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L F Povirk

Publications and source records attributed to L F Povirk.

At least 55 records · Page 3Linked to original sources
PubMed1990

Melphalan-induced mutagenesis in an SV40-based shuttle vector: predominance of A.T----T.A transversions.

The base and sequence specificity of mutagenesis by the carcinogenic antitumor agent melphalan (L-phenylalanine mustard) was examined by in vitro treatment of the plasmid pZ189 followed by replication in human 293 cells, rescue in bacteria, and sequence analysis of mutations in the supF gene. Melphalan was strongly mutagenic in this assay. The induced mutations were predominantly base substitutions, with a minor component of small deletions (3-80 base pairs). Surprisingly, A.T----T.A transversions were by far the most frequent substitutions, suggesting that modifications of adenine may play a major role in mutagenesis. More than half the substitutions were clustered in a C-T-A-A sequence in the anticodon loop. No base substitutions were detected at G-N-C sequences, which are thought to be potential sites of DNA interstrand cross-links. The results raise the possibility that the cytotoxic and mutagenic effects of melphalan may be separable.

Base Sequence↗
PubMed1990

Effect of in vitro cleavage of apurinic/apyrimidinic sites on bleomycin-induced mutagenesis of repackaged lambda phage.

Previous studies have revealed bleomycin to be a potent base-substitution mutagen in repackaged phage lambda. In order to assess the role of apurinic/apyrimidinic (AP) sites in bleomycin-induced mutagenesis, bleomycin-damaged lambda DNA was treated with putrescine or endonuclease IV to effect cleavage of bleomycin-induced AP sites. The DNA was then packaged, the phage grown in SOS-induced E. coli, and the frequency of clear-plaque mutants in the progeny was determined. Bleomycin-induced mutagenesis was decreased approx. 2-fold by treating the DNA with putrescine, but was unaffected by endonuclease IV. The results are consistent with the production of bleomycin-induced mutation at certain AP sites having a closely opposed single-strand break, since such sites are cleaved by putrescine but not by endonuclease IV.

Bacteriophage lambda↗
PubMed1990

Bleomycin-induced DNA lesions at mutational hot spots: implications for the mechanism of double-strand cleavage.

Using various end-labeled, defined-sequence DNA substrates, we examined bleomycin-induced damage at several G.C base pairs which correspond to mutational hot spots. The most frequent lesions detected were single-strand breaks and single apurinic/apyrimidinic (AP) sites at the C residue, suggesting that this was the primary site of damage. Strand breaks and AP sites also occurred, but less frequently, at a secondary damage site--i.e., the directly opposed G residue in the complementary strand. However, damage at the secondary site occurred only when a strand break was present at the primary site, and AP sites at the primary site were never accompanied by closely opposed damage in the complementary strand. Thus, formation of a strand break at the primary damage site was a necessary though not sufficient condition for attack at the secondary site. Similar patterns were seen at other sequences attacked by bleomycin, although primary and secondary sites were sometimes staggered by one nucleotide position rather than directly opposed. These and other results suggest a mechanism of double-strand cleavage in which bleomycin is reactivated during formation of the first strand break, and the reactivated drug subsequently attacks the complementary strand at a specific position which is not normally a site of bleomycin-induced cleavage. Regeneration of activated bleomycin could result from a reaction between Fe(III).bleomycin and a 4'-peroxyl derivative of deoxyribose, both produced during formation of the strand break.

Base Composition↗
PubMed1989

Structure of bleomycin-induced DNA double-strand breaks: predominance of blunt ends and single-base 5' extensions.

In order to examine the structure of bleomycin-induced DNA double-strand breaks, defined-sequence DNA was labeled in each strand at a single restriction site and treated with bleomycin. Various double-stranded fragments resulting from bleomycin-induced double-strand breaks were isolated, denatured, and run on sequencing gels to determine the sites of cleavage in each strand. For virtually every double-strand break, the cleavage site in one strand was a pyrimidine in a G-Py sequence, reflecting a specificity similar to that of bleomycin-induced single-strand cleavage. However, the cleavage site in the complementary strand was seldom a G-Py sequence, and was usually a site where single-strand cleavage was infrequent. When the sequence at the double-strand break was G-Py-Py', the break at Py was usually accompanied by a break at the base directly opposite Py, resulting in blunt ends. When the sequence was G-Py-Pu, the break at Py was usually accompanied by a break at the base opposite Pu, resulting in single-base 5' extensions. Double-strand breaks with 3' extensions, such as would result from cleavage of two C residues in a self-complementary G-C sequence, were conspicuously absent. These data provide further evidence that bleomycin-induced double-strand breaks do not result from coincidence of independent site-specific single-strand breaks.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibiotics, Antineoplastic↗
PubMed1989

Oxidized apurinic/apyrimidinic sites formed in DNA by oxidative mutagens.

Treatment of DNA with any of several agents, including ionizing radiation, hydrogen peroxide, bleomycin, neocarzinostatin and the copper (I) chelate complex of 1,10-phenanthroline, produces apurinic/apyrimidinic (AP) sites containing oxidized deoxyribose moieties. These AP sites, which are formed by specific or nonspecific free-radical attack on deoxyribose, have been shown to involve oxidation of deoxyribose at the C-1', C-2' or C-4' position. Oxidized AP sites are generally more susceptible to chemical cleavage than normal AP sites, but are in some cases resistant to cleavage by repair AP endonucleases. Nearly all of the AP sites produced by neocarzinostatin, and a fraction of those produced by bleomycin, are accompanied by closely opposed breaks in the complementary strand. Sequence specificity data strongly implicate oxidized AP sites in neocarzinostatin-induced mutagenesis. The role of AP sites in mutagenesis by the other oxidative mutagens is less clear, although there is in some cases suggestive evidence for such a role.

Apurinic Acid↗
PubMed1989

Expression of protein-associated DNA damage in the alkaline elution assay in the absence of enzymatic deproteinization.

In the alkaline elution assay, expression of protein-associated DNA damage induced by topoisomerase II antagonists is facilitated by proteinase K digestion of the drug-stabilized topoisomerase-II-DNA complex. In the absence of this enzymatic deproteinization step, drug-induced DNA strand breaks are masked by the binding of the topoisomerase-II-DNA complex to the synthetic filter from which DNA is eluted subsequent to alkaline denaturation. In this manuscript, we report that as the number of cells lysed on the filter is increased, binding of the topoisomerase-II-DNA complex to the filter is compromised, permitting expression of DNA damage in the absence of enzymatic deproteinization.

Animals↗
PubMed1988

Neocarzinostatin-induced DNA base release accompanied by staggered oxidative cleavage of the complementary strand.

Treatment of an end-labeled DNA restriction fragment with the nonprotein chromophore of neocarzinostatin induced lesions which, after treatment with endonuclease IV or putrescine, were expressed as site-specific double-strand breaks. Analysis of the termini at cleavage sites in each strand showed that the neocarzinostatin-induced lesions consisted of an apurinic/apyrimidinic site plus a closely opposed break in the complementary strand. The break always occurred opposite the base two positions upstream from the apurinic/apyrimidinic site and had the 3'-phosphate and 5'-aldehyde termini characteristic of neocarzinostatin-induced breaks. This positioning suggests that neocarzinostatin simultaneously attacks two DNA sugars on opposite edges of the minor groove. The sequence specificity for formation of apurinic/apyrimidinic sites with closely opposed breaks reflected that of neocarzinostatin-induced mutagenesis. The potent mutagenicity of these lesions may be attributable to the presence of closely opposed damage in both DNA strands.

Antibiotics, Antineoplastic↗
PubMed1988

Effect of apurinic/apyrimidinic endonucleases and polyamines on DNA treated with bleomycin and neocarzinostatin: specific formation and cleavage of closely opposed lesions in complementary strands.

Bleomycin and neocarzinostatin induce modified apurinic/apyrimidinic (AP) sites by oxidation of the sugar moiety in DNA. In order to quantitatively assess the susceptibility of these lesions to repair endonucleases, drug-treated 3H-labeled colE1 DNA was mixed with 14C-labeled heat-depurinated DNA, and endonuclease-susceptible sites in the mixture were titrated with various AP endonucleases or with polyamines. Single- and double-strand breaks were quantitated by determining the fractions of supercoiled, nicked circular, and linear molecules. Exonuclease III and endonucleases III and IV of Escherichia coli, as well as putrescine, produced a nearly 2-fold increase in single-strand breaks in bleomycin-treated DNA, indicating cleavage of drug-induced AP sites. The bleomycin-induced AP sites were comparable to heat-induced sites in their sensitivity to E. coli endonucleases III and IV but were cleaved by exonuclease III only at high concentrations. Bleomycin-induced AP sites were much more sensitive to cleavage by putrescine than heat-induced sites. Treatment with putrescine or very high concentrations of endonuclease III also increased the number of double-strand breaks in bleomycin-treated DNA, suggesting a minor class of lesion consisting of an AP site accompanied by a closely opposed break in the complementary strand. These complex lesions were resistant to cleavage by endonuclease IV. However, when colE1 DNA was treated with neocarzinostatin, subsequent treatment with putrescine, endonuclease IV, or very high concentrations of endonuclease III produced a dramatic increase in double-strand breaks but no detectable increase in single-strand breaks. These results suggest that virtually all neocarzinostatin-induced AP sites are accompanied by a closely opposed strand break.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibiotics, Antineoplastic↗
PubMed1988

Mutagenesis of bleomycin-damaged lambda phage in SOS-deficient and repair endonuclease-deficient Escherichia coli.

Previous DNA sequence analysis of bleomycin-induced forward mutations in repackaged lambda phage has suggested SOS-dependent replicative bypass of oxidized apyrimidinic sites as a possible mechanism of mutagenesis. In order to evaluate this hypothesis further, frequencies of mutation to a clear-plaque phenotype were compared for bleomycin-damaged phage grown in various repair-deficient strains of Escherichia coli. Survival of bleomycin-damaged phage was virtually identical in all host strains. Studies in SOS-deficient strains indicated specific requirements for functional recA+ and umuC+ alleles in the generation of the majority of bleomycin-induced mutations, as well as a less stringent requirement for induction of the SOS response by ultraviolet irradiation of the host cells. These results are expected for mutagenesis resulting from apyrimidinic sites. However, the mutation frequency for bleomycin-damaged phage was the same whether the phage were grown in a wild-type strain or in strains deficient in apurinic/apyrimidinic repair endonucleases; this was true even for an nth-nfo-xth- strain lacking all three major apurinic/apyrimidinic endonucleases (endonuclease III, endonuclease IV, and exonuclease III). Likewise, phage grown in an endonuclease IV-overproducing strain showed the same mutation frequency as those grown in wild-type cells. These data suggest that either i) bleomycin-induced mutagenesis results from SOS-dependent bypass of lesions other than apyrimidinic sites or ii) the number of apyrimidinic sites available for SOS processing is virtually independent of the level of apurinic/apyrimidinic endonuclease activity in the cell. It is possible that a fraction of the apyrimidinic sites induced by bleomycin either are intrinsically resistant to repair or undergo secondary reactions that render them resistant.

Bacterial Proteins↗
PubMed1987

Bleomycin-induced mutagenesis in repackaged lambda phage: base substitution hotspots at the sequence C-G-C-C.

DNA isolated from lambda phage was treated with bleomycin A2 plus Fe2+. The bleomycin-damaged DNA was added to lambda packaging extracts and the resulting phage were grown in SOS-induced E. coli. Under these conditions, treatment of the DNA with 0.8 microM bleomycin reduced the viability of the repackaged phage to 3% and increased the frequency of clear-plaque mutants in the progeny by a factor of 16. Bleomycin-induced mutations which mapped to the DNA-binding domain of the cI gene were subjected to DNA-sequence analysis. The most frequent events were single-base substitutions at G:C base pairs, nearly all of which occurred at cytosines in the sequence Py-G-C. Cytosines in the third position of the sequence C-G-C-C were particularly susceptible to mutation. At A:T base pairs, mutations were less frequent and were a mixture of single-base substitutions and -1 frameshifts, occurring primarily at G-T and A-T sequences. Thus, the overall specificity of bleomycin-induced mutations matches that of bleomycin-induced DNA lesions (strand breaks and apyrimidinic sites), which are formed at G-C (particularly Py-G-C), G-T and, to a lesser extent, A-T sequences. Furthermore, the frequency of various types of substitutions was consistent with selective incorporation of A and T residues opposite apyrimidinic sites at these sequences. The highly selective nature of bleomycin-induced mutations may explain the lack of mutagenesis by this compound in a number of reversion assays.

Bacteriophage lambda↗
PubMed1987

A role of oxidative DNA sugar damage in mutagenesis by neocarzinostatin and bleomycin.

The anti-tumor antibiotics neocarzinostatin and bleomycin specifically oxidize deoxyribose in DNA at the C-5' and C-4' positions, respectively. The resulting DNA lesions include strand breaks and apyrimidinic sites. Both agents are broad specificity mutagens, inducing, in various systems, base substitutions, frameshifts and deletions. Sequencing studies in bacterial systems have suggested that the base substitutions may result primarily from replicative bypass of the oxidized apyrimidinic sites.

Animals↗
PubMed1986

Base substitution mutations induced in the cI gene of lambda phage by neocarzinostatin chromophore: correlation with depyrimidination hotspots at the sequence AGC.

Treatment of intact lambda phage with the nonprotein chromophore of neocarzinostatin resulted in efficient phage inactivation and generation of clear-plaque mutants. Both effects required a preincubation at low pH to allow diffusion of chromophore into the phage head. Chromophore activation was then effected by addition of a sulfhydryl cofactor, followed by a shift to neutral pH. Sequence analysis of mutations mapped to the DNA-binding region of the cI gene revealed that nearly all were single base substitutions. Significant numbers of all possible base changes were found, with A:T to G:C transitions being the most frequent events. Of 11 G:C to A:T transitions, 7 were found at C residues in the trinucleotide sequence AGC, which has previously been shown to be a hotspot for chromophore-induced depyrimidination. This result, as well as the SOS dependence of mutagenesis and the overall distribution of various types of base substitutions, is consistent with the hypothesis that apurinic/apyrimidinic sites are important mutagenic lesions.

Antibiotics, Antineoplastic↗
PubMed1986

Molecular mechanism of novel DNA sugar damage by an antitumour protein antibiotic.

Neocarzinostatin (NCS) belongs to a family of antitumour protein antibiotics that selectively inhibit DNA synthesis. Replicon initiation in mammalian cells is selectively inhibited by NCS, and cells defective in DNA repair, such as ataxia telangiectasia fibroblasts, are especially sensitive to NCS as they are to X-ray. The holoantibiotic consists of a nonprotein chromophore (Mr = 659), tightly and specifically bound to an apoprotein (Mr = 10,700). The apoprotein protects the highly labile chromophore from degradation in aqueous solution; all the activity resides in the nonprotein chromophore. The latter binds specifically to DNA, especially to regions rich in T and A residues, with a tight binding site consisting of four base pairs. NCS chromophore consists of three main structural subunits: a naphthoic acid derivative, an amino-sugar and a connecting highly unsaturated middle component (C12H5) with a strained ether (probably epoxide) and cyclic carbonate. The authors have proposed that the naphthoic acid subunit intercalates DNA and the positively charged amino sugar binds electrostatically to the negatively charged sugar phosphate backbone of DNA; these two anchors serve to juxtapose the middle piece with the deoxyribose of mainly thymidylate residues in DNA. Upon activation of the drug by a thiol (which forms an adduct with the middle piece) and in the presence of O2, there is a selective oxidation of the 5'-C of deoxyribose to produce a DNA strand break with a phosphate at the 3'-end and a nucleoside 5'-aldehyde at the other. Kinetic analysis shows that one molecule of thiol adds to DNA-bound NCS chromophore even in the absence of oxygen; this is rapidly followed by the consumption of 1 mol of O2 and then another mol of thiol. The oxygen of the 5'-aldehyde is derived from O2, not H2O. Even in the absence of O2 the NCS chromophore abstracts a hydrogen from C-5' of deoxyribose in DNA, presumably generating a carbon-centred radical intermediate in the DNA (other mechanisms have not been eliminated) which can add O2 to form a peroxy derivative. The second molecule of thiol may be involved in the cleavage of this complex to form the 5'-aldehyde at the strand break. There is no evidence for the involvement of metals or a diffusible form of reduced oxygen.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Detection of neocarzinostatin chromophore-deoxyribose adducts as exonuclease-resistant sites in defined-sequence DNA.

A 5'-end-labeled DNA restriction fragment was treated with the nonprotein chromophore of neocarzinostatin under anoxia in the presence of dithiothreitol, conditions known to maximize formation of chromophore-deoxyribose adducts. Under conditions where unmodified DNA was digested to completion, chromophore-treated DNA was highly resistant to digestion by exonuclease III plus the 3'----5' exonucleolytic activity of T4 DNA polymerase and partially resistant to digestion by exonuclease III plus snake venom exonuclease. The electrophoretic mobilities of the products of exonucleolytic digestion suggested that (i) digestion by exonuclease III or T4 polymerase terminated one nucleotide before the nucleotide containing the adduct, (ii) the remaining nucleotide directly adjacent to the adduct (3' side) could be removed by snake venom phosphodiesterase, but at a slow rate, (iii) the covalently linked chromophore decreased the electrophoretic mobilities of the digestion products by the equivalent of approximately three nucleotides, and (iv) adducts formed under anaerobic conditions occurred at the same nucleotide positions as the strand breaks formed under aerobic conditions (primarily at T and, to a lesser extent, A residues). The close similarity in sequence specificity of adducts and strand breaks suggests that a common form of nascent DNA damage may be a precursor to both lesions. A chromophore-induced free radical on C-5' of deoxyribose, subject to competitive fixation by addition reactions with either oxygen or chromophore, is the most likely candidate for such a precursor. The base specificity of adduct formation does not reflect the reported base specificity of neocarzinostatin-induced mutagenesis, suggesting that lesions other than adducts may be responsible for at least some neocarzinostatin-induced mutations, particularly those occurring at G X C base pairs.

Animals↗
PubMed1985

Endonuclease-resistant apyrimidinic sites formed by neocarzinostatin at cytosine residues in DNA: evidence for a possible role in mutagenesis.

When defined-sequence DNA from the lacl region of plasmid pMC1 was treated with the nonprotein chromophore of neocarzinostatin in the presence of various thiols, the predominant lesions were direct strand breaks, occurring primarily at thymine and adenine residues. In the presence of glutathione, however, alkali-dependent strand breaks, occurring at certain cytosine residues, were also detected but were virtually absent when other thiols were used. Chromophore-induced release of free cytosine base from [3H]cytosine-labeled DNA was 2- to 3-fold greater with glutathione than with the other thiols. These results suggest that the alkali-dependent strand break is some form of apyrimidinic site. These sites were substrates for endonuclease IV of Escherichia coli, although a 5-fold greater concentration of enzyme was required for their cleavage than was required for cleavage of apurinic sites in depurinated DNA. These sites were also less sensitive to E. coli endonuclease VI (exonuclease III) by a factor of at least 5 and less sensitive to E. coli endonuclease III by a factor of at least 10. These and other results suggest that these sites are chemically different from normal apurinic/apyrimidine sites. When chromophore-induced apyrimidinic sites were quantitated as alkali-dependent breaks at 11 specific sites in the lacl gene, a correlation was found between occurrences of these lesions and the reported frequencies of G-C to A X T transitions at the same sites. All occurrences of the trinucleotide sequence A-G-C, including the ochre 21 mutational hot spot, were particularly prominent sites. The selective formation of endonuclease-resistant apyrimidinic sites at specific cytosine residues may explain the high frequency of G X C to A X T transitions in the mutational spectrum of neocarzinostatin.

Antibiotics, Antineoplastic↗
PubMed1984

Competition between anaerobic covalent linkage of neocarzinostatin chromophore to deoxyribose in DNA and oxygen-dependent strand breakage and base release.

Treatment of poly(dA-dT) X poly(dA-dT) with the nonprotein chromophore of neocarzinostatin in the presence of sulfhydryls resulted in both direct and alkali-dependent base release, indicative of DNA sugar oxidation. Covalent chromophore-DNA adducts were also formed. Under anaerobic conditions, base release was strongly inhibited; however, adduct formation was not inhibited and in some cases was markedly enhanced. In the presence of dithiothreitol, anoxia increased adduct formation by a factor of 2, and a particularly stable adduct species was formed, which was recovered from nuclease digests of the treated DNA as a highly fluorescent compound with structure chromophore-d(TpApT). Acid hydrolysis of chromophore-d(TpApT) released free adenine base and both 3'dTMP and 5'dTMP, leaving a compound that contained only chromophore and the deoxyadenosine sugar. These results conclusively confirm that the chromophore forms a covalent adduct with deoxyribose in DNA. Thus, even in the absence of oxygen, activation of the chromophore by sulfhydryls results in the formation of a species capable of reacting with deoxyribose. Several other adduct species were also formed, some of which were nonfluorescent and relatively hydrophilic, but all of which were produced in increased amounts under anoxia. This inverse relation between sugar oxidation and adduct formation suggests that the two lesions share a common precursor. In the presence of other thiols, the effects of anoxia were somewhat different. With glutathione, anoxia markedly enhanced adduct formation, but the total adduct formed was considerably less than with dithiothreitol. With 2-mercaptoethanol, anoxia had no effect on total adduct formation, but the distribution of adduct species was altered.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibiotics, Antineoplastic↗
PubMed1983

Stoichiometric uptake of molecular oxygen and consumption of sulfhydryl groups by neocarzinostatin chromophore bound to DNA.

In the presence of DNA, and under conditions which resulted in efficient DNA degradation, the reaction of the neocarzinostatin chromophore with sulfhydryl groups was accompanied by a rapid drop in the oxygen tension of the solution. The total extent of oxygen uptake indicated that, consistently, 1 mol of O2 was consumed/mol of chromophore. The rate of oxygen uptake, however, was strongly dependent on the sulfhydryl concentration, and uptake occurred within a few seconds of the sulfhydryl-induced increase in 420-nm fluorescence of the chromophore. Parallel experiments, in which the sulfhydryl concentration of the solution was monitored, showed that approximately 2 mol of sulfhydryl groups were consumed/mol of chromophore, with kinetics similar to those of O2 uptake. Under anaerobic conditions, only 1 mol of sulfhydryl was consumed, but the sulfhydryl-induced fluorescence increase was not inhibited. These results suggest that (i) a reaction with a single sulfhydryl group converts the chromophore to an activated form, (ii) in the presence of DNA this activated chromophore participates in a subsequent reaction which consumes 1 mol of O2 followed by an additional mole of sulfhydryl, and (iii) each chromophore molecule undergoes only one such reaction cycle. In the absence of sulfhydryl groups, the chromophore slowly degraded, giving a product with intense 490-nm fluorescence. This spontaneous degradation reaction, which does not result in DNA damage, was also accompanied by uptake of nearly 1 mol of O2/mol of chromophore.

Animals↗