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L F Povirk

Publications and source records attributed to L F Povirk.

At least 37 records · Page 2Linked to original sources
PubMed1996

Construction of a vector containing a site-specific DNA double-strand break with 3'-phosphoglycolate termini and analysis of the products of end-joining in CV-1 cells.

Previous studies have shown that linearized SV40-based shuttle vectors transfected into mammalian cells are efficiently recircularized by an error-prone end-joining pathway. To determine whether and with what specificity free radical-mediated double-strand breaks are rejoined by this pathway, a structural mimic of such a break was introduced at a specific site in an SV40-based shuttle vector, by ligating purified 3'-phosphoglycolate-terminated oligonucleotides into 3' recessed ends generated in the linearized vector. These terminally blocked linear vectors were efficiently repaired and replicated when transfected into simian CV-1 cells. Sequencing across the repair joints in progeny plasmid indicated that, for a blunt-ended vector, the most frequent mechanism of rejoining was splicing at a terminal 4-base homology; however, a significant fraction of the joints retained all bases from both ends of the break, consistent with a mechanism involving simple 3'-phosphoglycolate removal, followed by blunt-end ligation. For the analogous 3'-hydroxyl terminated break, the fraction of simple blunt-end ligations was considerably higher. For a phosphoglycolate-terminated vector with cohesive ends the most frequent repair mechanism was simple ligation of the annealed cohesive ends, presumably preceded by phosphoglycolate removal. For all these substrates, the remaining repair joints showed small or large deletions from one or both of the ends, usually with apparent annealing at short (1-4-base) homologies. The results suggest that while breaks with 3'-phosphoglycolates can be repaired, these blocked termini represent a significant barrier to DNA end-joining, and can significantly alter its specificity. The presence of cohesive ends appears to improve markedly the fidelity of rejoining for terminally blocked double-strand breaks.

Animals↗
PubMed1996

Deletion/insertion mutation that causes biotinidase deficiency may result from the formation of a quasipalindromic structure.

Biotinidase is responsible for recycling the vitamin biotin from biocytin that is formed after the proteolytic degradation of the biotin-dependent carboxylases. We have identified a deletion/insertion mutation within exon D of the human biotinidase gene in a child with biotinidase deficiency. The mutation causes a frame shift and premature termination which are predicted to result in a truncated protein. We propose that the mutation occurred during DNA replication by either of two mechanisms. Both mechanisms involve formation of a quasipalindromic hairpin loop in the template and dissociation of DNA polymerase alpha. This mutation supports the formation of palindromic structures as a possible cause of deletions in eukaryotes, and supports the proposal, derived from in vitro studies, that polymerase alpha may preferentially arrest or dissociate at specific template sequences.

Amidohydrolases↗
PubMed1995

Selective damage to the active X chromosome by camptothecin and amsacrine as determined by an allele-specific alkaline unwinding assay.

Previous studies with MCF-7 cells demonstrated that several agents induce greater strand breakage in active genes than in nontranscribed centromeric regions. To better assess the effects of gene activity and inactivity, an allele-specific DNA strand break assay was developed, which allowed direct comparison of damage at a specific genetic locus on the active and inactive X chromosomes. The ZP lymphoblastoid cell line is heterozygous at the glucose-6-phosphate dehydrogenase (G6PD) locus, and the unexpressed (A) allele on the inactive X chromosome contains a FokI restriction site that is lacking in the expressed (B) allele on the active X. ZP cells were treated with camptothecin or amsacrine, and subjected to alkaline-induced DNA unwinding. Following detergent lysis and digestion of single-stranded DNA with S1 nuclease, the remaining double-stranded DNA was isolated and subjected to polymerase chain reaction (PCR) with primers that flank the polymorphic FokI site, with [alpha-32P]dCTP being added in the last PCR cycle. The resulting labeled PCR product was cleaved with FokI to assess the A/B allele ratio in the double-stranded DNA fraction. Treatment with camptothecin and amsacrine increased the apparent A/B ratio by factors of 2-3 and 1.5-2 respectively, indicating that the active B allele is preferentially damaged by these agents.

Alleles↗
PubMed1995

Ionizing radiation-induced DNA strand breakage and rejoining in specific genomic regions as determined by an alkaline unwinding/Southern blotting method.

A recently developed, combined alkaline unwinding/Southern blotting assay was utilized to examine DNA damage and repair induced by ionizing radiation within specific large-scale genomic regions. Following treatment of MCF-7 breast tumour cells with 2-10-Gy gamma-rays, strand breakage and rejoining were measured in bulk DNA, in the centromeric alpha-satellite region of chromosome 17, and in the chromatin regions containing the unexpressed beta-globin gene and the expressed c-myc oncogene, which is known to be important for growth in the MCF-7 cell line. Damage in both the c-myc and beta-globin regions was markedly greater than in either alpha-satellite or bulk DNA. However, the kinetics of strand break repair were approximately the same in c-myc as in alpha-satellite or bulk DNA. Surprisingly, the radiomimetic antibiotic bleomycin, which also induces free-radical-mediated strand breakage, showed considerably less heterogeneity of DNA damage among the genomic regions examined than did radiation. The results suggest that actively transcribed genes, as well as at least some inactive genes, are surrounded by large-scale domains of radiosensitive chromatin. With no apparent enhancement of rejoining, the increased incidence of strand breaks in these regions persists until rejoining is essentially complete. Changes in the integrity of specific chromatin regions may be an important aspect of DNA damage-induced cell death.

Bleomycin↗
PubMed1994

DNA damage induced by bleomycin, neocarzinostatin, and melphalan in a precisely positioned nucleosome. Asymmetry in protection at the periphery of nucleosome-bound DNA.

The antitumor drugs bleomycin, neocarzinostatin, and melphalan all damage DNA by mechanisms which involve binding in the minor groove. In order to examine at high resolution the modulating effects of chromatin structure on the action of these drugs, an end-labeled DNA fragment from the Xenopus laevis 5 S rRNA gene was reconstituted with histone octamers to form a precisely positioned nucleosome. For each drug, DNA damage at specific sequence positions in the fragment was then compared for nucleosome-bound versus naked DNA. Reconstitution into nucleosomes resulted in a marked inhibition of the DNA cleavage induced by bleomycin (5-fold) and neocarzinostatin (2.4-fold) in the central region of nucleosomal DNA. However, at the periphery of nucleosome-bound DNA, a distinct asymmetry was apparent, with marked inhibition of cleavage toward the upstream side, but little if any inhibition toward the downstream side, which overlaps the binding site of the transcription factor TFIIIA. In the case of melphalan, alkylation at adenine N-3 was inhibited by nearly 2-fold throughout the nucleosome, whereas alkylation at guanine N-7 was either slightly inhibited or slightly enhanced, depending on sequence position. None of the drugs showed the 10-base pair periodicity characteristic of hydroxyl radical-induced cleavage of nucleosomal DNA. The results are consistent with a model in which minor groove sites in nucleosome-bound DNA remain relatively accessible to small molecules, even where the minor groove faces the histone core, and in which drug-induced DNA damage is inhibited by conformational constraints imposed on DNA by nucleosome structure. Furthermore, the degree of such constraints appears to be sequence-dependent, at least near the periphery of nucleosome-bound DNA.

Animals↗
PubMed1994

Single base-pair deletions induced by bleomycin at potential double-strand cleavage sites in the aprt gene of stationary phase Chinese hamster ovary D422 cells.

One possible mechanism for the generation of deletion mutations is inaccurate repair of DNA double-strand breaks. In an attempt to detect such aberrant repair events in intact cells, confluent stationary phase cultures of chinese hamster ovary D422 cells, which are hemizygous for aprt, were treated for two days with low concentrations of bleomycin, and aprt mutant clones were selected and analyzed by polymerase chain reaction and DNA sequencing. Bleomycin was quite mutagenic in stationary phase cells, increasing the mutant frequency by five to 40-fold at 5 to 50% survival. While spontaneous mutations generated under these conditions were predominantly base substitutions, the majority of the bleomycin-induced mutations were very small deletions, with lesser numbers of large deletions/rearrangements and base substitutions. Although the small deletions tended to be clustered in several short segments of the gene, nucleosome positioning studies indicated that there was no consistent phasing of nucleosomes in aprt, suggesting that the clustering was due to sequence specificity rather than chromatin structure. About half of the bleomycin-induced mutations were single-base-pair (-1) deletions, and the majority of these involved deletion of one C in a G-Cn sequence (n > or = 2). At such sites, bleomycin is known to induce double-strand breaks by fragmentation of deoxyribose moieties at the same sequence position in both strands, resulting in a blunt-ended double-strand break with 5'-phosphate and 3'-phosphoglycolate termini. Thus, this sequence specificity is consistent with a model in which bleomycin-induced -1 deletions are generated by a double-strand break rejoining process involving removal of phosphoglycolate moieties from both 3' ends, followed by blunt-end ligation. The results support the view that repair of free radical-mediated double-strand breaks in mammalian cells in G1/G0 phase can be effected by such simple end-joining mechanisms, without the need for homologous recombination.

Adenine Phosphoribosyltransferase↗
PubMed1994

Induction of apoptotic DNA fragmentation and cell death in HL-60 human promyelocytic leukemia cells by pharmacological inhibitors of protein kinase C.

The present studies were undertaken to characterize further the potential role of protein kinase C (PKC) in the regulation of apoptosis in HL-60 promyelocytic leukemia cells. The capacity of acute exposure to specific and nonspecific pharmacological inhibitors of PKC to promote apoptotic DNA fragmentation was examined both quantitatively and qualitatively and correlated with effects on cellular differentiation and proliferation. Incubation of HL-60 cells for 6 h with chelerythrine and calphostin C (highly specific inhibitors that act at the regulatory domain) or H7 and gossypol (nonspecific inhibitors that act at the PKC catalytic domain) produced concentration-dependent increases in DNA fragmentation. Induction of DNA fragmentation by chelerythrine, calphostin C, and gossypol was biphasic, resulting in a sharp decline in effect at concentrations above 5 microM, 0.1 microM, and 100 microM, respectively, whereas maximal and more stable effects were observed in response to H7 (100 microM). A 6-h exposure to staurosporine, a nonspecific but potent PKC inhibitor, failed to induce DNA fragmentation at concentrations generally used to achieve maximal inhibition of enzyme activity (e.g., 50 nM) but promoted fragmentation at considerably higher concentrations (e.g., > or = 200 nM). In contrast, 6-h exposures to the nonspecific protein kinase inhibitor hypericin (0.1 to 100 microM) or to the nonspecific inhibitor of protein kinase A, HA1004 (50 microM), were without effect on DNA fragmentation. DNA obtained from cells exposed to chelerythrine (5 microM), calphostin C (100 nM), H7 (50 microM), gossypol (50 microM), and staurosporine (200 nM)--but not hypericin (25 microM)--exhibited clear evidence of internucleosomal DNA cleavage on agarose gel electrophoresis; moreover, these cells exhibited the classical morphological features of apoptosis (cell shrinkage, nuclear condensation, and the formation of apoptotic bodies). All of the PKC inhibitors that induced apoptosis, and one of the inhibitors that did not (hypericin), substantially inhibited HL-60 cell clonogenicity at the concentrations evaluated. None of the agents tested induced cellular maturation as assessed by nonspecific esterase and nitro-blue tetrazolium positivity. DNA fragments obtained from cells exposed to specific and nonspecific PKC inhibitors possessed predominantly 5'-phosphate termini, consistent with the action of a Ca(2+)-/Mg(2+)-dependent endonuclease. Finally, Northern blot analysis revealed that exposure to calphostin C at a concentration that induced apoptosis (100 nM) failed to alter expression of bcl-2, an oncogene known to block apoptosis in both lymphoid and myeloid leukemia cells.(ABSTRACT TRUNCATED AT 400 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

Effects of bryostatin 1 and other pharmacological activators of protein kinase C on 1-[beta-D-arabinofuranosyl]cytosine-induced apoptosis in HL-60 human promyelocytic leukemia cells.

We have demonstrated previously that bryostatin 1, a macrocylic lactone with putative protein kinase C (PKC)-activating properties, synergistically augments the antileukemic actions of the deoxycytidine analog 1-[beta-D-arabinofuranosyl]cytosine (ara-C) in HL-60 human promyelocytic leukemia cells (Grant et al., Biochem Pharmacol 42: 853-867, 1991), and that this effect appears to be related to sensitization to ara-C-induced apoptosis (Grant et al., Cancer Res 52: 6270-6278, 1992). In the present studies, we have assessed the extent of this damage by quantitative spectrofluorophotometry of small molecular weight, double-stranded DNA fragments in order to provide: (a) a more complete characterization of the interaction between ara-C and bryostatin 1, and (b) a direct comparison of the relative effects of bryostatin 1 treatment with other pharmacological manipulations known to modulate protein kinase C activity. Exposure of cells to ara-C (10(-9) to 10(-4) M; 1-24 hr) induced time- and concentration-related increases in the extent of DNA fragmentation. Treatment with bryostatin 1 (10(-11) to 10(-7) M; 1-24 hr) alone failed to induce DNA damage, but promoted substantial time- and concentration-related increases in the extent of fragmentation induced by a subsequent 6-hr exposure to ara-C. Maximal potentiation of fragmentation (e.g. 2- to 3-fold greater than that obtained with ara-C alone) was observed following a 24-hr pretreatment with 10(-8) M or 10(-7) M bryostatin 1, and correlated closely with enhanced inhibition of HL-60 cell clonogenicity. The stage-1 tumor-promoter phorbol dibutyrate potentiated the effects of ara-C in a biphasic manner, maximally augmenting the response at 2.5 x 10(-8) M, but exerting no effect at 10(-7) M, whereas the stage-2 tumor-promoter mezerein failed to augment ara-C-related DNA fragmentation at low concentrations, and antagonized ara-C action at high concentrations. In contrast, ara-C-related DNA fragmentation was attenuated or abolished either by continual preexposure to synthetic diglyceride or by pretreatment with exogenous phospholipase C at all concentrations tested. Increased DNA fragmentation was not specifically related to recruitment of cells into S-phase or enhancement of ara-C-related cellular differentiation. Finally, concentrations of bryostatin 1 that maximally potentiated ara-C-related DNA fragmentation were associated with virtually complete down-regulation of total cellular PKC activity, whereas diglyceride and phospholipase C, which suppressed the response to ara-C, moderately increased total PKC activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Antineoplastic Agents↗
PubMed1994

Influence of amsacrine (m-AMSA) on bulk and gene-specific DNA damage and c-myc expression in MCF-7 breast tumor cells.

In the MCF-7 human breast tumor cell line, the aminoacridine, m-AMSA, induces protein-associated DNA strand breaks consistent with inhibition of topoisomerase II. However, neither single-strand nor double-strand breaks in DNA, determined using conventional assays, show a consistent relationship with m-AMSA-induced inhibition of growth. In contrast, when DNA strand breaks are determined by alkaline unwinding under the high salt conditions of the alkaline unwinding/Southern blotting (AU/SB) assay, developed by our laboratories, damage to DNA corresponds closely with growth inhibition. The AU/SB assay, which is capable of assessing breaks within large-scale domains (upwards of 1 megabase) surrounding genes of interest, was further utilized to explore the capacity of m-AMSA to induce damage within specific genomic regions that may regulate cell growth. Regions encompassing the transcriptionally active oncogenes, c-myc and c-fos, were found to be more susceptible to m-AMSA-induced strand breaks than the region encompassing the non-transcribed alpha-satellite DNA or the genome as a whole (bulk DNA). These findings demonstrate that m-AMSA may produce more pronounced damage within specific genomic regions than in bulk DNA, m-AMSA also preferentially altered expression of the c-myc oncogene; at an m-AMSA concentration where growth was inhibited by between 70 and 80%, steady-state c-myc mRNA levels declined to approximately 10-15% of control levels within 2-3 hr; furthermore, concentration-dependent reductions in c-myc expression appeared to coincide with growth inhibition. In addition, inhibition of [3H]thymidine incorporation after 2 hr directly paralleled inhibition of growth, suggesting an early effect at the level of DNA biosynthesis, possibly related to the down-regulation of c-myc expression. It is proposed that specific lesions, e.g., in regions surrounding the c-myc gene, as well as generalized lesions in DNA may lead to growth inhibition mediated by down-regulation of the expression of select growth regulatory genes, such as c-myc.

Amsacrine↗
PubMed1994

DNA damage and mutagenesis induced by nitrogen mustards.

The nitrogen mustards are bifunctional alkylating agents which, although used extensively in cancer chemotherapy, are themselves highly carcinogenic. All nitrogen mustards induce monofunctional guanine-N7 adducts, as well as interstrand N7-N7 crosslinks involving the two guanines in GNC.GNC (5'-->3'/5'-->3') sequences. In addition, the aromatic mustards melphalan and chlorambucil also induce substantial alkylation at adenine N3, while cyclophosphamide forms phosphotriesters with relatively high frequency. Nitrogen mustards are genotoxic in virtually every assay, and produce a wide array of mutations, including base substitutions at both G.C and A.T base pairs, intragenic as well as multilocus deletions, and chromosomal rearrangements. Mutational spectra generated by these agents in various model systems vary widely, and no single lesion has been implicated as being primarily responsible for mustard-induced mutagenesis. On the contrary, adducts of both adenine and guanine, and monofunctional as well as bifunctional adducts, appear to be involved. Further, it is still not known which types of mutation are responsible for mustard-induced cancers, since no genes have yet been identified which are consistently altered in these malignancies.

Animals↗
PubMed1994

Effect of distamycin on chlorambucil-induced mutagenesis in pZ189: evidence of a role for minor groove alkylation at adenine N-3.

Previous work has shown that the bifunctional alkylating agent chlorambucil induces thermolabile adenine adducts and that the predominant chlorambucil-induced mutations in shuttle vector pZ189 are transversions at AT base pairs. In order to assess the role of thermolabile adducts in generating these transversions, pZ189 was treated with chlorambucil in the presence of distamycin, which specifically blocks formation of thermolabile adenine adducts. Analysis of the mutations resulting from replication of the damaged vector in human 293 cells showed that base substitutions at AT base pairs were specifically suppressed in concert with suppression of thermolabile adducts at specific sites in the supF target gene, strongly supporting a role for these adducts in mutagenesis. Since there is considerable evidence that these adducts are N-3 alkylations, a computer graphics model of such an adduct was constructed. Modeling studies indicated that the adduct could be formed with little distortion of the DNA helix. Analysis of the adduct using the HINT (Hydropathic INTeractions) program was consistent with the proposal that favorable hydrophobic interactions of the phenyl ring of chlorambucil with the wall of the minor groove may promote adenine N-3 alkylation by this drug.

Adenine↗
PubMed1993

Dissociation between bulk damage to DNA and the antiproliferative activity of teniposide (VM-26) in the MCF-7 breast tumor cell line: evidence for induction of gene-specific damage and alterations in gene expression.

In the MCF-7 breast tumor cell line, induction of bulk damage to DNA (measured either as total strand breaks or as double-strand breaks) fails to correspond with the antiproliferative activity of the demethylepipodo-phyllotoxin derivative, VM-26. In contrast, VM-26 produces an early (within 2-3 h) concentration-dependent reduction in c-myc expression (and of DNA synthesis) which parallels inhibition of cell growth, suggesting the possibility of effects of VM-26 at the level of genomic regions which regulate DNA replicative function. Although VM-26 also produces a reduction in c-myc expression in K562 human leukemic cells, these alterations fail to correspond with the concentration-dependent effects on cell growth in this cell line. Utilizing the newly developed alkaline unwinding/Southern blotting assay in the MCF-7 breast tumor cell line, it was determined that VM-26 induces damage within regions surrounding the c-myc gene and the beta-globin gene which exceeds that induced in both alpha-satellite DNA and in L1 repeat sequences; damage within c-myc and beta-globin also exceeds that observed throughout the genome as a whole. These findings indicate that certain genomic regions incur preferential damage in MCF-7 cells exposed to VM-26. It appears possible that damage within such genomic regions could lead to alterations in expression of select genes associated with regulation of cellular proliferation, resulting in reduced DNA synthesis, compromised cell growth, and, ultimately, cell death.

Blotting, Southern↗
PubMed1993

Spontaneous cleavage of bleomycin-induced abasic sites in chromatin and their mutagenicity in mammalian shuttle vectors.

The stability of oxidized abasic sites induced by bleomycin and neocarzinostatin was examined in chromatin reconstituted from a supercoiled plasmid and core histones. Most of the drug-induced abasic sites were found to undergo spontaneous cleavage in chromatin, probably by reaction with histone amine groups. However, there was considerable heterogeneity in the rate of spontaneous cleavage, with some sites being cleaved almost immediately and some remaining intact even after 7 h. Bleomycin-induced abasic sites with closely opposed strand breaks were more unstable than lone abasic sites. Neocarzinostatin-induced abasic sites, which have a different chemical structure, were cleaved somewhat more slowly than those induced by bleomycin. To assess the mutagenic potential of bleomycin-induced abasic sites, bleomycin-treated shuttle vectors were transfected into mammalian cells, and mutations in progeny plasmids were sequenced. Bleomycin treatment resulted primarily in deletions of various sizes in the shuttle vectors, including a number of one-base deletions occurring at potential bleomycin damage sites. However, under certain conditions, substitutions occurring at expected sites of bleomycin attack were also observed. The results suggest that bleomycin-induced abasic sites have only a slight potential to produce base substitutions in mammalian cells and that a substantial fraction of the double-strand breaks induced by bleomycin and most of the double-strand breaks induced by neocarzinostatin are the result of spontaneous cleavage of abasic sites with closely opposed strand breaks. Inaccurate repair of these double-strand breaks may account for the large deletions, and perhaps the one-base deletions, induced by bleomycin.

Apurinic Acid↗
PubMed1993

Small deletion and insertion mutations induced by the topoisomerase II inhibitor teniposide in CHO cells and comparison with sites of drug-stimulated DNA cleavage in vitro.

Sixty-five teniposide-induced mutations at the hemizygous aprt locus of CHO D422 cells were analyzed by polymerase chain reaction and DNA sequencing. Most (63%) of the mutations were deletions, duplications and insertions of various sizes, with the majority being less than 20 base-pairs. The remaining mutations were base substitutions, the majority of which were transversions. A significant correspondence was found between the teniposide-induced small deletion/duplication mutations and sites of teniposide-stimulated DNA strand cleavage by topoisomerase II in vitro. In particular, sequences which were deleted in one or more of the mutants showed a much higher incidence of strong cleavage sites than sequences not involved in deletions. However, the exact positioning of the cleavage sites with respect to the deletion termini was variable. The data did not suggest any unified model to account for all the mutations, but most of the deletions and duplications could be accounted for by one of three mechanisms: (1) double-strand break repair nonhomologous end-joining; (2) replication slippage/misalignment; and (3) addition or deletion of a few nucleotides at free 3' ends left by topoisomerase II, as previously suggested for similar mutations in phage T4. There was no evidence that topoisomerase II subunit exchange was a significant mechanism of mutagenesis in this system.

Animals↗
PubMed1992

DNA sequence analysis of mutations induced by melphalan in the CHO aprt locus.

In previous work, we established that treatment with melphalan (L-phenylalanine mustard) produced a predominance of A.T-->T.A transversions in the Simian virus 40 (SV40)-based shuttle vector pZ189 during replication in human 293 cells. Mutations were induced with varying doses (4-12 microM) melphalan in the aprt gene of the hemizygous Chinese hamster ovary (CHO) cell line D422 to determine whether a similar mutation spectrum would be observed in an endogenous gene. DNA sequence alterations were determined for 39 spontaneous and 41 melphalan-induced independent mutant clones. Other than a predominance of transversions in both systems, the spectrum of melphalan-induced aprt mutations bears little resemblance to the spectrum observed in the supF gene of the shuttle plasmid pZ189. In aprt, mutations at G.C base pairs (bp) predominated (29 of 41 base substitutions). Significantly enhanced mutagenesis was observed at 5' G-G-C 3' and 5' G-G-C-C 3' sites in the aprt gene. Almost half of the melphalan-induced base substitutions occurred at 5' G-N-C 3' sequences, which are believed to be potential interstrand crosslink sites.

Adenine Phosphoribosyltransferase↗
PubMed1992

A combined alkaline unwinding/Southern blotting assay for measuring low levels of cellular DNA breakage within specific genomic regions.

A method is described which combines alkaline induced DNA unwinding and Southern blotting to measure DNA damage occurring in specific genomic regions. Damage induced by gamma-rays at levels as low as 2 Gy was measured in bulk DNA and in a one megabase region surrounding the actively transcribed oncogene, c-myc, as well as in the inactive alpha-satellite DNA of chromosome 17. Although the unwinding kinetics for bulk DNA were consistent with random strand breakage throughout the genome as a whole, measurements at specific loci indicated that the region encompassing c-myc was at least 2-fold more susceptible to damage than either the bulk of the genome or the alpha-satellite region. The results of this study indicate that the combined alkaline unwinding/Southern blotting assay is a sensitive method for the detection of DNA damage within specific chromatin regions, at biologically relevant doses.

Blotting, Southern↗
PubMed1991

Thermolabile adenine adducts and A.T base pair substitutions induced by nitrogen mustard analogues in an SV40-based shuttle plasmid.

It was previously shown that the predominant mutations induced by melphalan (L-phenylalanine mustard) in the supF gene of shuttle plasmid pZ189 during replication in human cells are A.T----T.A transversions. In order to determine whether adenine adducts were formed at sequence positions corresponding to these mutations, melphalan-induced thermolabile adducts were mapped in the supF gene by selective depurination followed by strand cleavage in alkali. All A.T base pairs which were frequent sites for melphalan-induced A.T----T.A transversions were also prominent sites for formation of thermolabile adenine adducts. Although no mutations were detected at some prominent adduct sites, there was a significant correlation between adduct sites and mutation sites. While runs of two or more adenines were particularly prominent adduct sites, comparison of results obtained with 3'- and 5'-end-labeled DNA gave no evidence for intrastrand cross-links between adjacent adenines. Chlorambucil, another aromatic nitrogen mustard, showed sequence specificities for both mutagenesis and adenine adduct formation nearly identical to those seen with melphalan. The nonaromatic analogues mechlorethamine and phosphoramide mustard were much less efficient in inducing thermolabile adenine adducts, and mechlorethamine induced significantly fewer transversions at A.T base pairs than chlorambucil or melphalan. Formation of thermolabile adenine adducts by the aromatic nitrogen mustards was markedly reduced by blockage of the minor groove with distamycin, or by prior heat denaturation of the DNA. These results suggest that alkylation occurs primarily at the N-3 rather than N-7 position of adenine, probably as a consequence of the affinity of the aromatic rings of melphalan and chlorambucil for the minor groove.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗