Effects of lead acetate on DNA and RNA synthesis by intact HeLa cells, isolated nuclei and purified polymerases

The effects of lead acetate on DNA and RNA synthesis have been investigated with intact HeLa cells, isolated nuclei, and purified DNA and RNA polymerases. No inhibition of DNA or RNA synthesis in intact cells was found even after exposure to 0.5 mM lead acetate for 18 hr. In contrast, both DNA and RNA synthesis in isolated nuclei were inhibited by lead (with 50% inhibition at approximately 150 and 80 microM respectively). Similarly, both HeLa DNA polymerase alpha and RNA polymerase II were inhibited, with 50% inhibition obtained at approximately 150 and 20 microM lead acetate respectively. The inhibition of nucleic acid synthesis in isolated nuclei can thus be accounted for by inhibition of the polymerases. The sensitivity of Escherichia coli DNA polymerase I to lead acetate was found to be significantly greater than the HeLa DNA polymerase alpha (50% inhibition at only 10 microM), but the sensitivity of the E. coli RNA polymerase was the same as that of the HeLa enzyme.     

Inhibition of DNA synthesis by chromium compounds

Cr(VI) irreversibly inhibited DNA synthesis in cultured mouse L cells to 50% of controls at 10 microM; 3.3 mM Cr(III) did not. At 0.3 mM, Cr(III) and Cr(VI) inhibited DNA synthesis in permeabilized L cells to 50% of control values. Cr(III) was a stronger inhibitor of DNA synthesis in the DNA-Escherichia coli DNA polymerase I system than was Cr(VI). The inhibitory effect of Cr(VI) depended on the ratio of Cr/DNA and Cr/enzyme; on the other hand, the increase in the concentration of DNA polymerase did not affect the inhibition of Cr(III), Cr(III), below the inhibitory concentration, produced an increase in the incorporation of [3H]dTMP into DNA; this was not observed with Cr(VI).     

Inhibition of Escherichia coli DNA polymerase I catalysed DNA polymerization by trans-imidazolium-bisimidazoletetrachlororuthenate(III).

The tumor-inhibiting metal complex trans-imidazolium-bisimidazoletetrachlororuthenate(III) (ICR) reacts with DNA and inhibits template-primer properties for DNA synthesis catalysed by Escherichia coli DNA polymerase I. The reaction with DNA depends on the aging (half-life 6.8 h) of the aqueous solution containing ICR. The kinetics of the reaction with DNA are reminiscent of those for cisplatin.     

Synthesis and biochemical properties of cyanuric acid nucleoside-containing DNA oligomers.

1-(2-Deoxy-beta-D-erythro-pentofuranosyl)cyanuric acid (cyanuric acid nucleoside, dY) (1) has been shown to be formed upon exposure of DNA components to ionizing radiation and excited photosensitizers. To investigate the biological and structural significance of dY residue in DNA, the latter modified 2'-deoxynucleoside was chemically prepared and then site-specifically incorporated into oligodeoxyribonucleotides (ODNs). This was achieved in good yields using the phosphoramidite approach. For this purpose, a convenient glycosylation method involving 3,5-protected 2-deoxyribofuranoside chloride and cyanuric acid (2,4,6-trihydroxy-1,3,5-triazine) was devised. The anomeric mixture of modified 2'-deoxyribonucleosides (1/2 alpha/beta) was resolved by silica gel purification of the 5'-O-dimethoxytritylated derivatives, and then, phosphitylation afforded the desired beta-phosphoramidite monomer (5). After solid-phase condensation and final deprotection, the purity and the integrity of the modified synthetic DNA fragments were checked using different complementary techniques such as HPLC and polyacrylamide gel electrophoresis, together with electrospray ionization and MALDI-TOF mass spectrometry. The presence of cyanuric acid nucleoside in a 14-mer was found to have destabilizing effects on the double-stranded DNA fragment as inferred from melting temperature measurements. The piperidine test applied to dY-containing ODNs supported the high stability of cyanuric acid nucleoside inserted into the oligonucleotide chain. Several enzymatic experiments aimed at determining the biological features of such a DNA lesion were carried out. Thus, processing of dY by nuclease P(1), snake venom phosphodiesterase (SVPDE), calf spleen phosphodiesterase (CSPDE), and repair enzymes, including Escherichia coli endonuclease III (endo III) and Fapy glycosylase (Fpg), was investigated. Finally, a 22-mer ODN bearing a cyanuric acid residue was used as a template to study the in vitro nucleotide incorporation opposite the damage by the Klenow fragment of E. coli polymerase I.     

Repair and coding properties of 5-hydroxy-5-methylhydantoin nucleosides inserted into DNA oligomers

1-(2-Deoxy-beta-D-erythro-pentofuranosyl)-5-hydroxy-5-methylhydantoin (5-OH-5-Me-dHyd) (3) has been shown to be a major oxidation product of thymidine formed upon exposure of DNA to (*)OH-radical and excited photosensitizers. To investigate the biological and structural significance of the 5-OH-5-Me-dHyd residue to DNA, the latter modified 2'-deoxyribonucleoside was chemically prepared and then site-specifically incorporated into oligodeoxyribonucleotides. This was efficiently achieved using the phosphoramidite approach that involved mild deprotection conditions. The purity and the integrity of the modified synthetic DNA fragments were checked using different complementary techniques such as HPLC and polyacrylamide gel electrophoresis, together with electrospray ionization and MALDI-TOF mass spectrometry. The piperidine test applied to 5-OH-5-Me-dHyd containing oligonucleotides showed a weak instability of hydantoin nucleoside inserted into the oligonucleotide chain. Several enzymatic experiments aimed at determining the biochemical features of such a DNA lesion were carried out. Thus, processing of 5-OH-5-Me-dHyd by nuclease P(1), snake venom phosphodiesterase, and calf spleen phosphodiesterase was investigated. The specificity and the mechanism of excision of the lesion by several bacterial and yeast DNA N-glycosylases, namely, endonuclease III (endo III), endonuclease VIII (endo VIII), formamidopyrimidine DNA N-glycosylase (Fpg), Ntg1 protein (Ntg1), Ntg2 protein (Ntg2), and Ogg1 protein (yOgg1), were also determined. These repair studies clearly showed that all these enzymes, with the exception of the yOgg1 protein, are able to recognize and remove 5-hydroxy-5-methylhydantoin from the double-stranded DNA fragment. Finally, a 22-mer DNA oligomer bearing a 5-OH-5-Me-dHyd residue was used as a template to study the in vitro nucleotide incorporation opposite the damage by the Klenow fragment of Escherichia coli polymerase I, Taq DNA polymerase, and DNA polymerase beta. Thus, it may be concluded that the oxidized thymine residue is a strongly blocking lesion for the three studied DNA polymerases.     

A study of DNA bases sequence preference of aclarubicin B to bind DNA and inhibit DNA-dependent RNA synthesis in vitro

采用荧光淬灭法和体外无细胞ＲＮＡ合成系统研究了阿柔比星Ｂ结合ＤＮＡ和抑制ＤＮＡ依赖性ＲＮＡ合成的ＤＮＡ碱基顺序选择性。结果表明阿柔比星Ｂ与小牛胸腺ＤＮＡ，ｐｏｌｙ［ｄ（Ａ－Ｔ）］和ｐｏｌｙ［ｄ（Ｇ－Ｃ）］有明显结合；结合ＲＮＡ的活性小；与天然ＤＮＡ的结合力较与变性ＤＮＡ的结合力大。Ｓｃａｔｃｈａｒ分析显示阿柔比星Ｂ结合３种ＤＮＡ的亲和性依次为ｐｏｌｙ［ｄ（Ａ－Ｔ）］＞ｐｏｌｙ［ｄ（Ｇ－Ｃ）］＞小牛胸腺ＤＮＡ。同样，用大肠杆菌ＲＮＡ多聚酶和大鼠肝细胞核游离ＲＮＡ多聚酶实验均显示阿柔比星Ｂ的抑制力依次为ｐｏｌｙ［ｄ（Ａ－Ｔ）］＞ｐｏｌｙ［ｄ（Ｇ－Ｃ）］＞ｐｏｌｙ［ｄ（Ｉ－Ｃ）］。上述结果证明抑制ＲＮＡ合成的碱基顺序选择性与其结合ＤＮＡ碱基顺序的选择性有关     

Non-random effect on RNA synthesis in liver chromatin by administration of dimethylnitrosamine to mice

The effect of dimethylnitrosamine on functional activities of liver chromatin was studied in mice. After a single dose of dimethylnitrosamine injected i.v. (25 mg/kg body wt, 45 min before sacrifice) liver nuclei were isolated and incubated with micrococcal nuclease (EC 3.1.4.7) to an acid-solubility of 2.5% of total DNA. Chromatin was fractionated into a 1,200g pellet P1, 102,000g pellet P2 and supernatant fraction S2. Chromatin-bound RNA polymerase I plus III activity decreased 15% in the P1 and 25% in the P2 fraction. No changes in activity were observed in the S2 fraction. Chromatin-bound RNA polymerase II activity decreased 19% in the P1, 49% in the P2 and 32% in the S2 fraction. Heparin stimulated RNA polymerase II activity decreased 10% in the P1 and 44% in the P2 fraction. Formation of initiation in nuclear lysates with RNA polymerase fromEscherichia coli increased after administration of dimethylnitrosamine suggesting an increase in the number of sites available for the start of new RNA chains. The results show that limited digestion of nuclei with endonuclease cleaves chromatin regions which are more affected by dimethylnitrosamine than the total chromatin suggesting a non-random effect of the hepatotoxin on chromatin. Modifications of the DNA template by dimethylnitrosamine is indicated by the change in number of initiation complexes.     

Oligopyrrole carboxamides linked with a nucleobase as potential DNA minor groove binding ligands: synthesis, DNA binding and biological evaluation

The synthesis of a series of new oligopyrrole carboxamides closely related to netropsin and distamycin A, linked with a nucleobase is reported. The new compounds possess similar structure elements as the known peptide nucleic acids which are interesting sequence reading DNA ligands. Cytotoxicity in vitro, the DNA binding characteristics and the inhibition of topoisomerase I were studied. Four of the compounds, 27, 31, 33 and 37 bind to DNA probably at AT sequences like netropsin or distamycin A in the minor groove. Surprisingly, no cytotoxicity and no inhibition of topoisomerase I was found.     

Mitochondrial DNA D-loop as a new target of Saporin 6 nuclease activity.

The single-chain ribosome-inactivating proteins (RIPs) from plant origin, including Saporin 6 from the seeds of Saponaria officinalis, are ribotoxins known to act as N-glycosidases which depurinate the conserved alpha sarcin loop of large rRNAs. As a consequence, the eukaryotic ribosomes become inactivated, thereby arresting the protein synthesis at the elongation step. RIPs are currently under study as antiviral and antiproliferative agents. Additional in vitro activities of RIPs against either RNA or DNA have been recently described. A specific nuclease activity on plasmidic DNA was demonstrated by either purified or bacterial-recombinant molecules. We report here that human mitochondrial DNA (mtDNA) is a new specific target of Saporin 6 nuclease activity. A unique site of cleavage has been identified and mapped within the most variable part of the D-loop region of the covalently closed circular mtDNA molecule.     

DNA alkylation with N-methylquinolinium quinone methide to N2-dG adducts resulting in extensive stops in primer extension with DNA polymerases and subsequent suppression of GFP expression in A549 cells

Quinone methides (QMs) are involved in the metabolism of many drugs and carcinogens as reactive intermediates to form covalent nucleobase adducts in DNA that associate with high mutagenicity. Recently, a plethora of synthetic QM DNA alkylating agents have been developed to form various nucleobase adducts as potential antitumor agents. However, the mutagenic potential of these synthetic QM alkylating agents has not been fully investigated. In this report, N-methylquinolinium QM was developed as a synthetic model to study biological consequences of the formation of nucleobase adducts in a DNA target. N-Methylquinolinium QM was generated in situ via an elimination process from a bis-quaternary ammonium precursor that was synthesized from a quinoline derivative. Alkylation with N-methylquinolinium QM on a DNA target produced mostly a stable N(2)-dG adduct as revealed by gel electrophoresis and DNA digestion assays and confirmed by mass and NMR analyses. The formation of N(2)-dG adducts of a DNA target was found to cause extensive stops in the primer extension with high fidelity DNA polymerase T7 and even low fidelity error prone Dpo4. The direct biological impact of a prealkylated green fluorescence protein plasmid with N-methylquinolinium QM was demonstrated as significant suppression of protein expression in A549 cells. Overall, our results suggested that nucleobase-QM adducts could potentially block nucleobase mismatch/translesion in the error-prone process to reduce the mutagenic potential if designed carefully.     

Inhibition of RNA polymerase by captan at both DNA and substrate binding sites.

RNA synthesis carried out in vitro by Escherichia coli RNA polymerase was inhibited irreversibly by captan when T7 DNA was used as template. An earlier report and this one show that captan blocks the DNA binding site on the enzyme. Herein, it is also revealed that captan acts at the nucleoside triphosphate (NTP) binding site, and kinetic relationships of the action of captan at the two sites are detailed. The inhibition by captan via the DNA binding site of the enzyme was confirmed by kinetic studies and it was further shown that [14C]captan bound to the beta' subunit of RNA polymerase. This subunit contains the DNA binding site. Competitive-like inhibition by captan versus UTP led to the conclusion that captan also blocked the NTP binding site. In support of this conclusion, [14C]captan was observed to bind to the beta subunit which contains the NTP binding site. Whereas, preincubation of RNA polymerase with both DNA and NTPs prevented captan inhibition, preincubation with either DNA or NTPs alone was insufficient to protect the enzyme from the action of captan. Furthermore, the interaction of [14C]captan with the beta and beta' subunits was not prevented by a similar preincubation. Captan also bound, to a lesser extent, to the alpha and sigma subunits. Therefore, captan binding appears to involve interaction with RNA polymerase at sites in addition to those for DNA and NTP; however, this action does not inhibit the polymerase activity.     

The effects of Penicillium roqueforti toxin on the activity of rat hepatic DNA polymerases

PR toxin, a mycotoxin from cultures of Penicillium roqueforti, inhibited the in vitro activities of rat liver DNA polymerase alpha, beta, and gamma irrespectively of the nature of template-primer used. The concentration required for 50% inhibition of DNA polymerase alpha was 5-6 X 10(-6) M, while those for DNA polymerase beta and gamma were several times higher. By using DNA polymerase beta as a model, and based on the enzyme and template-primer concentration effects and also from the kinetic analysis on PR toxin inhibition, we concluded that two action mechanisms of PR toxin inhibition on in vitro DNA synthesis are operative. Inhibition of the in vitro DNA synthesis directed by DNA template was mediated primarily through alteration of the enzyme itself, whereas in the DNA synthesis reaction directed by RNA template DNA primer, the impairment of template or primer function due to PR toxin treatment probably had occurred. The inhibition of DNA polymerase by PR toxin persisted even after exhaustive dialysis. Addition of PR toxin to an ongoing reaction also inhibited DNA synthesis. Inactivation of DNA polymerase activity of PR toxin likely involved some essential amino acid residues other than sulfhydryl groups.     

Nucleobase modifications in peptide nucleic acids

Peptide nucleic acid (PNA) is an oligonucleotide mimic originally designed upon a repeating N-(2-aminoethyl)glycine polyamide backbone to which nucleobase heterocycles are attached through a methylene carbonyl linkage to the alpha-amino group. These molecules possess remarkable hybridization properties with DNA or RNA forming complexes with high stability and with excellent sequence discrimination despite the substantial structural divergence from natural nucleic acids. Since the disclosure of PNA, a vibrant research community with interest in the chemistry and applications of polyamide-based nucleic acid analogs has developed. This has led to the synthesis and evaluation of a wide variety of modified polyamide nucleic acids. The focus of this report is a comprehensive review of nucleobase modifications in aminoethylglycine (aeg) PNA with reference, where appropriate, to the same modification in DNA or RNA.     

Regulation of bleomycin-induced DNA breakage and chromatin structure in lung endothelial cells by integrins and poly(ADP-ribose) polymerase

Activation of endothelial cell integrins inhibits DNA breakage by diverse agents, including the DNA-damaging agent bleomycin. DNA breaks activate nuclear poly(ADP-ribose) polymerase (PARP), which regulates chromatin structure and DNA repair. We determined the role of PARP in suppression of bleomycin genotoxicity by integrins using wild-type and PARP knockout mouse lung endothelial cells (MLEC), and the PARP inhibitor, 3-aminobenzamide (3AB). Activation of beta1 integrins by antibody clustering enhanced the sensitivity of wild-type nuclei to digestion with micrococcal nuclease and deoxyribonuclease I, indicating that chromatin structure was altered. 3AB blocked this effect. Knockout and 3AB-treated wild-type MLEC were hypersensitive to deoxyribonuclease I compared with wild-type cells, demonstrating that PARP regulates chromatin structure. Integrin clustering reduced the hypersensitivity of knockout cells, suggesting additional, PARP-independent mechanisms that inhibit nuclease interaction with chromatin. Bleomycin caused DNA breakage in wild-type and knockout MLEC. Breaks were eliminated after 60 min incubation of wild-type cells in drug-free medium, whereas 3AB or PARP knockout inhibited DNA repair. Integrin clustering protected wild-type cells from DNA breakage, and 3AB and PARP knockout inhibited this protection. Bleomycin caused large increases in PARP activity in wild-type but not knockout MLEC, and integrin clustering inhibited the activation of PARP. The results indicate that the antigenotoxic effects of integrin activation require PARP and that integrins alter chromatin structure by PARP-dependent and -independent mechanisms.     

Effects of the antitumor drugs 3-nitrobenzothiazolo[3,2-alpha]quinolinium and fagaronine on nucleic acid and protein synthesis

3-Nitrobenzothiazolo[3,2-alpha]quinolinium perchlorate (NBQ) has been shown to be active against in vivo experimental tumors of P388 and Ehrlich ascites cells. Furthermore, it has been established that NBQ binds to DNA by intercalation. In this work we describe its effects on DNA, RNA and protein syntheses both in KB cells and in cell-free synthesizing systems. Fagaronine, an alkaloid structurally related to NBQ, was studied also in an attempt to establish the basis for future studies on structure-activity relationships. Both NBQ and fagaronine inhibited DNA, RNA and protein syntheses in KB cells, with essentially equal effectiveness. Exposure of KB cells to NBQ for 2 hr caused irreversible inhibition of DNA, RNA and protein syntheses. Studies in cell-free systems showed that NBQ strongly inhibited Escherichia coli DNA polymerase I, whereas RNA polymerase activities were moderately affected. Furthermore, both drugs inhibited protein synthesis in cell-free systems derived from rabbit reticulocytes and Saccharomyces cerevisiae. Our results indicate that NBQ and fagaronine exert their cytotoxic activity by at least two independent mechanisms: inhibition of DNA activity by binding to this molecule, and inhibition of protein synthesis probably by interacting with the ribosomal system.     

Effects of coordinated gold compounds on in vitro and in situ DNA replication

Auranofin, a coordinated gold compound, inhibits in vitro DNA synthesis and displays in vivo antitumor activity. To understand the mechanisms of inhibition of DNA replication, we have examined the effects of auranofin and other gold complexes on the activities of purified cellular and herpesvirus-induced DNA polymerases, and on in situ DNA replication in permeabilized S phase KB cells. Evaluation of the data suggests the following conclusions. (1) The gold compounds varied in their abilities to inhibit DNA polymerase activities. DNA polymerase alpha was more sensitive to inhibition by gold compounds than DNA polymerase beta; (2) Inhibition of purified DNA polymerases by gold (I) compounds was noncompetitive with both DNA template and triphosphate substrates. Inhibition by SKF 101675, a gold (III) complex was competitive with DNA. (3) None of the gold compounds tested preferentially inhibited herpesvirus-induced DNA polymerases. (4) The gold complexes that inhibited in vitro DNA replication also inhibited in situ DNA synthesis. However, the potency and order of potency of the compounds varied between the in vitro and in situ systems. (5) Auranofin and other gold compounds inhibited the clonogenic capacity of KB cells in a concentration-dependent manner. The IC50 values measured in the clonogenic assay were significantly lower than those obtained from the in vitro and in situ DNA replication assays.     

异靛甲体外抑制DNA和RNA生物合成的抗癌作用机理

以同位素参入法及光谱法观察异靛甲对核酸合成的抑制作用和药物与DNA相互作用。结果表明,异靛甲对DNA,RNA合成有较强的抑制作用,其IC_(50)分别为9和15μmol/L,药物作用非常迅速。30μmol/L异靛甲作用15μmin后DNA,RNA合成被抑制95％以上。实验还表明,异靛甲不能损伤DNA模板,也不抑制DNA拓扑异构酶及DNA多聚酶Ⅰ,但能明显抑制T7 RNA多聚酶,在’100μmol/L浓度下对mRNA的合成抑制达70％以上。