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S M Hecht

Publications and source records attributed to S M Hecht.

At least 19 recordsLinked to original sources
PubMed1995

Phosphoryl migration during the chemical synthesis of RNA.

By the use of high sensitivity assay systems, we have measured the occurrence of strand scission and phosphoryl migration that accompany the deblocking of chemically synthesized oligoribonucleotides. Substantial phosphoryl migration was observed for both enzymatically derived poly(uridylic acid) and synthetic uridine oligoribonucleotides 2'-O-protected with the 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp) group, when these species were subjected to the acidic conditions suggested for Fpmp deprotection. Strand scission occurred in parallel and could be demonstrated readily by 5'-32P end labeling, but not by 3'-32P end labeling, of the acid-treated oligoribonucleotides. Increasing the pH of the deprotection solution and decreasing the temperature at which the deprotection was accomplished diminished both phosphoryl migration and strand scission. A mechanism that can rationalize these results is discussed.

Base Sequence↗
PubMed1995

Sequence-specific hydrolysis of yeast tRNA(Phe) mediated by metal-free bleomycin.

Bleomycin A2 (BLM) was found to mediate sequence specific hydrolysis of tRNA(Phe) in the absence of added metal ions. BLM A2 promoted phosphodiester bond hydrolysis 3' to the pyrimidine residue at all resolved Py-Pu sites not involving modified bases, as demonstrated by high-resolution electrophoretic analysis of 5'- and 3'-32P-end-labeled substrates. The reaction proceeded with surprising facility, approaching efficiency that of oxidative strand scission mediated by the FeII.BLM A2 complex. By the use of a number of BLM congeners, as well as a study of the time, temperature, and salt dependence of the hydrolysis, it was shown that in many respects the hydrolytic reaction parallels the oxidative degradation of RNA and DNA mediated by metallobleomycins. Thus, in contrast to the well-characterized oxidative degradation of DNA and RNA by bleomycin studied for two decades, the present report documents the ability of certain metal-free bleomycins to mediate RNA hydrolysis.

Antibiotics, Antineoplastic↗
PubMed1995

A model for topoisomerase I-mediated insertions and deletions with duplex DNA substrates containing branches, nicks, and gaps.

The ability of DNA topoisomerase I to promote insertions and deletions in vitro has been studied at nucleotide resolution for structurally diverse DNA substrates that uncouple the cleavage and ligation reactions of the enzyme. Topoisomerase I-mediated ligations afforded DNA duplexes having deletions and insertions with "branched" substrates and deletions up to 18 nucleotides in length with substrates containing nicks or gaps. In addition, a number of the acceptor substrates altered the preferred site of DNA cleavage, thereby increasing the diversity of accessible ligation products. Also demonstrated by the production of two "recombinant" duplexes from a single set of reactants was the potential for amplification of such alterations. These findings illustrate plausible mechanisms by which topoisomerase I-mediated illegitimate recombination may obtain at a molecular level.

Base Sequence↗
PubMed1994

Iron(II) bleomycin-mediated degradation of a DNA-RNA heteroduplex.

The effect of iron(II) bleomycin on a DNA-RNA heteroduplex was investigated using a substrate formed by reverse transcription of Escherichia coli 5S ribosomal RNA. Both strands of the heteroduplex were cleaved by FeII.BLM A2 at comparable concentrations; complete digestion of both strands was observed using 5 microM FeII.BLM A2. The DNA strand of the heteroduplex was cleaved predominantly at 5'-G-pyr-3' sites; the sites of cleavage of the DNA strand were a subset of those observed for the corresponding DNA strand of a DNA duplex of identical sequence. The sites of cleavage of the RNA strand of the heteroduplex involved both purines and pyrimidines and were found to be different than the sites of cleavage of the 5S rRNA alone, demonstrating that cleavage of the former must actually have involved heteroduplex recognition by FeII.BLM A2. Both the DNA and RNA strands of the heteroduplex were cleaved by FeII.BLM A2 in the presence of physiological concentrations of Mg2+, consistent with the possibility that DNA-RNA heteroduplexes may be therapeutically relevant targets for bleomycin.

Base Sequence↗
PubMed1994

On the role of the bithiazole moiety in sequence-selective DNA cleavage by Fe.bleomycin.

A recent study of Fe(II).bleomycin-mediated DNA strand scission suggested that the metal binding domain of the drug is also the primary determinant of the observed sequence selectivity of strand scission (Carter, B. J., Murty, V. S., Reddy, K. S., Wang, S.-N., and Hecht, S. M. (1990) J. Biol. Chem. 265, 4193-4196). Although it is well established that the bithiazole moiety+C-terminal substituent of bleomycin are required for DNA binding, the role of the bithiazole in sequence-selective DNA recognition remains unclear. To determine whether the bithiazole moiety exhibits an intrinsic DNA binding selectivity, three synthetic EDTA-conjugated bithiazole derivatives were used to mediate DNA cleavage in the presence of Fe2+ and dithiothreitol. Incubation of these Fe(II).EDTA-bithiazoles in the presence of a 5'-32P end-labeled DNA duplex resulted in strand scission at every position to essentially the same extent. The relative cleavage efficiencies among the bithiazoles were a strong function of their ionic state. These findings imply that the bithiazoles can bind to many sites on the DNA; they support a model of bleomycin-DNA interaction in which the bithiazole moiety+C-terminal substituent are required only for DNA binding, whereas the metal binding domain is responsible for metal ion coordination and oxygen activation as well as being the primary determinant of sequence-selective DNA cleavage.

Binding Sites↗
PubMed1993

Fe.bleomycin cleaves a transfer RNA precursor and its "transfer DNA" analog at the same major site.

Previously, Fe.bleomycin (BLM) has been shown to mediate RNA cleavage in a fashion more highly selective than that of DNA. Because RNAs often assume secondary and tertiary structures not commonly encountered with DNAs, it was not clear whether the greater selectivity of RNA cleavage was a consequence of differences in the mononucleotide constituents of RNA and DNA, or of the three-dimensional structures of the individual substrates. Accordingly, we prepared a "tDNA" identical in sequence with Bacillus subtilis tRNA(His) precursor, the latter of which is known to be a good substrate for Fe(II).BLM A2 and which undergoes oxidative cleavage predominantly at U35. Remarkably, the tDNA underwent cleavage predominantly at T35. At higher concentrations of Fe(II).BLM A2, the tDNA was extensively degraded, while the tRNA(His) precursor was not. Competition experiments suggested that this was not due to more efficient binding of Fe.BLM to the tDNA; in fact the tRNA precursor appeared to be bound more efficiently. The lesser cleavage of the tRNA(His) may be due to limitations in the facility of chemical transformation following Fe.BLM binding, or else to the formation of RNA lesions that do not lead directly to RNA strand scission.

Bacillus subtilis↗
PubMed1993

Characterization of iron (II).bleomycin-mediated RNA strand scission.

The ability of iron(II).bleomycin to mediate RNA degradation was further characterized. At micromolar concentrations, FeII.BLM was shown to effect cleavage of Escherichia coli tRNA(1His) and a Schizosaccharomyces pombe amber suppressor tRNA construct in an efficient fashion. In contrast, E. coli tRNA(Cys) and yeast mitochondrial tRNA(Asp) and tRNA(fMet) precursors were not substrates for FeII.BLM. Also shown to be a good substrate for cleavage by FeII.BLM was yeast 5S ribosomal RNA. Since HIV-1 reverse transcriptase mRNA has previously been shown to be degraded by Fe.BLM (Carter et al., 1990a), members of the three major classes of RNA have now been shown to undergo Fe.BLM-mediated strand scission. For each of the substrate RNAs, cleavage occurred at sites unique to that substrate. Although RNA cleavage occurred at numerous sequences, 5'-G-pyr-3' sites were prominent. Likewise, while cleavage was noted in regions anticipated to be double-stranded, as well as in single-stranded regions, a disproportionate number of cleavages were noted at the junction between single- and double-stranded regions. As found in earlier studies, RNA cleavage was much more selective than DNA cleavage. Further, when RNA cleavage was carried out in the presence of reagents such as Mg2+, spermidine, and NaCl, the selectivity of cleavage was further enhanced. The highly selective and efficient cleavage of a number of RNA molecules reinforces our earlier suggestion that RNA may constitute a therapeutically relevant target for bleomycin.

Base Sequence↗
PubMed1992

Amplification of protein expression in a cell free system.

Large quantities of a catalytically active protein have been produced in a cell free system. More than 10(9) copies of protein were produced from each DNA plasmid containing DNAfol, the bacterial gene encoding dihydrofolate reductase (DHFR). The strategy employed, denoted gene amplification with transcription/translation (GATT), involves sequential coupling of (i) DNA amplification by the polymerase chain reaction (PCR) and (ii) in vitro RNA transcription by T7 RNA polymerase, followed by (iii) translation of the run-off transcripts in a rabbit reticulocyte system. The protein product had the expected size (18 kDa) and catalyzed the NADPH-dependent reduction of 7,8-dihydrofolic acid to 5,6,7,8-tetrahydrofolic acid as efficiently as authentic DHFR. Potential applications of the strategy include large scale production of enzymes containing synthetic amino acids and facilitation of the characterization of the function of genes encountered in genomic mapping studies.

Cell-Free System↗
PubMed1992

A strategy for identifying novel, mechanistically unique inhibitors of topoisomerase I.

While the design of molecules that inhibit or antagonize the functions of specific macromolecules is now well precedented, in many cases the structural information requisite to the design process is lacking. The tools of molecular biology can now furnish the target macromolecules for use in mechanism-based exploration; highly defined assays can be devised based upon the known biochemistry of these macromolecules to permit the discovery of novel inhibitors or antagonists present in chemical collections. Presently, we describe a set of assays directed toward the discovery of novel inhibitors of eukaryotic topoisomerase I, an enzyme critical to maintenance of chromosomal DNA topology and therefore essential for normal replication and transcription. The identification of chebulagic acid as an extraordinarily potent and mechanically novel inhibitor of topoisomerase I illustrates the potential of this approach.

Animals↗
PubMed1991

Site-specific DNA cleavage by antisense oligonucleotides covalently linked to phenazine di-N-oxide.

Site-specific degradation of DNA was achieved by the use of DNA oligonucleotides covalently tethered to phenazine 5,10-di-N-oxide. When annealed to a complementary DNA target strand, the antisense oligonucleotide effected alkylation of guanosine residues in proximity to the phenazine di-N-oxide prosthetic group. Admixture of dithiothreitol to the formed duplex resulted in reductive activation of the phenazine di-N-oxide moiety with concomitant generation of diffusible oxygen radicals; the latter effected strand scission of the target DNA oligonucleotide. Several parameters of DNA degradation were studied, including the effect on DNA degradation of chain length in the tether connecting the oligonucleotides and prosthetic group, the relative efficiencies of DNA cleavage when the prosthetic group was in the middle or at the end of the antisense oligonucleotide, and the effect of O2 on DNA degradation. Also studied was the actual chemistry of DNA oligonucleotide degradation and the ability of individual diastereomers of the modified oligonucleotides to mediate degradation of the target DNA.

Base Sequence↗
PubMed1991

Synthesis of water-soluble (aminoalkyl)camptothecin analogues: inhibition of topoisomerase I and antitumor activity.

Water-soluble analogues of the antitumor alkaloid camptothecin (1) were prepared in which aminoalkyl groups were introduced into ring A or B. Most of the analogues were prepared by oxidation of camptothecin to 10-hydroxycamptothecin (2) followed by a Mannich reaction to give N-substituted 9-(aminomethyl)-10-hydroxycamptothecins (4-12) or by subsequent modification of Mannich product 4 (13, 15, 17, 19, 21). Others were obtained by modification of the hydroxyl group of 2 (25,26) or by total synthesis (35,42,43). These analogues, as well as some of their synthetic precursors, were evaluated for inhibition of topoisomerase I, cytotoxicity, and antitumor activity. Although there was not a quantitative correlation between these assays, compounds that inhibited topoisomerase I were also cytotoxic and demonstrated antitumor activity in vivo. Further evaluation of the most active water-soluble analogue led to the selection of 9-[(dimethylamino)methyl]-10-hydroxycamptothecin (4, SK&F 104864) for development as an antitumor agent. In addition to its water solubility, ease of synthesis from natural camptothecin, and high potency, 4 demonstrated broad-spectrum activity in preclinical tumor models and is currently undergoing Phase I clinical trials in cancer patients.

Animals↗
PubMed1990

Construction and processing of transfer RNA precursor models.

Several "dimeric" tRNA molecules were constructed as potential substrates for ribonuclease P (RNase P) and for M1 RNA, the catalytic subunit of RNase P. Construction was affected by the T4 RNA ligase-mediated coupling of a mature Escherichia coli tRNA (acceptor substrate) and nucleotides 1-36 of yeast tRNAPhe (donor substrate), followed by annealing of the 3'-half of yeast tRNAPhe (nucleotides 38-76). E. coli RNase P and M1 RNA were both found to cleave the dimeric tRNA precursor model constructed from E. coli tRNAPhe (5'-tRNA) and yeast tRNAPhe (3'-tRNA) in a reaction that was dependent on the presence of the annealed 3'-half molecule derived from yeast tRNAPhe, or on some conformation imposed by the presence of this species; the product had the same mobility as authentic E. coli tRNAPhe on a polyacrylamide gel. By utilizing tRNA precursor models radiolabeled at phosphodiesters immediately preceding or following the putative site of processing, cleavage of the substrate by both M1 RNA and the holoenzyme was demonstrated to occur at the expected phosphate ester linkage. The results obtained here suggest that the endonucleolytic separation of two tRNAs by RNase P is dependent on one or more structural features in the 3'-half of the 3'-tRNA, and thus are consistent with the report of McClain et al. (McClain, W. H., Guerrier-Takada, C., and Altman, S. (1987) Science 238, 527-530) that identifies the T stem and loop as a possible recognition site.

Base Sequence↗
PubMed1990

Metal ion and substrate structure dependence of the processing of tRNA precursors by RNase P and M1 RNA.

A synthetic tRNA precursor analog containing the structural elements of Escherichia coli tRNA(Phe) was characterized as a substrate for E. coli ribonuclease P and for M1 RNA, the catalytic RNA subunit. Processing of the synthetic precursor exhibited a Mg2+ dependence quite similar to that of natural tRNA precursors such as E. coli tRNA(Tyr) precursor. It was found that Sr2+, Ca2+, and Ba2+ ions promoted processing of the dimeric precursor at Mg2+ concentrations otherwise insufficient to support processing; very similar behavior was noted for E. coli tRNA(Tyr). As noted previously for natural tRNA precursors, the absence of the 3'-terminal CA sequence in the synthetic precursor diminished the facility of processing of this substrate by RNase P and M1 RNA. A study of the Mg2+ dependence of processing of the synthetic tRNA dimeric substrate radiolabeled between C75 and A76 provided unequivocal evidence for an alteration in the actual site of processing by E. coli RNase P as a function of Mg2+ concentration. This property was subsequently demonstrated to obtain (Carter, B. J., Vold, B.S., and Hecht, S. M. (1990) J. Biol. Chem. 265, 7100-7103) for a mutant Bacillus subtilis tRNAHis precursor containing a potential A-C base pair at the end of the acceptor stem.

Base Sequence↗
PubMed1990

Irreversible trapping of the DNA-topoisomerase I covalent complex. Affinity labeling of the camptothecin binding site.

Camptothecin (CPT) binds reversibly to, and thereby stabilizes, the cleavable complex formed between DNA and topoisomerase I. The nature of the interaction of CPT with the DNA-topoisomerase I binary complex was studied by the use of two affinity labeling reagents structurally related to camptothecin: 10-bromoacetamidomethylcamptothecin (BrCPT) and 7-methyl-10-bromoacetamidomethylcamptothecin (BrCPTMe). These compounds have been shown to trap the DNA-topoisomerase I complex irreversibly. Although cleavage of DNA plasmid mediated by topoisomerase I and camptothecin was reduced significantly by treatment with high salt or excess competitor DNA, enzyme-mediated DNA cleavage stabilized by BrCTPMe persisted for at least 4 h after similar treatment. The production of irreversible topoisomerase I-DNA cleavage was time-dependent, suggesting that BrCPTMe first bound noncovalently to the enzyme-DNA complex and, in a second slower step, alkylated the enzyme or DNA in a manner that prevented DNA ligation. The formation of a covalent linkage was supported by experiments that employed [3H]BrCPT, which was shown to label topoisomerase I within the enzyme-DNA complex. [3H]BrCPT labeling of topoisomerase I was enhanced greatly by the presence of DNA; very little labeling of isolated topoisomerase I or isolated DNA occurred. Even in the presence of DNA, [3H]BrCPT labeling of topoisomerase I was inhibited by camptothecin, suggesting that both CPT and BrCPT bound to the same site on the DNA-topoisomerase I binary complex. These studies provide further evidence that a binding site for camptothecin is created as the DNA-topoisomerase I complex is formed and suggest that the A-ring of camptothecin is proximate to an enzyme residue.

Affinity Labels↗
PubMed1990

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)

Base Sequence↗
PubMed1990

Control of the position of RNase P-mediated transfer RNA precursor processing.

Two Bacillus subtilis tRNA(His) precursors (Green, C. J., and Vold, B. S. (1988) J. Biol. Chem. 263, 652-657) were processed by Escherichia coli RNase P in the presence of varying [Mg2+]. The wild type precursor was processed under all conditions to afford a single tRNA product containing 8 base pairs in the acceptor stem. In contrast, the position of processing of a mutant tRNA(His) precursor (containing a G27----A27 alteration) was shown to be condition-dependent. Processing occurred at A27 under conditions consistent with formation of an A27-C100 base pair in the acceptor stem but at G28 under conditions that disfavored base pair formation. The ability to control the site of RNase P-mediated tRNA precursor processing is unprecedented and permits analysis of the chemical factors that promote processing.

Bacillus subtilis↗