DNA strand scission by benzo[a]pyrene diol epoxides.

Syn-and anti-benzo[a]pyrene diol epoxides elicit a concentration-dependent nicking of superhelical Col E1 DNA in an in vitro reaction monitored by agarose gel electrophoresis and electron microscopy. This strand scission represents less than 1 percent of the DNA modification by diol epoxide. Kinetic analysis implicates the formation of unstable phosphotriesters, hydrolysis of which nick the DNA.

The DNA strand of chimeric RNA/DNA oligonucleotides can direct gene repair/conversion activity in mammalian and plant cell-free extracts.

Chimeric oligonucleotides (chimeras), consisting of RNA and DNA bases folded by complementarity into a double hairpin conformation, have been shown to alter or repair single bases in plant and animal genomes. An uninterrupted stretch of DNA bases within the chimera is known to be active in the sequence alteration while RNA residues aid in complex stability. In this study, the two strands were separated in the hope of defining the role each plays in conversion. Using a series of single-stranded oligonucleotides, comprised of all RNA or DNA residues and various mixtures, several new structures have emerged as viable molecules in nucleotide conversion. When extracts from mammalian and plant cells and a genetic readout assay in bacteria are used, single-stranded oligonucleotides, containing a defined number of thioate backbone modifications, were found to be more active than the original chimera structure in the process of gene repair. Single-stranded oligonucleotides containing fully modified backbones were found to have low repair activity and in fact induce mutation. Molecules containing various lengths of modified RNA bases (2'-O-methyl) were also found to possess low activity. Taken together, these results confirm the directionality of nucleotide conversion by the DNA strand of the chimera and further present a novel, modified single-stranded DNA molecule that directs conversion in plant and animal cell-free extracts.

Targeted mutagenesis of DNA with alkylating RecA assisted oligonucleotides.

Site-specific mutation was demonstrated in a shuttle vector system using nitrogen mustard-conjugated oligodeoxyribonucleotides (ODNs). Plasmid DNA was modified in vitro by ODNs containing all four DNA bases in the presence of Escherichia coli RecA protein. Up to 50% of plasmid molecules were alkylated in the targeted region of the supF gene and mutations resulted upon replication in mammalian cells. ODNs conjugated with either two chlorambucil moieties or a novel tetrafunctional mustard caused interstrand crosslinks in the target DNA and were more mutagenic than ODNs that caused only monoadducts.

Benzo(alpha)pyrene effects on mouse epithelial cells in culture.

The effect of benzo (a) pyrene on the growth in culture of 5 mouse epithelial cell strains was examined. These epithelial cells are highly sensitive to the cytotoxic action of benzo (a)-pyrene. In addition, the activity of the benzol (a) pyrene-metabolizing system, aryl hydrocarbon hydroxylase, is low but highly iducible by the carcinogen. As the sensitivity of a cell strain to the cytotoxic action of benjo (a) pyrene decreased, the inudcibility of the hydroxylase also decreased,. However, a strong correlation could not be found between cytotoxicity and the level of uninduced or induced hydroxylase when the values from different cell strains were compared. These experiments suggest that thehydroxylase is important in determining the sensitivity of epithelial cells to the cytotoxic action of benzo (a) pyrene, but other factors may also modulate this sensitivity.

Discovery of short, 3'-cholesterol-modified DNA duplexes with unique antitumor cell activity.

A new class of modified oligodeoxynucleotides with unique, selective cytotoxic properties has been discovered. Self-complementary, 3'-cholesterol-modified oligodeoxynucleotides caused morphology changes and death in certain cancer cell lines, whereas other cell lines were unaffected. Susceptible cells were killed in a dose-dependent manner at submicromolar concentrations. Optimum potency was exhibited by phosphodiester duplexes approximately 10 base pairs in length, and base composition was important only in the context of duplex stability. Phosphorothioate analogues were less potent. Although the molecular mechanism of action of these unique compounds is not yet known, they offer potential applications in cancer therapy and in studies of cell death. In addition, the path toward elucidation of the structure-based biological activity of these oligonucleotides should be especially instructive for researchers studying sequence-specific effects.

Interaction of Escherichia coli RNA polymerase with DNA in an elongation complex arrested at a specific psoralen crosslink site.

We have probed the interaction of Escherichia coli RNA polymerase with DNA in an elongation complex arrested by a site-specifically placed psoralen crosslink using DNase I footprinting techniques. The psoralen derivative 4'-hydroxymethyl-4,5',8-trimethylpsoralen was first placed at a specific site in the middle of a chemically synthesized double-stranded DNA fragment containing an E. coli RNA polymerase promoter at one end. The psoralen molecule was photochemically attached to two adjacent thymidine residues on opposite strands as a diadduct. Using this crosslinked DNA as the template for transcription, we found that the E. coli RNA polymerase was blocked at the psoralen diadduct, yielding a transcript 29 nucleotides long. The arrested elongation complex inhibited DNase I digestion of both the coding strand and the non-coding strand from about 22 nucleotides upstream to 15 nucleotides downstream from the diadduct. These results, which suggest that the unwindase and the catalytic sites of the polymerase are very close to each other, have been incorporated into a model of the transcription elongation complex.

Solution hybridization of crosslinkable DNA oligonucleotides to bacteriophage M13 DNA. Effect of secondary structure on hybridization kinetics and equilibria.

Several DNA oligonucleotides have been photochemically modified with the furocoumarin 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) such that each contained a single HMT furan side monoadduct to thymidine at a unique 5' TpA 3' sequence. When these oligonucleotides were hybridized to their respective complements, the HMT adduct could be driven to form an interstrand crosslink by irradiation of the hybrid with 360 nm light. The ability to crosslink probe-target complexes has allowed us to determine the kinetics and the extent of hybridization in solution between these oligonucleotides and their complementary sequences in single-stranded bacteriophage M13 DNA. Our data indicate that these parameters are strongly influenced by the existence of local as well as global secondary structure in the viral DNA. During hybridization, rearrangement of this secondary structure so as to expose the target sequence can be rate-limiting. Upon attainment of equilibrium, only a portion of the target sequence may be hybridized to the probe with the remainder involved in intrastrand base-pairing. Using crosslinkable oligonucleotide probes hybridized and irradiated near the melting temperature of the respective probe-target complex one can partially overcome these secondary structure effects.

Base specificity in the binding of benzo[a]pyrene diol epoxide to simian virus 40 DNA.

[14C]Simian virus 40 (SV40) DNA was reacted with [3H]7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene to give 0.60 adducts per genome. The modified DNA was digested to completion with Hind III restriction endonuclease and the 6 fragments isolated by polyacrylamide gel electrophoresis. Hydrocarbon binding to the fragments was proportional to their guanine--cytosine (G--C) content, reflecting selective reaction of the hydrocarbon with deoxyguanosine residues. No sites unusually susceptible to alkylation were detected.

Biochemical basis for the acquisition of resistance to benzo[a]pyrene in clones of mouse liver cells in culture.

In a Namru mouse liver epithelial cell strain designated NMuLi, aryl hydrocarbon hydroxylase (AHH) activity peaked at 12 h post-induction with 1 microgram/ml of benzo(a)pyrene (BaP) in both confluent and growing cells. Maximal levels of AHH activity were reached on day two post-plating. This induced activity was inhibited in vitro 78% by gassing the incubation mixture with carbon monoxide for 15 s, and inhibited 93% by addition of 40 microgram/ml of 7,8 benzoflavone(BF). Induced AHH levels were higher in epithelial clones that were sensitive to the toxicity of BaP than in resistant clones. The survival fraction of clones from NMuLi and of subclones derived from a sensitive clone of NMuLi after BaP treatment was a negative exponential function of the maximal induced AHH activity in the clones. One of the clones, NMuLi cl 8, was extremely susceptible to the toxic effects of BaP, the +/-(trans)-7alpha, 8beta-dihydroxy-7,8-dihydro-BaP(7,8-diol), and the (+/-)-7alpha, 8beta-dihydroxy-9beta, 10beta-epoxy-7,8,9,10-tetrahydro-BaP (diol-epoxide), known metabolites of BaP. The toxicity of BaP and the 7,8 diol to this clone was inhibited by BF, suggesting that these cells possessed an enzyme activity inhibitable by BF that could epoxidize BaP to the 7,8 oxide and then epoxidize the resultant 7,8 diol to the diol-epoxide. Another clone derived from NMuLi, clone 7, was relatively resistant to the toxic effects of BaP and the 7,8-diol, but still extremely susceptible to the toxic effects of the diol-epoxide. The slight toxicity to BaP in this clone was inhibited by BF, but the toxicity of the 7,8-diol to this clone was not inhibited by BF. A typical cytochrome P450 inhibitor, metyrapone, had no effect on the toxicity of BaP, the 7,8-diol, or the diol-epoxide to either clone 7 or clone 8. The results suggest that these liver cells possess two enzymes that play some role in polycyclic hydrocarbon-induced toxicity. Enzyme A, a BaP-inducible enzyme that is inhibitable by BF, efficiently metabolizes BaP to the 7,8-diol and the 7,8-diol to the diol-epoxide. It is responsible for most of the hydrocarbon toxicity. Enzyme B is not inhibitable by BF and metabolizes the 7,8-diol less efficiently to the diol-epoxide or efficiently to other, less toxic products.

[Selective stabilization of AT-rich DNA duplexes by oligodeoxyribonucleotide conjugates with distamycin analogues]. / Selektivnaya stabilizatsiya AT-bogatykh DNK-dupleksov kon''iugatami oligodezoksiribonuleotidov i analogov distamitsina.

Oligodeoxyribonucleotide conjugates with distamycin analogues containing up to five pyrrolecarboxamide moieties were synthesized. The stability of duplexes formed by these conjugates was shown to depend directly upon the number of pyrrolecarboxamide moieties in the ligand molecule. For the duplexes formed by octaadenylate and octathymidilate conjugates with the distamycin pentapyrrole analogue, stability was demonstrated to be achieved by either one or two ligand molecules; however, duplexes containing two ligand molecules are more stable.

Efficient synthesis of a supercoiled M13 DNA molecule containing a site specifically placed psoralen adduct and its use as a substrate for DNA replication.

We report a simple method for the in vitro synthesis of large quantities of site specifically modified DNA. The protocol involves extension of an oligonucleotide primer annealed to M13 single-stranded DNA using part of the T4 DNA polymerase holoenzyme. The resulting nicked double-stranded circles are ligated and supercoiled in the same tube, producing good yields of form I DNA. When the oligonucleotide primer is chemically modified, the resultant product contains a site-specific lesion. In this study, we report the synthesis of an M13 mp19 form I DNA which contains a psoralen monoadduct or cross-link at the KpnI site. We demonstrate the utility of these modified substrates by assessing the ability of the bacteriophage T4 DNA replication complex to bypass the damage and show that the psoralen monoadduct poses a severe block to the holoenzyme when attached to the template strand.

Inhibition of tRNA aminoacylation by 2'-O-methyl oligonucleotides.

A 2'-O-methyl oligonucleotide complementary to 18 nucleotides in the dihydrouridine stemloop of Escherichia coli tRNA(Cys) has been shown to stably bind to the tRNA. The binding inhibits aminoacylation of the tRNA by cysteine tRNA synthetase. The same oligonucleotide sequence but with the DNA deoxy backbone does not bind to the tRNA. This provides the basis for the design and test of a series of 2'-O-methyl oligonucleotides for their ability to bind to E. coli tRNA(Cys) and inhibit aminoacylation. We show here that different regions of the tRNA have different sensitivities to oligonucleotides. A 10-mer that targets G15 forms a stable complex with the tRNA. The Kd of the complex is several orders of magnitude lower than that of the tRNA-synthetase complex. Measurements of dissociation rate constants indicate that the stronger affinity of the 10-mer to tRNA(Cys) is due to a significantly slower rate of dissociation (by a factor of 10(6)) than that of the synthetase from the tRNA. Only a stoichiometric amount of the 10-mer is necessary to completely inhibit aminoacylation. Because tRNA aminoacylation is fundamental to cell growth, these results provide the rationale for the 10-mer and its derivatives as pharmaceutical agents that target specific cell growth.

A topological model for transcription based on unwinding angle analysis of E. coli RNA polymerase binary, initiation and ternary complexes.

DNA unwinding induced by Escherichia coli RNA polymerase is measured for binary, initiation and ternary complexes formed from a unique promoter sequence on simian virus 40 DNA. At 37 degrees C the complexes all have an unwinding angle of 17 +/- 1 base pairs (580 degrees +/- 30 degrees). This unwinding is attributed to an enzyme-stabilized separation of the double helix at the promoter site, which is maintained throughout initiation and elongation. There is no heterogeneity in the unwinding angle of the ternary complex as it progresses down the helical template. The constant DNA unwinding during all phases of transcription leads us to propose the existence of unwindase and rewindase activities on the enzyme that allow it to travel down the helix like a nut on a DNA bolt. During elongation, the unwindase unwinds the DNA helix while the rewindase, lagging by 17 base pairs, displaces the RNA transcript and reseals the helix. Both activities induce a rotation in the DNA double helix relative to the polymerase. The RNA-DNA hybrid also rotates, maintaining both ends of that helix fixed relative to the catalytic and windase sites. Formation of an RNA-DNA hybrid which spans the distal end of the DNA unwound region is proposed as a possible mechanism for polymerase pausing and termination. This model requires that the polymerase direct the transcript past the noncoding DNA strand. Pausing occurs 16-20 nucleotides downstream from the centers of appropriately sized dyad symmetry elements.

Interaction of T7 RNA polymerase with DNA in an elongation complex arrested at a specific psoralen adduct site.

We have probed the interaction of T7 RNA polymerase with DNA in an elongation complex arrested by a site specifically placed psoralen diadduct or furanside monoadduct using DNase I footprinting techniques. The psoralen derivative, HMT (4'-hydroxy-methyl-4,5',8-trimethylpsoralen), was first placed at a specific site in the middle of a chemically synthesized double-stranded DNA fragment containing a T7 RNA polymerase promoter at one end. The psoralen molecule was photochemically attached either to 2 adjacent thymidine residues on opposite strands as a diadduct or to only 1 thymidine residue on the coding strand as a furan-side monoadduct. Using these psoralen-modified DNAs as templates for transcription, we found that T7 RNA polymerase was blocked at the psoralen adduct site and that the arrested elongation complex protected about 15 nucleotides upstream from the adduct on the coding strand and 20 nucleotides around the adduct on the noncoding strand from DNase I digestion. The two psoralen-modified DNA templates yielded identical RNA transcripts and DNase I footprints. In contrast, T7 polymerase protected only the coding strand from -20 to +8 in the initiation complex. These results suggest that the RNA polymerase undergoes a marked conformational change upon converting from an initiation complex to an elongation complex.

The effects of covalent additions of a psoralen on transcription by E. coli RNA polymerase.

Synthetic DNA substrates containing a site-specifically engineered psoralen monoadduct or diadduct were used to characterize the response of the E. coli RNA polymerase elongation complex to these lesions. The psoralen derivative HMT (4'-hydroxymethyl-4,5', 8-trimethylpsoralen) was site specifically placed into two synthetic double-stranded DNA fragments each of which contained an E. coli RNA polymerase promoter at one end. The HMT molecule was attached to the middle of the DNA fragments as either a furan-side monoadduct or an interstrand diadduct. Transcription off the HMT crosslinked DNA templates showed that E. coli RNA polymerase terminated at the HMT diadduct site, i. e., one nucleotide before the modified thymidine residue on the template strand. The furan-side monoadduct when on the template strand also blocked transcription by the polymerase. However, no effect on transcription was observed when the monoadduct was located on the non-template strand.

G/C-modified oligodeoxynucleotides with selective complementarity: synthesis and hybridization properties.

Modified oligodeoxyribonucleotides (ODNs) that have unique hybridization properties were designed and synthesized for the first time. These ODNs, called selective binding complementary ODNs (SBC ODNs), are unable to form stable hybrids with each other, yet are able to form stable, sequence specific hybrids with complementary unmodified strands of nucleic acid. To make SBC ODNs, deoxyguanosine (dG) and deoxycytidine (dC) were substituted with deoxyinosine (dI) and 3-(2'-deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one (dP), respectively. The hybridization properties of several otherwise identical complementary ODNs containing one or both of these nucleoside analogs were studied by both UV monitored thermal denaturation and non-denaturing PAGE. The data showed that while dI and dP did form base pairs with dC and dG, respectively, dI did not form a stable base pair with dP. A self-complementary ODN uniformly substituted with dI and dP acquired single-stranded character and was able to strand invade the end of a duplex DNA better than an unsubstituted ODN. This observation implies that SBC ODNs should effectively hybridize to hairpins present in single-stranded DNA or RNA.