myb DNA Binding Is Abrogated by a Point Mutation in the v-mybOncogene

The viral Myb (v-Myb) oncoprotein of the avian myeloblastosis virus (AMV) is an activated form of the cellular transcription factor c-Myb causing acute monoblastic leukemia in chicken. Oncogenic v-Myb alterations include N- and C-terminal deletions as well as point mutations. Whereas truncations in Myb cause loss of various protein modifications, none of the point mutations in v-Myb has been directly linked to protein modifications. Here we show that the DNA-binding domain of c-Myb can be phosphorylated on serine 116 by the catalytic subunit of protein kinase A. Phosphorylation of Ser(116) differentially destabilizes a subtype of c-Myb-DNA complexes. The V117D mutation of the AMV v-Myb oncoprotein abolishes phosphorylation of the adjacent Ser(116) residue. Modification of Ser(116) was also detected in live cells in c-Myb, but not in AMV v-Myb. Phosphorylation-mimicking mutants of c-Myb failed to activate the resident mim-1 gene. Our data imply that protein kinase A or a kinase with similar specificity negatively regulates c-Myb function, including collaboration with C/EBP, and that the leukemogenic AMV v-Myb version evades inactivation by a point mutation that abolishes a phosphoacceptor consensus site. This suggests a novel link between Myb, a signal transduction pathway, cooperativity with C/EBP, and a point mutation in the myb oncogene.

An Infrared Spectroscopic Study of the Effect of Hydration on Cationic Lipid/DNA Complexes

Infrared spectroscopy was used to examine the effect of dehydration on the structure of DNA and cationic lipid/DNA complexes (CLDCs). Information regarding the effect of hydration on the interface between the cationic lipids and DNA was obtained by following subtle but reproducible changes in vibrational bands arising from the DNA bases and phosphate backbone as well as bands from the lipid ester groups within the interfacial region of the bilayer. Dehydration of supercoiled plasmid DNA induces a transition from a B-conformation in solution to a mixed conformation in the dried state. Changes in vibrations of the bases upon drying suggest a change to an A-conformation whereas vibrations from the phosphate moieties suggest A- or C-forms. Vibrational changes in the ribose ring suggest adoption of a C-conformation. When CLDCs composed of either DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) or DDAB (dioctadecyldimethylammonium bromide) cationic lipids with or without equimolar amounts of the helper lipids cholesterol or DOPE (1,2-dioleoylphosphatidylethanolamin) are dried, the DNA is still able to undergo these structural transitions suggesting a nonrigid CLDC structure. The effect of dehydration on these interfacial interactions was found to be dependent on the type of cationic lipid used as well as the type of helper lipid. In addition, this work provides a simple spectroscopic analytical approach that can be used for the characterization of nonviral vectors that has potential pharmaceutical utility.

Label‐Free and Label Based Electrochemical Detection of Hybridization by Using Methylene Blue and Peptide Nucleic Acid Probes at Chitosan Modified Carbon Paste Electrodes

AbstractA chitosan modified carbon paste electrode (ChiCPE) based DNA biosensor for the recognition of calf thymus double stranded DNA (dsDNA), single stranded DNA (ssDNA) and hybridization detection between complementary DNA oligonucleotides is presented. DNA and oligonucleotides were electrostatically attached by using chitosan onto CPE. The amino groups of chitosan formed a strong complex with the phosphate backbone of DNA. The immobilized probe could selectively hybridize with the target DNA to form hybrid on the CPE surface. The detection of hybridization was observed by using the label‐free and label based protocols. The oxidation signals of guanine and adenine greatly decreased when a hybrid was formed on the ChiCPE surface. The changes in the peak currents of methylene blue (MB), an electroactive label, were observed upon hybridization of probe with target. The signals of MB were investigated at dsDNA modified ChiCPE and ssDNA modified ChiCPE and the increased peak currents were observed, in respect to the order of electrodes. The hybridization of peptide nucleic acid (PNA) probes with the DNA target sequences at ChiCPE was also investigated. Performance characteristics of the sensor were described, along with future prospects.

The structural transition of DNA–Tris(1,10-phenanthroline) cobalt(III) complexes in ethanol–water solution

The interaction of DNA with Tris(1,10-phenanthroline) cobalt(III) was studied by means of atomic force microscopy. Changes in the morphologies of DNA complex in the presence of ethanol may well indicate the crucial role of electrostatic force in causing DNA condensation. With the increase of the concentration of ethanol, electrostatic interaction is enhanced corresponding to a lower dielectric constant. Counterions condense along the sugar phosphate backbone of DNA when ε is lowered and the phosphate charge density can thus be neutralized to the level of DNA condensation. Electroanalytical measurement of DNA condensed with Co(phen) 3 3+ in ethanol solution indicated that intercalating reaction remains existing. According to both the microscopic and spectroscopic results, it can be found that no secondary structure transition occurs upon DNA condensing. B–A conformation transition takes place at more than 60% ethanol solution.

Activator recruitment by the general transcription machinery: X-ray structural analysis of the Oct-1 POU domain/human U1 octamer/SNAP190 peptide ternary complex.

Transcriptional activation of the human U1 snRNA genes is dependent on a noncanonical octamer element contained within an upstream enhancer. The U1 octamer only weakly recruits the Oct-1 POU domain, although recruitment is stimulated by a peptide containing the Oct-1-binding domain of SNAP190. Structural analysis of the Oct-1 POU domain/U1 octamer/SNAP190 peptide complex revealed that SNAP190 makes extensive protein contacts with the Oct-1 POU-specific domain and with the DNA phosphate backbone within the enhancer. Although SNAP190 and OCA-B both interact with the Oct-1 POU domain through the same Oct-1 interface, a single nucleotide within the U1 octamer ablates OCA-B recruitment without compromising activator recruitment by SNAP190.

A putative DNA-binding domain in the NUCKS protein

We have studied the DNA-binding properties of a NUCKS-derived, synthetic peptide containing an extended GRP motif. This peptide binds to random-sequence DNA, but did not bind preferentially to poly(dA–dT). A synthetic peptide with the same amino acid composition but with a random sequence did not bind to DNA, suggesting that the structure of the DNA-binding domain plays a pivotal role in the interaction with DNA. NMR and graphic modeling were employed to investigate the structure of the synthetic peptide. It was shown that the DNA-binding peptide constituted an α helix in phosphate buffer at pH 5.5. Docking results indicated an almost perfect fit for this small, helical peptide into the major groove of DNA with the possibility of four basic residues interacting with the phosphate backbone of DNA. One consensus site for phosphorylation by Cdk1 is located in the N-terminal end of the DNA-binding peptide. Upon phosphorylation of this site, the binding to DNA was completely prohibited. Immunofluorescence experiments showed that NUCKS was located in the nuclei in proliferating cells in interphase of the cell cycle, but was distributed throughout the cytoplasm in mitotic cells.

Interaction of the ocr gene 0.3 protein of bacteriophage T7 with EcoKI restriction/modification enzyme.

The ocr protein, the product of gene 0.3 of bacteriophage T7, is a structural mimic of the phosphate backbone of B-form DNA. In total it mimics 22 phosphate groups over approximately 24 bp of DNA. This mimicry allows it to block DNA binding by type I DNA restriction enzymes and to inhibit these enzymes. We have determined that multiple ocr dimers can bind stoichiometrically to the archetypal type I enzyme, EcoKI. One dimer binds to the core methyltransferase and two to the complete bifunctional restriction and modification enzyme. Ocr can also bind to the component subunits of EcoKI. Binding affinity to the methyltransferase core is extremely strong with a large favourable enthalpy change and an unfavourable entropy change. This strong interaction prevents the dissociation of the methyltransferase which occurs upon dilution of the enzyme. This stabilisation arises because the interaction appears to involve virtually the entire surface area of ocr and leads to the enzyme completely wrapping around ocr.

Plasmid promiscuity: meeting the challenge of DNA immigration control.

Plasmid promiscuity: meeting the challenge of DNA immigration control.

Spectroscopic and voltammetric study on the binding of aluminium(III) to DNA.

Cyclic voltammetry (CV) and ultraviolet (UV) spectroscopy were used, for the first time, to study the interaction between aluminium(III) and calf thymus DNA under neutral pH conditions. Thus obtained data confirmed the existence of a relatively strong interaction between Al(III) and DNA. The binding site for aluminium(III) on DNA chains is not the bases, but the phosphate groups on the DNA backbones, the same as that for [Co(phen)3](3+/2+) that binds non-specifically and electrostatically to the deoxyribose phosphate backbone of DNA. When coexisting, Al(III) binds more favorably to DNA than [Co(phen)3](3+/2+), which implies the relatively strong binding of Al(III) to the phosphate backbone of DNA under neutral pH conditions. The nature of the binding of Al(III) to DNA is also discussed.

Structural basis of transcription initiation: an RNA polymerase holoenzyme-DNA complex.

The crystal structure of Thermus aquaticus RNA polymerase holoenzyme (alpha2betabeta'omegasigmaA) complexed with a fork-junction promoter DNA fragment has been determined by fitting high-resolution x-ray structures of individual components into a 6.5-angstrom resolution map. The DNA lies across one face of the holoenzyme, completely outside the RNA polymerase active site channel. All sequence-specific contacts with core promoter elements are mediated by the sigma subunit. A universally conserved tryptophan is ideally positioned to stack on the exposed face of the base pair at the upstream edge of the transcription bubble. Universally conserved basic residues of the sigma subunit provide critical contacts with the DNA phosphate backbone and play a role in directing the melted DNA template strand into the RNA polymerase active site. The structure explains how holoenzyme recognizes promoters containing variably spaced -10 and -35 elements and provides the basis for models of the closed and open promoter complexes.

Structure of Ocr from Bacteriophage T7, a Protein that Mimics B-Form DNA

We have solved, by X-ray crystallography to a resolution of 1.8 Å, the structure of a protein capable of mimicking approximately 20 base pairs of B-form DNA. This ocr protein, encoded by gene 0.3 of bacteriophage T7, mimics the size and shape of a bent DNA molecule and the arrangement of negative charges along the phosphate backbone of B-form DNA. We also demonstrate that ocr is an efficient inhibitor in vivo of all known families of the complex type I DNA restriction enzymes. Using atomic force microscopy, we have also observed that type I enzymes induce a bend in DNA of similar magnitude to the bend in the ocr molecule. This first structure of an antirestriction protein demonstrates the construction of structural mimetics of long segments of B-form DNA.

Fluorine‐18 labeling of peptide nucleic acids

AbstractPeptide nucleic acids (PNAs) are a unique class of synthetic macromolecules, originally designed as ligands for the recognition of double stranded DNA. From a chemical point of view, the deoxyribose phosphate backbone of DNA is replaced by a pseudo‐peptide N‐(2‐aminoethyl)glycyl backbone, while the nucleobases of DNA (adenine, guanine, cytosine and thymine) are retained. Due to the increasing interest in the labeling of peptide nucleic acids (PNAs) as potent diagnostic agents in nuclear medicine, we have used and adapted the reliable methodology developed for the fluorine‐18 labeling of oligonucleotides and have now demonstrated that it is possible to label PNAs in sufficient quantity and with high specific radioactivity for PET studies in a time compatible with the half life of fluorine‐18. Copyright © 2002 John Wiley & Sons, Ltd.