Cationic 5,10,15,20-tetrakis(n-methylpyridinium-4-yl)porphyrin Fully Intercalates At 5 \u27-cg-3 \u27 Steps Of Duplex Dna In Solution

The interaction of 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)p (T4MPyP(4+)) with the oligonucleotide DNA duplex [d(GCACGTGC)](2) was studied by two-dimensional (1)H NMR spectroscopy, optical absorbance, circular dichroism, and molecular dynamics simulation employing particle mesh Ewald methods. T4MPyP(4+) is one of the largest aromatic molecules for which intercalative binding to DNA has been proposed, although this has been called into question by recent X-ray crystallographic work [Lipscomb et al. (1996) Biochemistry 35, 2818-2823]. T4MPyP(4+) binding to [d(GCACGTGC)](2) produced a single set of (mostly) upfield-shifted DNA resonances in slow exchange with the resonances of the free DNA. Intra- and intermolecular NOEs observed in the complex showed that the porphyrin intercalates at the central 5\u27-CG-3\u27 step of the DNA duplex without disrupting the flanking base pairs. Absorption and circular dichroism spectra of the complex also support intercalative binding. Molecular dynamics simulations (using explicit solvent and PME methods), carried out for fully and partially intercalated complexes, yielded stable trajectories and plausible structures, but only the symmetrical, fully intercalated model agreed with NOESY data. Stable hydrogen bonding was observed during 600 ps of MD simulation for the base pairs flanking the binding site

Photo Processes On Self-associated Cationic Porphyrins And Plastocyanin Complexes 1. Ligation Of Plastocyanin Tyrosine 83 Onto Metalloporphyrins And Electron-transfer Fluorescence Quenching

The spectroscopic properties of the self-associated complexes formed between the anionic surface docking site of spinach plastocyanin and the cationic metalloporphyrins, in which the tyrosine 83 (Y83) moiety is placed just below the docking site, tetrakis(N-methyl-4-pyridyl)porphyrin (Pd(II)TMPyP4+ and Zn(II)TMPyP4+), have been studied and reported herein. The fluorescence quenching phenomenon of the self-assembled complex of Zn(II)TMPyP4+/plastocyanin has also been discovered. The observed red-shifting of the Soret and Q-bands of the UV-visible spectra, ca. 9 nm for Pd(II)TMPyP4+/plastocyanin and ca. 6 nm for the Zn(II)TMPyP4+/ plastocyanin complexes, was explained in terms of exciton theory coupled with the Gouterman model. Thus, the hydroxyphenyl terminus of the Y83 residue of the self-associated plastocyanin/cationic porphyrin complexes was implicated in the charge-transfer ligation with the central metal atoms of these metalloporphyrins. Moreover, ground-state spectrometric-binding studies between Pd(II)TMPyP4+ and the Y83 mutant plastocyanin (Y83F-PC) system proved that Y83 moiety of plastocyanin played a critical role in the formation of such ion-pair complexes. Difference absorption spectra and the Job\u27s plots showed that the electrostatic attractions between the cationic porphyrins and the anionic patch of plastocyanin, bearing the nearby Y83 residue, led to the predominant formation of a self-associated 1:1 complex in the ground-state with significantly high binding constants (K = (8.0 +/- 1.1) x 10(5) M-1 and (2.7 +/- 0.8) x 10(6) M-1 for Pd(II)TMPyP4+ and zinc variant, respectively) in low ionic strength buffer, 1 mM KCl and 1 mM phosphate buffer (pH 7.4). Molecular modeling calculations supported the formation of a 1: 1 self-associated complex between the porphyrin and plastocyanin with an average distance of ca. 9 A between the centers of mass of the porphyrin and Y83 positioned just behind the anionic surface docking site on the protein surface. The photoexcited singlet state of Zn(II)-TMPyP4+ was quenched by the Y83 residue of the self-associated plastocyanin in a static mechanism as evidenced by steady-state and time-resolved fluorescence experiments. Even when all the porphyrin was complexed (more than 97%), significant residual fluorescence from the complex was observed such that the amplitude of quenching of the singlet state of uncomplexed species was enormously obscured

DNA damage: Alkylation

Alkylation is the reaction of electrophilic chemical compounds or alkylating agents with the nucleophilic centers in organic macromolecules. Alkylating agents include a large variety of chemicals, some of which are potent mutagens or carcinogens

Cadmium(II) inhibition of human uracil-DNA glycosylase by catalytic water supplantation.

Toxic metals are known to inhibit DNA repair but the underlying mechanisms of inhibition are still not fully understood. DNA repair enzymes such as human uracil-DNA glycosylase (hUNG) perform the initial step in the base excision repair (BER) pathway. In this work, we showed that cadmium [Cd(II)], a known human carcinogen, inhibited all activity of hUNG at 100 μM. Computational analyses based on 2 μs equilibrium, 1.6 μs steered molecular dynamics (SMD), and QM/MM MD determined that Cd(II) ions entered the enzyme active site and formed close contacts with both D145 and H148, effectively replacing the catalytic water normally found in this position. Geometry refinement by density functional theory (DFT) calculations showed that Cd(II) formed a tetrahedral structure with D145, P146, H148, and one water molecule. This work for the first time reports Cd(II) inhibition of hUNG which was due to replacement of the catalytic water by binding the active site D145 and H148 residues. Comparison of the proposed metal binding site to existing structural data showed that D145:H148 followed a general metal binding motif favored by Cd(II). The identified motif offered structural insights into metal inhibition of other DNA repair enzymes and glycosylases

Metal inhibition of human alkylpurine-DNA-N-glycosylase activity in base excision repair

Cadmium (Cd{sup 2+}), nickel (Ni{sup 2+}) and cobalt (Co{sup 2+}) are human and/or animal carcinogens. Zinc (Zn{sup 2+}) is not categorized as a carcinogen, and rather an essential element to humans. Metals were recently shown to inhibit DNA repair proteins that use metals for their function and/or structure. Here we report that the divalent ions Cd{sup 2+}, Ni{sup 2+}, and Zn{sup 2+} can inhibit the activity of a recombinant human N-methylpurine-DNA glycosylase (MPG) toward a deoxyoligonucleotide with ethenoadenine (var epsilonA). MPG removes a variety of toxic/mutagenic alkylated bases and does not require metal for its catalytic activity or structural integrity. At concentrations starting from 50 to 1000 {micro}M, both Cd{sup 2+} and Zn{sup 2+} showed metal-dependent inhibition of the MPG catalytic activity. Ni{sup 2+} also inhibited MPG, but to a lesser extent. Such an effect can be reversed with EDTA addition. In contrast, Co{sup 2+} and Mg{sup 2+} did not inhibit the MPG activity in the same dose range. Experiments using HeLa cell-free extracts demonstrated similar patterns of inactivation of the var epsilonA excision activity by the same metals. Binding of MPG to the substrate was not significantly affected by Cd{sup 2+}, Zn{sup 2+}, and Ni{sup 2+} at concentrations that show strong inhibition of the catalytic function, suggesting that the reduced catalytic activity is not due to altered MPG binding affinity to the substrate. Molecular dynamics (MD) simulations with Zn{sup 2+} showed that the MPG active site has a potential binding site for Zn{sup 2+}, formed by several catalytically important and conserved residues. Metal binding to such a site is expected to interfere with the catalytic mechanism of this protein. These data suggest that inhibition of MPG activity may contribute to metal genotoxicity and depressed repair of alkylation damage by metals in vivo

Structural insights by molecular dynamics simulations into specificity of the major human AP endonuclease toward the benzene-derived DNA adduct, pBQ-C

The benzetheno exocyclic adduct of the cytosine (C) base (pBQ-C) is a product of reaction between DNA and a stable metabolite of the human carcinogen benzene, p-benzoquinone (pBQ). We reported previously that the pBQ-C-containing duplex is a substrate for the human AP endonuclease (APE1), an enzyme that cleaves an apurinic/apyrimidinic (AP) site from double stranded DNA. In this work, using molecular dynamics simulation (MD), we provided a structural explanation for the recognition of the pBQ-C adduct by APE1. Molecular modeling of the DNA duplex containing pBQ-C revealed significant displacement of this adduct toward the major groove with pronounced kinking of the DNA at the lesion site, which could serve as a structural element recognized by the APE1 enzyme. Using 3 ns MD it was shown that the position of the pBQ-C adduct is stabilized by two hydrogen bonds formed between the adduct and the active site amino acids Asp 189 and Ala 175. The pBQ-C/APE1 complex, generated by MD, has a similar hydrogen bond network between target phosphodiester bond at the pBQ-C site and key amino acids at the active site, as in the crystallographically determined APE1 complexed with an AP site-containing DNA duplex. The position of the adduct at the enzyme active site, together with the hydrogen bond network, suggests a similar reaction mechanism for phosphodiester bond cleavage of oligonucleotide containing pBQ-C as reported for the AP site

Chloroethyinitrosourea-derived ethano cytosine and adenine adducts are substrates for escherichia coli glycosylases excising analogous etheno adducts

Exocyclic ethano DNA adducts are saturated etheno ring derivatives formed mainly by therapeutic chloroethylnitrosoureas (CNUs), which are also mutagenic and carcinogenic. In this work, we report that two of the ethano adducts, 3,N{sup 4}-ethanocytosine (EC) and 1,N{sup 6}-ethanoadenine (EA), are novel substrates for the Escherichia coli mismatch-specific uracil-DNA glycosylase (Mug) and 3-methyladenine DNA glycosylase II (AlkA), respectively. It has been shown previously that Mug excises 3,N{sup 4}-ethenocytosine ({var_epsilon}C) and AlkA releases 1,N{sup 6}-ethenoadenine ({var_epsilon}A). Using synthetic oligonucleotides containing a single ethano or etheno adduct, we found that both glycosylases had a {approx}20-fold lower excision activity toward EC or EA than that toward their structurally analogous {var_epsilon}C or {var_epsilon}A adduct. Both enzymes were capable of excising the ethano base paired with any of the four natural bases, but with varying efficiencies. The Mug activity toward EC could be stimulated by E. coli endonuclease IV and, more efficiently, by exonuclease III. Molecular dynamics (MD) simulations showed similar structural features of the etheno and ethano derivatives when present in DNA duplexes. However, also as shown by MD, the stacking interaction between the EC base and Phe 30 in the Mug active site is reduced as compared to the {var_epsilon}C base, which could account for the lower EC activity observed in this study

Cadmium(II) inhibition of human uracil-DNA glycosylase by catalytic water supplantation.

Toxic metals are known to inhibit DNA repair but the underlying mechanisms of inhibition are still not fully understood. DNA repair enzymes such as human uracil-DNA glycosylase (hUNG) perform the initial step in the base excision repair (BER) pathway. In this work, we showed that cadmium [Cd(II)], a known human carcinogen, inhibited all activity of hUNG at 100 μM. Computational analyses based on 2 μs equilibrium, 1.6 μs steered molecular dynamics (SMD), and QM/MM MD determined that Cd(II) ions entered the enzyme active site and formed close contacts with both D145 and H148, effectively replacing the catalytic water normally found in this position. Geometry refinement by density functional theory (DFT) calculations showed that Cd(II) formed a tetrahedral structure with D145, P146, H148, and one water molecule. This work for the first time reports Cd(II) inhibition of hUNG which was due to replacement of the catalytic water by binding the active site D145 and H148 residues. Comparison of the proposed metal binding site to existing structural data showed that D145:H148 followed a general metal binding motif favored by Cd(II). The identified motif offered structural insights into metal inhibition of other DNA repair enzymes and glycosylases