Synthesis of 5-(pyridinyl and pyridiniumyl)-2'-deoxyuridine nucleosides: reversible electron traps for DNA

The desire to produce reversible electron traps for direct, room temperature studies of excess electron transport in DNA duplexes and hairpins motivated our efforts first to link pyridines to 2'-deoxyuridine (pyridinyl-dU) and then to convert these new conjugates into pyridiniumyl-dU nucleosides. Base sensitivity studies presented here rule out general use of bipyridinediiumyl compounds, but show that pyridiniumyl compounds are suitable for use under the strand cleavage and base deprotection procedures required for automated solid-phase oligonucleotide synthesis. This paper presents the synthesis of four 5'-O-DMT-protected 5-(N-methylpyridiniumyl)-dU conjugates using either ethynyl or ethylenyl linkers to join the pyridiniumyl and dU subunits.     

SYNTHESIS OF 5-(PYRIDINYL AND PYRIDINIUMYL)-2′-DEOXYURIDINE NUCLEOSIDES: REVERSIBLE ELECTRON TRAPS FOR DNA

The desire to produce reversible electron traps for direct, room temperature studies of excess electron transport in DNA duplexes and hairpins motivated our efforts first to link pyridines to 2′-deoxyuridine (pyridinyl-dU) and then to convert these new conjugates into pyridiniumyl-dU nucleosides. Base sensitivity studies presented here rule out general use of bipyridinediiumyl compounds, but show that pyridiniumyl compounds are suitable for use under the strand cleavage and base deprotection procedures required for automated solid-phase oligonucleotide synthesis. This paper presents the synthesis of four 5′-O-DMT-protected 5-(N-methylpyridiniumyl)-dU conjugates using either ethynyl or ethylenyl linkers to join the pyridiniumyl and dU subunits.     

Electrostatic Suppression Allows Tyrosine Site-specific Recombination in the Absence of a Conserved Catalytic Arginine

The active site of the tyrosine family site-specific recombinase Flp contains a conserved catalytic pentad that includes two arginine residues, Arg-191 and Arg-308. Both arginines are essential for the transesterification steps of strand cleavage and strand joining in DNA substrates containing a phosphate group at the scissile position. During strand cleavage, the active site tyrosine supplies the nucleophile to form a covalent 3′-phosphotyrosyl intermediate. The 5′-hydroxyl group produced by cleavage provides the nucleophile to re-form a 3′-5′ phosphodiester bond in a recombinant DNA strand. In previous work we showed that substitution of the scissile phosphate (P) by the charge neutral methylphosphonate (MeP) makes Arg-308 dispensable during the catalytic activation of the MeP diester bond. However, in the Flp(R308A) reaction, water out-competes the tyrosine nucleophile (Tyr-343) to cause direct hydrolysis of the MeP diester bond. We now report that for MeP activation Arg-191 is also not required. In contrast to Flp(R308A), Flp(R191A) primarily mediates normal cleavage by Tyr-343 but also exhibits a weaker direct hydrolytic activity. The cleaved MeP-tyrosyl intermediate formed by Flp(R191A) can be targeted for nucleophilic attack by a 5′-hydroxyl or water and channeled toward strand joining or hydrolysis, respectively. In collaboration with wild type Flp, Flp(R191A) promotes strand exchange between MeP- and P-DNA partners. Loss of a catalytically crucial positively charged side chain can thus be suppressed by a compensatory modification in the DNA substrate that neutralizes the negative charge on the scissile phosphate.     

Electron-Transfer-Induced Acidity/Basicity and Reactivity Changes of Purine and Pyrimidine Bases. Consequences of Redox Processes for Dna Base Pairs

Changes in the oxidation state of the DNA bases, induced by oxidation (ionization) or by reduction (electron capture), have drastic effects on the acidity or basicity, respectively, of the molecules. Since in DNA every base is connected to its complementary base in the other strand, any change of the electric charge status of a base in one DNA strand that accompanies its oxidation or reduction may affect also the other strand via proton transfer across the hydrogen bonds in the base pairs. The free energies for electron transfer to or from a base can be drastically altered by the proton transfer processes that accompany the electron transfer reactions. Electron-transfer (ET) induced proton transfer sensitizes the base opposite to the ET-damaged base to redox damage, i.e., damage produced by separation of charge (ionization) has an increased change of being trapped in a base pair. Of the two types of base pair in DNA, A-T and C-G, the latter is more sensitive to both oxidative and reductive processes than the former.

Proton transfer induced by ET does not only occur between the heteroatoms (o and N) of the base pairs (intra-pair proton transfer), but also to and from adjacent water molecules in the hydration shell of DNA (extra-pair proton transfer). These proton transfers can involve carbon and as such are likely to be irreversible. It is the A-T pair which appears to be particularly prone to such irreversible reactions.     

Electron transfer in peptides and proteins

Proteins and peptides use their amino acids as medium for electron-transfer reactions that occur either in single-step superexchange or in multistep hopping processes. Whereas the rate of the single-step electron transfer dramatically decreases with the distance, a hopping process is less distance dependent. Electron hopping is possible if amino acids carry oxidizable side chains, like the phenol group in tyrosine. These side chains become intermediate charge carriers. Because of the weak distance dependency of hopping processes, fast electron transfer over very long distances occurs in multistep reactions, as in the enzyme ribonucleotide reductase.     

The ethylene/metal(0) and ethylene/metal(I) redox system: model ab initio calculations

Ab initio calculations (coupled cluster with single and double excitations; CCSD) have been used to investigate the model redox systems ethylene:M(0) (M = Li, Na, K, Rb, Cs) and ethylene:M(I) (M = Be, Mg, Ca, Sr, Ba, Zn, Cd, Hg). Within C2v symmetry, the ground (2A1) states correspond to the charge distribution given in the title. The lowest (2B2) excited states correspond, somewhat counter intuitively, to the ethylene*-/=M(II)ion pair. These trends can be rationalized on the basis of simple electrostatic and configuration-mixing arguments that lead to two simple equations for predicting the electron-transfer energies for oxidation or reduction of the ethylene. The electron-transfer energies to the 2B2 ion pairs are dominated by the electrostatic ion-pairing energies.     

Oligonucleotide-directed triple helix formation at adjacent oligopurine and oligopyrimidine DNA tracts by alternate strand recognition.

A significant limitation to the practical application of triplex DNA is its requirement for oligopurine tracts in target DNA sequences. The repertoire of triplex-forming sequences can potentially be expanded to adjacent blocks of purines and pyrimidines by allowing the third strand to pair with purines on alternate strands, while maintaining the required strand polarities by combining the two major classes of base triplets, Py.PuPy and Pu.PuPy. The formation of triplex DNA in this fashion requires no unusual bases or backbone linkages on the third strand. This approach has previously been demonstrated for target sequences of the type 5'-(Pu)n(Py)n-3' in intramolecular complexes. Using affinity cleaving and DNase I footprinting, we show here that intermolecular triplexes can also be formed at both 5'-(Pu)n(Py)n-3' and 5'-(Py)n(Pu)n-3' target sequences. However, triplex formation at a 5'-(Py)n(Pu)n-3' sequence occurs with lower yield. Triplex formation is disfavored, even at acid pH, when a number of contiguous C+.GC base triplets are required. These results suggest that triplex formation via alternate strand recognition at sequences made up of blocks of purines and pyrimidines may be generally feasible.     

Effects of Holliday junction position on Xer-mediated recombination in vitro.

Site-specific recombination mediated by XerC and XerD functions in the segregation of circular replicons in Escherichia coli. A key feature of most models of recombination for the family of recombinases to which XerC and XerD belong is that a Holliday junction forms at the position of the first pair of recombinase-mediated strand exchanges and then branch migrates 6-8 bp to the position of the second pair of strand exchanges. We have tested this hypothesis for Xer recombination by studying the effects of junction position on XerC-mediated strand exchange in vitro. Recombination of synthetic Holliday junction substrates in which junction mobility was constrained to a region extending over or removed away from the normal cleavage and exchange point was analysed. All substrates undergo strand cleavage at the normal position. We infer that the Holliday junction need not be at this position during strand cleavage and exchange. With substrates in which the Holliday junction is constrained to a region away from the XerC-mediated cleavage point, strand exchange generates products with the predicted mispaired bases.     

Oxidative dimerization of proteins: role of tyrosine accessibility

PURPOSE: To investigate the importance of two possible mechanisms of tyrosine oxidation on the yield of protein dimerization. The model chosen is hen and turkey egg-white lysozymes, which differ by seven amino acids, among which one tyrosine is in the 3 position. MATERIALS AND METHODS: Aqueous solutions of proteins were oxidized by OH(*) or N(*)(3) free radicals produced by gamma or pulse irradiation in an atmosphere of N(2)O. Protein dimers were quantified by SDS-PAGE and reverse-phase HPLC. Dityrosines were identified by absorption and fluorescence. RESULTS: Using N(*)(3) free radicals, the initial yields of dimerization are equal to (8.6 +/- 0.7) x 10(-9) mol J(-1) for both proteins. Using OH(*) free radicals, they become equal to (1.23 +/- 0.1) x 10(-8) and (4.42 +/- 0.1) x 10(-8) mol J(-1) for hen and turkey egg-white lysozymes, respectively (gamma radiolysis). DISCUSSION. N(*)(3) radicals react primarily with tryptophan residues only. Tyrosine gets oxidized by intramolecular long-range electron migration, whereas OH(*) may react directly with tyrosines. We propose a low participation of Tyr3 in turkey protein in the intramolecular process, because Tyr3 is far from all tryptophans. On the other hand, Tyr3 is very accessible to solvent and in a flexible area; thus collisions with OH(*) could easily be followed by intermolecular dimerization.     

Primary light-energy conversion in tetrameric chlorophyll structure of photosystem II and bacterial reaction centers: II. Femto- and picosecond charge separation in PSII D1/D2/Cyt b559 complex

In Part I of the article, a review of recent data on electron-transfer reactions in photosystem II (PSII) and bacterial reaction center (RC) has been presented. In Part II, transient absorption difference spectroscopy with 20-fs resolution was applied to study the primary charge separation in PSII RC (DI/DII/Cyt b 559 complex) excited at 700 nm at 278 K. It was shown that the initial electron-transfer reaction occurs within 0.9 ps with the formation of the charge-separated state P680(+)Chl(D1)(-), which relaxed within 14 ps as indicated by reversible bleaching of 670-nm band that was tentatively assigned to the Chl(D1) absorption. The subsequent electron transfer from Chl(D1)(-) within 14 ps was accompanied by a development of the radical anion band of Pheo(D1) at 445 nm, attributable to the formation of the secondary radical pair P680(+)Pheo(D1)(-). The key point of this model is that the most blue Q(y) transition of Chl(D1) in RC is allowing an effective stabilization of separated charges. Although an alternative mechanism of charge separation with Chl(D1)* as a primary electron donor and Pheo(D1) as a primary acceptor can not be ruled out, it is less consistent with the kinetics and spectra of absorbance changes induced in the PSII RC preparation by femtosecond excitation at 700 nm.     

DNA microstructural requirements for neocarzinostatin chromophore-induced direct strand cleavage.

The microstructural requirements for optimal interaction of neocarzinostatin chromophore (NCS-C) with DNA have been investigated using a series of hexadeoxyribonucleotides with modified bases such as O6-methyl G (MeG), I, 5-methyl C (MeC), U, or 5-Bromo U (BrU) at specific sites in its preferred trinucleotide 5'GNaNb3':5'Na,Nb,C3' (Na = A, C, or T). Results show that MeG:C and G:MeC in place of G:C improve direct strand cleavage at the target Nb (Nb = T greater than A much greater than C greater than G), whereas MeC:G and C:MeG in place of Na:Nb, hinder cleavage. The optimal base target at Nb appears to be determined by its ability to form T:A type base pairing instead of C:G type. The observed differences in DNA strand cleavage patterns can be rationalized by induced changes in target site structure and are compatible with a model for NCS-C:DNA interaction in which the naphthoate moiety intercalates between 5'GNa3', and the activated tetrahydro-s-indacene, lying in the minor groove, abstracts a hydrogen atom from C-5' of Nb.     

Antitumor agents 4. Characterization of free radicals produced during reduction of the antitumor drug 5H-pyridophenoxazin-5-one: an EPR study

5H-Pyridophenoxazin-5-one (PPH), a new anticancer iminoquinone, is able to inhibit a large number of lymphoblastoid and solid tumor-derived cells at submicromolar concentrations. Molecular modeling calculations indicated that this compound might intercalate into the DNA double strand. This was also supported by nuclear magnetic resonance studies. Since free radicals arising from anticancer quinonic drugs have been proposed to be key species responsible for DNA cleavage, we have aimed to intercept and identify free radicals from PPH generated under bioreductive conditions. The first and second monoelectronic reduction potentials of PPH were measured by means of cyclic voltammetry: the reduction potential of PPH is compatible with its reduction by compounds such as NADH, and suggested that reduction of PPH may play a role in its cytotoxicity. The radical anion PPH(*)(-) was detected by means of electron paramagnetic resonance spectroscopy, and its identification was supported by DFT calculations. EPR experiments in the presence of spin traps 5,5-dimethylpyrroline N-oxide and 5-(diethoxyphosphoryl)-5-methylpyrroline N-oxide suggested the occurrence of an electron transfer between the radical anion of the drug and oxygen resulting in the formation of the superoxide anion (O(2)(*)(-)). The enthalpy of the reaction of PPH(*)(-) with O(2) was determined both in the gas phase and in solution at the B3LYP/6-31+G level using the isodensity PCM method, and the overall process in dimethyl sulfoxide was predicted to be slightly exothermic. We propose that the monoelectronic reduction of PPH in the proximity of DNA may eventually lead to radicals that could cause considerable damage to DNA, thus accounting for the high cytotoxic activity of the drug. Indeed, a comet assay (alkaline single-cell electrophoresis) showed that PPH causes free radical-induced DNA damage.     

Developing a programmed restriction endonuclease for highly specific DNA cleavage

Specific cleavage of large DNA molecules at few sites, necessary for the analysis of genomic DNA or for targeting individual genes in complex genomes, requires endonucleases of extremely high specificity. Restriction endonucleases (REase) that recognize DNA sequences of 4-8 bp are not sufficiently specific for this purpose. In principle, the specificity of REases can be extended by fusion to sequence recognition modules, e.g. specific DNA-binding domains or triple-helix forming oligonucleotides (TFO). We have chosen to extend the specificity of REases using TFOs, given the combinatorial flexibility this fusion offers in addressing a short, yet precisely recognized restriction site next to a defined triple-helix forming site (TFS). We demonstrate here that the single chain variant of PvuII (scPvuII) covalently coupled via the bifunctional cross-linker N-(gamma-maleimidobutryloxy) succinimide ester to a TFO (5'-NH2-[CH2](6 or 12)-MPMPMPMPMPPPPPPT-3', with M being 5-methyl-2'-deoxycytidine and P being 5-[1-propynyl]-2'-deoxyuridine), cleaves DNA specifically at the recognition site of PvuII (CAGCTG) if located in a distance of approximately one helical turn to a TFS (underlined) complementary to the TFO ('addressed' site: 5'-TTTTTTTCTCTCTCTCN(approximately 10)CAGCTG-3'), leaving 'unaddressed' PvuII sites intact. The preference for cleavage of an 'addressed' compared to an 'unaddressed' site is >1000-fold, if the cleavage reaction is initiated by addition of Mg2+ ions after preincubation of scPvuII-TFO and substrate in the absence of Mg2+ ions to allow triple-helix formation before DNA cleavage. Single base pair substitutions in the TFS prevent addressed DNA cleavage by scPvuII-TFO.     

Water oxidation chemistry of photosystem II

The O(2)-evolving complex of photosystem II catalyses the light-driven four-electron oxidation of water to dioxygen in photosynthesis. In this article, the steps leading to photosynthetic O(2) evolution are discussed. Emphasis is given to the proton-coupled electron-transfer steps involved in oxidation of the manganese cluster by oxidized tyrosine Z (Y(*)(Z)), the function of Ca(2+) and the mechanism by which water is activated for formation of an O-O bond. Based on a consideration of the biophysical studies of photosystem II and inorganic manganese model chemistry, a mechanism for photosynthetic O(2) evolution is presented in which the O-O bond-forming step occurs via nucleophilic attack on an electron-deficient Mn(V)=O species by a calcium-bound water molecule. The proposed mechanism includes specific roles for the tetranuclear manganese cluster, calcium, chloride, Y(Z) and His190 of the D1 polypeptide. Recent studies of the ion selectivity of the calcium site in the O(2)-evolving complex and of a functional inorganic manganese model system that test key aspects of this mechanism are also discussed.     

Oligonucleotide-minor groove binder conjugates and their complexes with complementary DNA: effect of conjugate structural factors on the thermal stability of duplexes

Synthetic polycarboxamide minor groove binders (MGB) consisting of N-methylpyrrole (Py), N-methylimidazole (Im), N-methyl-3-hydroxypyrrole (Hp) and beta-alanine (beta) show strong and sequence-specific interaction with the DNA minor groove in side-by-side antiparallel or parallel orientation. Two MGB moieties covalently linked to the same terminal phosphate of one DNA strand stabilize DNA duplexes formed by this strand with a complementary one in a sequence-specific manner, similarly to the corresponding mono-conjugated hairpin structures. The series of conjugates with the general formula Oligo-(L-MGB-R)m was synthesized, where m = 1 or 2, L = linker, R = terminal charged or neutral group, MGB = -(Py)n-, -(Im)n- or -[(Py/Im)n-(CH2)3CONH-(Py/Im)n-] and I < n < 5. Using thermal denaturation, we studied effects of structural factors such as m and n, linker L length, nature and orientation of the MGB monomers, the group R and the backbone (DNA or RNA), etc. on the stability of the duplexes. Structural factors are more important for linear and hairpin monophosphoroamidates than for parallel bis-phosphoroamidates. No more than two oligocarboxamide strands can be inserted into the duplex minor groove. Attachment of the second sequence-specific parallel ligand [-L(Py)4R] to monophosphoroamidate conjugate CGTTTATT-L(Py)4R leads to the increase of the duplex Tm, whereas attachment of [-L(Im)4R] leads to its decrease. The mode of interaction between oligonucleotide duplex and attached ligands could be different (stacking with the terminal A:T pair of the duplex or its insertion into the minor groove) depending on the length and structure of the MGB.     

The Flp recombinase cleaves Holliday junctions in trans

The Flp site-specific recombinase is encoded by the 2 µm plasmid of Saccharomyces cerevisiae and is a member of the integrase family of recombinases. Like all members of the integrase family studied, Flp mediates recombination in two steps. First, a pair of strand exchanges creates a Holliday-like intermediate; second, this intermediate is resolved to recombinant products by a second pair of strand exchanges.
Evidence derived from experiments using linear substrates indicates that Flp's active site is composed of two Flp protomers. One binds to the Flp recognition target site (FRT site) and activates the scissile phosphodiester bond for cleavage. Another molecule of Flp bound elsewhere in the synaptic complex ( in trans ) donates the nucleophilic tyrosine that executes cleavage and thereby becomes covalently attached to the 3' phosphoryl group at the cleavage site.
It has previously been shown that Flp efficiently resolves synthetic, Holliday-like (χ) structures to linear products. In this paper, we examined whether resolution of χ structures by Flp also occurs via the trans cleavage mechanism. We used in vitro complementation studies of mutant Flp proteins as well as nicked χ structures to show that Flp resolves χ structures by trans cleavage. We propose a model for Flp-mediated recombination that incorporates trans cleavage at both the initial and resolution steps of strand exchange.     

Coupled electron transfers in artificial photosynthesis

Light-induced charge separation in molecular assemblies has been widely investigated in the context of artificial photosynthesis. Important progress has been made in the fundamental understanding of electron and energy transfer and in stabilizing charge separation by multi-step electron transfer. In the Swedish Consortium for Artificial Photosynthesis, we build on principles from the natural enzyme photosystem II and Fe-hydrogenases. An important theme in this biomimetic effort is that of coupled electron-transfer reactions, which have so far received only little attention. (i) Each absorbed photon leads to charge separation on a single-electron level only, while catalytic water splitting and hydrogen production are multi-electron processes; thus there is the need for controlling accumulative electron transfer on molecular components. (ii) Water splitting and proton reduction at the potential catalysts necessarily require the management of proton release and/or uptake. Far from being just a stoichiometric requirement, this controls the electron transfer processes by proton-coupled electron transfer (PCET). (iii) Redox-active links between the photosensitizers and the catalysts are required to rectify the accumulative electron-transfer reactions, and will often be the starting points of PCET.