Guanine versus deoxyribose damage in DNA oxidation mediated by vanadium(IV) and vanadium(V) complexes

Vanadyl sulfate reacts with the peroxy acid oxidant KHSO5 to produce guanine-selective oxidation of a 167-bp restriction fragment of DNA. The oxidized lesions result in strand scission after hot piperidine treatment. Although several reactive intermediates are possible, quenching studies with ethanol and tert-butyl alcohol suggest that a monoperoxysulfate radical or a caged sulfate radical are the likely species responsible for oxidation of guanine. Several oxidants and various vanadium complexes (including insulin mimetic compounds) were studied with DNA for comparison. None of the other vanadium complexes showed modification of the double-stranded 167-bp fragment of DNA in the presence of KHSO5. The reactivity of VOSO4 may be due to its irreversible oxidation potential of 0.77 V (vs. Ag+/AgCl, pH 7.0, 10 mM phosphate), making it an appropriate catalyst for decomposition of monoperoxysulfate.     

On the chemical nature of DNA and RNA modification by a hemin model system.

In order to model the interaction of hemin with DNA and other polynucleotides, we have studied the degradation of DNA, RNA, and polynucleotides of defined structure by [meso-tetrakis(N-methyl-4-pyridyl)porphinato]manganese(III) (MnTMPP) + KHSO5. The activated porphyrin was shown to release adenine, thymine, and cytosine from DNA; RNA degradation afforded adenine, uracil, and cytosine. The same products were obtained from single- and double-stranded DNA oligonucleotides of defined sequence, and also from single-stranded DNA and RNA homopolymers. The overall yield of bases from the dode-canucleotide d(CGCT3A3GCG) was equal to 14% of the nucleotides present initially, indicating that each porphyrin catalyzed the release of approximately 4 bases. Although no guanine was detected as a product from any of the substrates studied, the ability of MnTMPP + KHSO5 to degrade guanine nucleotides was verified by the destruction of pGp, and by the appearance of bands corresponding to guanosine cleavage following treatment of 32P end labeled DNA restriction fragments with activated MnTMPP. Inspection of a number of sites of MnTMPP-promoted cleavage indicated that the process was sequence-selective, occurring primarily at G residues that were part of 5'-TG-3' or 5'-AG-3' sequences, or at T residues. Also formed in much greater abundance were alkali-labile lesions; these were formed largely at guanosine residues. Also studied was the degradation of a 47-nucleotide RNA molecule containing two hairpins. Degradation of the 5'-32P end labeled RNA substrate afforded no distinct, individual bands, suggesting that multiple modes of degradation may be operative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Magnesium monoperoxophtalate: an efficient single oxygen atom donor in DNA cleavage catalyzed by metalloporphyrin

The peracid compound magnesium monoperoxophtalate (MMPP), water-soluble at physiological pH, behaves as a promising oxygen donor in the oxidative cleavage of DNA catalyzed by meso-tetrakis-(4-N-methylpyridyl)porphyrinatomanganese(III) pentaacetate. Shown on cleavage of supercoiled phi X174 DNA, MMPP is significantly more efficient than KHSO5, another recently developed single oxygen atom donor (Fouquet et al., J. Chem. Soc., Chem. Commun., 1987, 1169).     

31P NMR characterization of terminal phosphates induced on DNA by the artificial nuclease ''Mn-TMPyP/KHSO5'' in comparison with DNases I and II.

Phosphorus-31 NMR has been applied to the characterization of terminal phosphates on fragments of calf thymus DNA induced by three different nuclease systems: DNase I, DNase II and the artificial nuclease 'Mn-TMPyP/KHSO5'. In this last case, the oxidative damage to deoxyribose leads to two monophosphates esters (at the 3' and 5' ends) on both sides of the cleavage site. This method constitutes a promising approach to visualise the phosphate termini generated in DNA or RNA cleavage by cytotoxic drugs or chemical nucleases and provides a novel insight into the molecular aspects of their mechanism of action.     

Oxidative cleavage of DNA mediated by hybrid metalloporphyrin-ellipticine molecules and functionalized metalloporphyrin precursors

The nuclease activity of functionalized metalloporphyrins 1-8 and hybrid metalloporphyrin-ellipticine molecules 10-16 in the presence of potassium monopersulfate (KHSO5) or magnesium monoperoxyphthalate (MMPP), water-soluble oxygen atom donors at physiological pH, toward double-stranded phi X174 DNA is reported. The DNA cleavage efficiency as a function of the nature of functionalized metalloporphyrins, the length of the linkage between the two parts of the hybrid molecule, viz., metalloporphyrin and 9-methoxyellipticine, the nature of the central metal atom (Mn, Fe, or Zn) the ionic strength, and the nature of the oxygen donor has been studied. Single-strand breaks (SSBs) are observed on double-stranded DNA with a short incubation time of 2 min in the presence of manganese derivatives of both metalloporphyrins and hybrid molecules. Owing to their cytotoxic and nuclease activity, these new water-soluble hybrid molecules may be considered as efficient bleomycin models based on cationic metalloporphyrins.     

Oxidative damage generated by an oxo-metalloporphyrin onto the human telomeric sequence

The cationic metalloporphyrin Mn-TMPyP activated by KHSO(5) has been used as cleaver of an oligonucleotide containing the four human telomere repeats of 5'-GGGTTA. This oligonucleotide formed an intramolecular quadruplex DNA under 200 mM KCl as probed by DMS footprinting and could fold into different quadruplex structures under 200 mM NaCl. We found that the oxo-metalloporphyrin was able to mediate efficient oxidative cleavage of the quadruplex. The location of damage showed that the metalloporphyrin was able to bind to the last G-tetrad of the quadruplex structure via an external interaction. This metalloporphyrin-G-tetrad interaction needs a relatively high flexibility of the single-stranded linker regions to allow the partial stacking of the metalloporphyrin with the last G-tetrad planar structure. The oxidative damage consisted of guanine oxidation within the interacting G-tetrad together with an 1'-carbon hydroxylation of deoxyribose residues of the thymidine residues located on the neighboring single-stranded loop. So the high-valent oxo-metalloporphyrin is able to mediate both electron-abstraction or H-abstraction on G or T residues, respectively, within the DNA quadruplex target.     

DNA methylation by N-methyl-N-nitrosourea: methylation pattern changes in single- and double-stranded DNA, and in DNA with mismatched or bulged guanines.

The detection of abnormal DNA base pairing arrangements and conformations is chemically probed in synthetic 32P-end-labeled deoxyribonucleotide oligomers using N-methyl-N-nitrosourea (MNU) and 2,12,-dimethyl-3,7,11,17-tetraazabicyclo-[11.3.1]heptadeca-1 -[17],2,11,13,15 pentaene-Ni (II) (Ni-complex) with KHSO5. The DNA targets studied are single-stranded (s-s) DNA, double-stranded (d-s) DNA, d-s DNA with G-G, G-A and G-T mismatches, d-s DNA with a single bulged G and d-s DNA with two bulged G's. The effect of the non-Watson--Crick structures on the formation of N7-methylguanine (N7-MeG) by MNU and the oxidation of G by Ni-complex is reported along with the Tm's and circular dichroism spectra of the different duplex oligomers. The results for MNU and Ni-complex show that the qualitative and quantitative character of the cleavage patterns at a G3 run change with the nature of the abnormal base pairing motif. Based on the DNA substrates studied, the results indicate that a combination of reagents which report electronic and steric perturbations can be a useful approach to monitor DNA mismatches and bulges.     

Synthesis of a metallopeptide-PNA conjugate and its oxidative cross-linking to a DNA target

A nickel(II)-PNA bioconjugate was prepared by formation of a salicylaldimine complex with the amino terminus of a peptide-PNA hybrid with the sequence Arg-His-Gly-[TACCTAGCAT]PNA-Arg-CONH2. Hybridization to complementary oligodeoxynucleotides was demonstrated, and covalent adduct formation was observed upon addition of KHSO5 as oxidant. In the absence of PNA, the reactivity of the phenolic radical generated as an intermediate was found to be G > T > C, A; by inclusion of the PNA delivery agent, cross-links between the two oligomers could be observed with T and C bases in the vicinity of the nickel complex, although G was still the most reactive site. The metal complex could be removed by treatment with EDTA following which the Schiff base linkage was readily hydrolyzed. The final result in this case is a salicylaldehyde moiety appended at the target site in DNA.     

Perferryl complex of nitric oxide synthase: role in secondary free radical formation

Neuronal nitric oxide synthase (NOS I) has been shown to generate nitric oxide (NO*) and superoxide (O(2)*-)during enzymatic cycling, the ratio of each free radical is dependent upon the concentration of L-arginine. Using spin trapping and electron paramagnetic resonance (EPR) spectroscopy, we recently reported that NOS I can oxidize ethanol (EtOH) to alpha-hydroxyethyl radical (CH(3)*CHOH). We speculated that the perferryl complex of NOS, (NOS-[Fe(5+)[double bond]O](3+)) was responsible for the generation of CH(3)*CHOH. Using potassium monopersulfate (KHSO(5)) to oxidize the heme of NOS I to NOS-[Fe(5+)[double bond]O](3+), we were able to demonstrate that this perferryl complex can oxidize L-arginine to L-citrulline and NO*. Even in the absence of L-arginine, EtOH was oxidized to CH(3)*CHOH by NOS-[Fe(5+)[double bond]O](3+). Sodium cyanide (NaCN), a heme blocker, inhibited the formation of CH(3)*CHOH by NOS.     

Assessment of synthetic methods for the preparation of N-beta-d-glucopyranosyl-N'-substituted ureas, -thioureas and related compounds

Preparation of O-peracetylated N-beta-d-glucopyranosyl-N'-acyl urea derivatives resulted in the formation of anomeric mixtures under the following conditions: acylation of O-peracetylated beta-d-glucopyranosyl urea by acyl chlorides in the presence of ZnCl(2) in refluxing CHCl(3); addition of O-peracetylated beta-d-glucopyranosylamine to acyl isocyanates in acetonitrile at rt; addition of carboxamides to in situ prepared O-peracetylated beta-d-glucopyranosyl isocyanate in refluxing toluene. Deprotection of O-peracetylated N-beta-d-glucopyranosyl-N'-acyl ureas either under base (NaOMe in MeOH at or below rt) or under acid (KHSO(4) or AcCl in MeOH at rt) catalyzed transesterification conditions resulted in unavoidable partial cleavage of the N'-acyl moieties. Reaction of beta-d-glucopyranosylammonium carbamate with an isocyanate, isothiocyanate or isoselenocyanate in dry pyridine at rt appears as a general method for the preparation of the corresponding beta-d-glucopyranosyl ureas, -thio- and -selenoureas, respectively, inclusive N'-acyl derivatives.     

Effects of humic substances on the oxidation of pentachlorophenol by peroxosulfate catalyzed by iron(III)-phthalocyanine-tetrasulfonic acid

In an attempt to enhance the oxidation of pentachlorophenol (PCP) in the Fe(III)-PcTS/KHSO5 system, the presence of added humic substances (HSs) was studied, investigating the chemical properties of HSs related to the enhancement in PCP oxidation by correlations with the degree of enhancement in PCP oxidation (%delta(PCP)60). The %delta(PCP)60 value increased with a decrease in the content of oxygen-containing functional groups, such as carboxylic acids. This indicated that HSs with a lower content of oxygen-containing functional groups would be useful for enhancing the oxidation of PCP. A negative correlation between %delta(PCP)60 and the kinetic constants of Fe(III)-PcTS self-oxidation indicated that the enhancement by added HSs could be attributed to the suppression of Fe(III)-PcTS deactivation by self-oxidation. Such a stabilization of Fe(III)-PcTS could be attributed to hydrophobic interactions between the catalyst and HSs.     

Interaction between macrocyclic nickel complexes and the nucleotides GMP,AMP and ApG

Reactions between the nucleotides GMP, AMP and ApG and the complexes Ni(tren), Ni(cyclam) and NiCR in aqueous solution have been monitored by (1)H, (15)N NMR and UV spectroscopy. The three nickel complexes display different properties in reactions with nucleotides. Ni(tren) which has a pseudo-octahedral coordination geometry was shown to bind to all three nucleotides. Ni(cyclam) and NiCR, both with four nitrogen atoms in a square planar arrangement are not able to bind to nucleotides efficiently because of steric hindrance. Oxidation of Ni(cyclam) by KHSO(5) to produce trivalent Ni(III)(cyclam) improves the coordination capacity, while oxidation of NiCR does not produce a similar effect. The nucleotides interact with trivalent nickel complexes to different extent. Ni(III)CR is seen to oxidize GMP gradually but does not affect AMP significantly. Ni(III)(cyclam), on the other hand, does not oxidize either GMP or AMP at the 1:1 concentration of oxidant used. This result is probably due to the lower redox potential of Ni(cyclam). ApG binds less efficiently to the Ni complexes but is easier oxidized than the mononucleotides.     

Deoxyribonucleic acid polymerase of bacteriophage T7. Characterization of the exonuclease activities of the gene 5 protein and the reconstituted polymerase.

Homogeneous gene 5 protein of bacteriophage T7, a subunit of T7 DNA polymerase, catalyzes the stepwise hydrolysis of single-stranded DNA in a 3' leads to 5' direction to yield nucleoside 5'-monophosphates. The gene 5 protein itself does not hydrolyze duplex DNA. However, in the presence of Escherichia coli thioredoxin, the host-specified subunit of T7 DNA polymerase, duplex DNA is hydrolyzed in a 3' leads to 5' direction to yield nucleoside 5'-monophosphates. The apparent Km for thioredoxin in the reaction is 4.8 x 10(-8) M, a value similar to that for the apparent Km of thioredoxin in the complementation assay with gene 5 protein to restore T7 DNA polymerase activity. Both exonuclease activities require Mg2+ and a sulfhydryl reagent for optimal activity, and both activities are sensitive to salt concentration. Deoxyribonucleoside 5'-triphosphates inhibit hydrolysis by both exonuclease activities; hydrolysis of single-stranded DNA by the gene 5 protein is inhibited even in the absence of thioredoxin where there is less than 2% active T7 DNA polymerase. E. coli DNA binding protein (helix destabilizing protein) stimulates the hydrolysis of duplex DNA up to 9-fold under conditions where the hydrolysis of the single-stranded DNA is inhibited 4-fold.     

2'',3''-Dideoxy-3'' aminonucleoside 5''-triphosphates are the terminators of DNA synthesis catalyzed by DNA polymerases.

It is shown that 2',3'-dideoxy-3'-aminonucleoside 5'-triphosphates with adenine, guanine, cytosine and thymine bases are effective inhibitors of DNA polymerase I, calf thymus DNA polymerase alpha and rat liver DNA polymerase beta. The effect of the above-mentioned compounds is markedly higher than corresponding action of the well-known DNA synthesis inhibitors arabinonucleoside 5'-triphosphates and 2',3'-dideoxynucleoside 5'-triphosphates. 2',3'-dideoxy-3'-aminonucleoside 5'-monophosphate residues incorporate into the 3'-terminus of the primer and terminate the DNA chain elongation. The possibility of using 2',3'-dideoxy-3'-aminonucleoside 5'-triphosphates as terminators for DNA sequencing by the polymerization method is demonstrated.     

DNA double-strand breaks with 5′ adducts are efficiently channeled to the DNA2-mediated resection pathway

DNA double-strand breaks (DSBs) with 5′ adducts are frequently formed from many nucleic acid processing enzymes, in particular DNA topoisomerase 2 (TOP2). The key intermediate of TOP2 catalysis is the covalent complex (TOP2cc), consisting of two TOP2 subunits covalently linked to the 5′ ends of the nicked DNA. In cells, TOP2ccs can be trapped by cancer drugs such as etoposide and then converted into DNA double-strand breaks (DSBs) that carry adducts at the 5′ end. The repair of such DSBs is critical to the survival of cells, but the underlying mechanism is still not well understood. We found that etoposide-induced DSBs are efficiently resected into 3′ single-stranded DNA in cells and the major nuclease for resection is the DNA2 protein. DNA substrates carrying model 5′ adducts were efficiently resected in Xenopus egg extracts and immunodepletion of Xenopus DNA2 also strongly inhibited resection. These results suggest that DNA2-mediated resection is a major mechanism for the repair of DSBs with 5′ adducts.     

DNA activates the Nse2/Mms21 SUMO E3 ligase in the Smc5/6 complex

Modification of chromosomal proteins by conjugation to SUMO is a key step to cope with DNA damage and to maintain the integrity of the genome. The recruitment of SUMO E3 ligases to chromatin may represent one layer of control on protein sumoylation. However, we currently do not understand how cells upregulate the activity of E3 ligases on chromatin. Here we show that the Nse2 SUMO E3 in the Smc5/6 complex, a critical player during recombinational DNA repair, is directly stimulated by binding to DNA. Activation of sumoylation requires the electrostatic interaction between DNA and a positively charged patch in the ARM domain of Smc5, which acts as a DNA sensor that subsequently promotes a stimulatory activation of the E3 activity in Nse2. Specific disruption of the interaction between the ARM of Smc5 and DNA sensitizes cells to DNA damage, indicating that this mechanism contributes to DNA repair. These results reveal a mechanism to enhance a SUMO E3 ligase activity by direct DNA binding and to restrict sumoylation in the vicinity of those Smc5/6‐Nse2 molecules engaged on DNA.     

Role for the adenovirus IVa2 protein in packaging of viral DNA

Although it has been demonstrated that the adenovirus IVa2 protein binds to the packaging domains on the viral chromosome and interacts with the viral L1 52/55-kDa protein, which is required for viral DNA packaging, there has been no direct evidence demonstrating that the IVa2 protein is involved in DNA packaging. To understand in greater detail the DNA packaging mechanisms of adenovirus, we have asked whether DNA packaging is serotype or subgroup specific. We found that Ad7 (subgroup B), Ad12 (subgroup A), and Ad17 (subgroup D) cannot complement the defect of an Ad5 (subgroup C) mutant, pm8001, which does not package its DNA due to a mutation in the L1 52/55-kDa gene. This indicates that the DNA packaging systems of different serotypes cannot interact productively with Ad5 DNA. Based on this, a chimeric virus containing the Ad7 genome except for the inverted terminal repeats and packaging sequence from Ad5 was constructed. This chimeric virus replicates its DNA and synthesizes Ad7 proteins, but it cannot package its DNA in 293 cells or 293 cells expressing the Ad5 L1 52/55-kDa protein. However, this chimeric virus packages its DNA in 293 cells expressing the Ad5 IVa2 protein. These results indicate that the IVa2 protein plays a role in viral DNA packaging and that its function is serotype specific. Since this chimeric virus cannot package its own DNA, but produces all the components for packaging Ad7 DNA, it may be a more suitable helper virus for the growth of Ad7 gutted vectors for gene transfer.