High resolution footprinting of a type I methyltransferase reveals a large structural distortion within the DNA recognition site.

The type I DNA methyltransferase M.EcoR124I is a multi-subunit enzyme that binds to the sequence GAAN6RTCG, transferring a methyl group from S-adenosyl methionine to a specific adenine on each DNA strand. We have investigated the protein-DNA interactions in the complex by DNase I and hydroxyl radical footprinting. The DNase I footprint is unusually large: the protein protects the DNA on both strands for at least two complete turns of the helix, indicating that the enzyme completely encloses the DNA in the complex. The higher resolution hydroxyl radical probe shows a smaller, but still extensive, 18 bp footprint encompassing the recognition site. Within this region, however, there is a remarkably hyper-reactive site on each strand. The two sites of enhanced cleavage are co-incident with the two adenines that are the target bases for methylation, showing that the DNA is both accessible and highly distorted at these sites. The hydroxyl radical footprint is unaffected by the presence of the cofactor S-adenosyl methionine, showing that the distorted DNA structure induced by M.EcoR124I is formed during the initial DNA binding reaction and not as a transient intermediate in the reaction pathway.     

Interaction of a four-way junction in DNA with T4 endonuclease VII

The binding of a synthetic four-way junction in DNA by T4 endonuclease VII has been studied using gel retardation and footprint analysis. Two specific protein-DNA complexes have been observed, but only one is stable in the presence of moderate concentrations of salt. The footprint of T4 endonuclease VII in the salt-resistant complex has been probed using hydroxyl radicals generated by the reaction of iron(II)/EDTA with hydrogen peroxide. The hydroxyl radical cleavage pattern indicates protection of approximately 5 residues in two strands that are diametrically opposed across the junction point.     

DNA and protein footprinting analysis of the modulation of DNA binding by the N-terminal domain of the Saccharomyces cerevisiae TATA binding protein

Recombinant full-length Saccharomyces cerevisiae TATA binding protein (TBP) and its isolated C-terminal conserved core domain (TBPc) were prepared with measured high specific DNA-binding activities. Direct, quantitative comparison of TATA box binding by TBP and TBPc reveals greater affinity by TBPc for either of two high-affinity sequences at several different experimental conditions. TBPc associates more rapidly than TBP to TATA box bearing DNA and dissociates more slowly. The structural origins of the thermodynamic and kinetic effects of the N-terminal domain on DNA binding by TBP were explored in comparative studies of TBPc and TBP by "protein footprinting" with hydroxyl radical (*OH) side chain oxidation. Some residues within TBPc and the C-terminal domain of TBP are comparably protected by DNA, consistent with solvent accessibility changes calculated from core domain crystal structures. In contrast, the reactivity of some residues located on the top surface and the DNA-binding saddle of the C-terminal domain differs between TBP and TBPc in both the presence and absence of bound DNA; these results are not predicted from the crystal structures. A strikingly different pattern of side chain oxidation is observed for TBP when a nonionic detergent is present. Taken together, these results are consistent with the N-terminal domain actively modulating TATA box binding by TBP and nonionic detergent modulating the interdomain interaction.     

Footprint analysis of the bsp RI DNA methyltransferase-DNA interaction.

The interaction between the GGCC-specific Bsp RI DNA methyltransferase (M. Bsp RI) and substrate DNA was studied with footprinting techniques using a DNA fragment that was unmodified on both strands. Footprinting with DNase I revealed an approximately 14 bp protected region. Footprinting with dimethylsulfate detected major groove interactions with the guanine bases of the recognition sequence. Reaction with 1,10-phenanthroline-copper did not show protection, suggesting that minor groove interactions play little role in sequence-specific recognition by M. Bsp RI. Hydroxyl radical footprinting revealed a protected stretch of 6 nt. The hydroxyl radical footprint of M. Bsp RI differs markedly from the the footprint reported for the Hha I and Sss I methyltransferases. The pattern of protection from dimethylsulfate and hydroxyl radicals suggests that the interactions of M. Bsp RI with DNA are similar to those detected in the co-crystal structure of the Hae III methyltransferase.     

Detection of 2-deoxy-D-ribose radicals generated by the reaction with the hydroxyl radical using a rapid flow-ESR method

ESR spectrum of the short-lived radicals derived from 2-deoxy-D-ribose by the reaction with the hydroxyl radical (HO*) was measured using a rapid flow method. A dielectric mixing resonator was used for the measurement, which made it possible to measure the highly sensitive ESR spectra of the radicals with a lifetime of the order of milliseconds. A complex spectrum was obtained and the spectral simulation was done to show that it was the superposition of the signals due to five radicals (I-V). Three of them were those formed by the dehydrogenation with the HO* at C-1 (I), C-3 (II), and C-4 (III) positions of the 2-deoxy-D-ribose molecule. The other two (IV and V) were carbonyl-conjugated radicals formed by the elimination of a water molecule from III and II. The results showed that dehydrogenation occurred randomly at the positions where hydroxyl groups are attached, but the most preferred position was C-3 and the radical position moved from C-3 to C-4 by the elimination of water molecule.     

Molecular modeling of the reactive configuration of peroxidized lipid and α-tocopherol in the membrane

The mutual arrangement of a phospholipid molecule containing a peroxyl radical and a molecule of membrane-acting antioxidant α-tocopherol (vitamin E) in the lipid bilayer has been studied by molecular dynamics simulation. The geometry of molecules in the membrane is revealed at which the hydrogen atom can be transferred from the exocyclic hydroxyl of α-tocopherol to the peroxyl lipid radical. It is shown that, under equilibrium conditions, the peroxidized fatty acid segment rises nearer to the polar surface of the membrane, while α-tocopherol submerges into the hydrophobic part of the lipid bilayer.     

Fermentation micromethod for the quantitative determination of thiamine diphosphate in biological fluids]

An enzymatic micromethod is proposed for quantification of thiamine biphosphate (TBP) at concentrations from 0.5 ng in 0.1-0.2 ml samples of blood or other biological liquids. The dynamics of TBP degradation in blood was studied depending on the time and conditions of storage. A high efficient complex of alcohol dehydrogenase and apopyruvate decarboxylase was isolated from baker's yeasts that can be successfully used for quantitative detection of TBP. The complex was stabilized for further application to biochemical kits for diagnosis of B1-deficiency.     

Detection of drug binding to DNA by hydroxyl radical footprinting. Relationship of distamycin binding sites to DNA structure and positioned nucleosomes on 5S RNA genes of Xenopus.

We report the use of hydroxyl radical footprinting to analyze the interaction of distamycin and actinomycin with the 5S ribosomal RNA genes of Xenopus. There is a qualitative difference in the hydroxyl radical footprints of the two drugs. Distamycin gives a conventional (albeit high-resolution) footprint, while actinomycin does not protect DNA from hydroxyl radical attack, but instead induces discrete sites of hyperreactivity. We find concentration-dependent changes in the locations of distamycin binding sites on the somatic 5S gene of Xenopus borealis. A high-affinity site, containing a G.C base pair, is replaced at higher levels of bound drug by a periodic array of different lower affinity sites that coincide with the places where the minor groove of the DNA would face in toward a nucleosome core that is known to bind to the same sequence. These results suggest that distamycin recognizes potential binding sites more by the shape of the DNA than by the specific sequence that is contained in the site and that structures of many sequences are deformable to a shape that allows drug binding. We discuss the utility of hydroxyl radical footprinting of distamycin for investigating the underlying structure of DNA.     

Architecture of Protein and DNA Contacts within the TFIIIB-DNA Complex

The RNA polymerase III factor TFIIIB forms a stable complex with DNA and can promote multiple rounds of initiation by polymerase. TFIIIB is composed of three subunits, the TATA binding protein (TBP), TFIIB-related factor (BRF), and B". Chemical footprinting, as well as mutagenesis of TBP, BRF, and promoter DNA, was used to probe the architecture of TFIIIB subunits bound to DNA. BRF bound to TBP-DNA through the nonconserved C-terminal region and required 15 bp downstream of the TATA box and as little as 1 bp upstream of the TATA box for stable complex formation. In contrast, formation of complete TFIIIB complexes required 15 bp both upstream and downstream of the TATA box. Hydroxyl radical footprinting of TFIIIB complexes and modeling the results to the TBP-DNA structure suggest that BRF and B" surround TBP on both faces of the TBP-DNA complex and provide an explanation for the exceptional stability of this complex. Competition for binding to TBP by BRF and either TFIIB or TFIIA suggests that BRF binds on the opposite face of the TBP-DNA complex from TFIIB and that the binding sites for TFIIA and BRF overlap. The positions of TBP mutations which are defective in binding BRF suggest that BRF binds to the top and N-terminal leg of TBP. One mutation on the N-terminal leg of TBP specifically affects the binding of the B" subunit.     

Selective binding of the TATA box-binding protein to the TATA box-containing promoter: analysis of structural and energetic factors.

We report the results of an energy-based exploration of the components of selective recognition of the TATA box-binding protein (TBP) to a TATA box sequence that includes 1) the interaction between the hydrophobic Leu, Pro, and Phe residues of TBP with the TA, AT, AA, TT, and CG steps, by ab initio quantum mechanical calculations; and 2) the free energy penalty, calculated from molecular dynamics/potential of mean force simulations, for the conformational transition from A-DNA and B-DNA into the TA-DNA form of DNA observed in a complex with TBP. The GTAT, GATT, GAAT, and GTTT tetramers were explored. The results show that 1) the discrimination of TA, AT, AA, TT, or CG steps by TBP cannot rest on their interaction with the inserting Phe side chains; 2) the steric clash between the bulky and hydrophobic Pro and Leu residues and the protruding -NH2 group of guanine is responsible for the observed selectivity against any Gua-containing basepair; 3) the Pro and Leu residues cannot selectively discriminate among TA, AT, AA, or TT steps; and 4) the calculated energy required to achieve the TA-DNA conformation of DNA that is observed in the complex with TBP appears to be a key determinant for the observed selectivity against the AT, AA, and TT steps. The simulations also indicate that only the TA step can form a very efficient interbase hydrogen bond network in the TA-DNA conformation. Such an energetically stabilizing network is not achievable in the AA and TT steps. While it is viable in the AT step, structural constraints render the hydrogen bonding network energetically ineffective there.     

On the Information Content of Protein Sequences

Abstract

TATA-box binding protein (TBP) in a monomelic form and the complexes it forms with DNA have been elucidated with molecular dynamics simulations. Large TBP domain motions (bend and twist) are detected in the monomer as well as in the DNA complexes; these motions can be important for TBP binding of DNA. TBP interacts with guanine bases flanking the TATA element in the simulations of the complex; these interactions may explain the preference for guanine observed at these DNA positions. Side chains of some TBP residues at the binding interface display significant dynamic flexibility that results in ‘flipflop’ contacts involving multiple base pairs of the DNA. We discuss the possible functional significance of these observations.     

Photogeneration of hydroxyl radicals for footprinting.

Hydroxyl radicals yield footprints of DNA-ligand interactions that are uniform in intensity and display single base pair resolution. It is shown here that brief illumination of dilute aqueous solutions of hydrogen peroxide with a standard uv transilluminator can be used to generate hydroxyl radicals for footprinting studies. Photogenerated hydroxyl radicals are used to footprint netropsin, a drug that interacts with the minor groove of DNA. The method presented eliminates two of the reagents used in conventional Fenton-reaction-based hydroxyl radical footprinting. It has the further advantage that the extent of cleavage of the DNA can be precisely regulated by controlling the illumination time. Because light is used to drive the reaction, photogenerated hydroxyl radicals can be used to footprint DNA-ligand interactions under experimental conditions of temperature and pressure inaccessible to Fenton-reaction chemistry.     

Conformational Properties of the TATA-Box Binding Sequence of DNA

Abstract

Nanosecond scale molecular dynamics simulations in water demonstrate that the DNA oligomer, GCGTATATAAAACGC, which contains a target site for the TATA-box binding protein (TBP), has an intrinsic preference to adopt an A-like conformation in the region of the TATA-box and undergoes bending related to that seen within in the TBP complex. This result is obtained from two independent simulations using different starting structures. In line with earlier suggestions of Guzikevich-Guerstein and Shakked, these simulations imply that an A-DNA conformation may be an important intermediate step in forming the strongly distorted DNA observed within the crystallographically determined complex with TBP. These results also support modeling studies by Lebrun et al. which suggest that the TBP binding mechanism can be broken down into a backbone transition to an A-like form coupled with a mechanical distortion which locally stretches and unwinds the DNA.     

Nadph-Cytochrome-P450 Reductase Promotes Hydroxyl Radical Production by the Iron Complex of ADR-925, the Hydrolysis Product of ICRF-187 (Dexrazoxane)

ICRF-187 (dexrazoxane) is currently in clinical trials as a cardioprotective agent for the prevention of doxorubicin-induced cardiotoxicity. ICRF-187 likely acts through its strongly metal ion-binding rings-opened hydrolysis product ADR-925 by removing iron from its complex with doxorubicin or by chelating free iron. The ability of NADPH-cytochrome-P450 reductase to promote hydroxyl radical formation by iron complexes of ADR-925 and EDTA was compared by EPR spin trapping. The iron-EDTA complex produced hydroxyl radicals at six times the rate that the iron-ADR-925 complex did. The aerobic oxidation of ferrous complexes of ADR-925, its tetraacid analog, EDTA and DTPA was followed spectropho-tometrically. The iron(II)-ADR-925 complex was aerobically oxidized 700 times slower than was the EDTA complex. It is concluded that even though ADR-925 does not completely eliminate iron-based hydroxyl radical production, it likely protects by preventing site-specific hydroxyl radical damage by the iron-doxorubicin complex.     

Cu(II) complexes with a sulfonamide derived from benzoguanamine. Oxidative cleavage of DNA in the presence of H2O2 and ascorbate

Reaction between benzoguanamine (2,4-diamino-6-phenyl-1,3,5-triazine) and 2-mesitylenesulfonyl chloride leads to formation of a sulfonamide able to form two mononuclear Cu(II) complexes with a CuL(2) stoichiometry. The local environment of the metal cation is a distorted octahedron, with two ligands and two solvent molecules; both complexes crystallize in the monoclinic structure, space group P2(1), with Z=2. In the presence of ascorbate/H(2)O(2,) the two complexes significantly cleavage double-strand pUC18 DNA plasmid. Both complexes exhibit more nuclease efficiency that the copper phenantroline complex. From scavenging reactive oxygen studies we conclude that the hydroxyl radical and a singlet oxygen-like entity, such a peroxide copper complex, are the radical species involved in the DNA damage.