Biomedical subjects

M Z Humayun

Publications and source records attributed to M Z Humayun.

At least 37 records · Page 2Linked to original sources

PubMed1995

Functional recA, lexA, umuD, umuC, polA, and polB genes are not required for the Escherichia coli UVM response.

The Escherichia coli UVM response is a recently described phenomenon in which pretreatment of cells with DNA-damaging agents such as UV or alkylating agents significantly enhances mutation fixation at a model mutagenic lesion (3,N4-ethenocytosine; epsilon C) borne on a transfected M13 single-stranded DNA genome. Since UVM is observed in delta recA cells in which SOS induction should not occur, UVM may represent a novel, SOS-independent, inducible response. Here, we have addressed two specific hypothetical mechanisms for UVM: (i) UVM results from a recA-independent pathway for the induction of SOS genes thought to play a role in induced mutagenesis, and (ii) UVM results from a polymerase switch in which M13 replication in treated cells is carried out by DNA polymerase I (or DNA polymerase II) instead of DNA polymerase III. To address these hypotheses, E. coli cells with known defects in recA, lexA, umuDC, polA, or polB were treated with UV or 1-methyl-3-nitro-1-nitrosoguanidine before transfection of M13 single-stranded DNA bearing a site-specific ethenocytosine lesion. Survival of the transfected DNA was measured as transfection efficiency, and mutagenesis at the epsilon C residue was analyzed by a quantitative multiplex DNA sequencing technology. Our results show that UVM is observable in delta recA cells, in lexA3 (noninducible SOS repressor) cells, in LexA-overproducing cells, and in delta umuDC cells. Furthermore, our data show that UVM induction occurs in the absence of detectable induction of dinD, an SOS gene. These results make it unlikely that UVM results from a recA-independent alternative induction pathway for SOS gene.

Bacterial Proteins↗

PubMed1994

UVM, an ultraviolet-inducible RecA-independent mutagenic phenomenon in Escherichia coli.

Most mutagenic DNA lesions are noninstructive in the sense that template instruction is either missing or inaccessible during DNA replication, leading to replication arrest. According to the SOS hypothesis, arrested replication induces the expression of SOS factors that force replication past stalled sites at the cost of mutagenesis. We have recently shown that prior UV irradiation of delta recA cells, in which the SOS pathway does not function, enhances mutagenesis at an ethenocytosine residue borne on a circular gapped duplex DNA vector, indicating the existence of an SOS-independent inducible mutagenic phenomenon termed UVM (UV modulation of mutagenesis). In the previous experiments, mutation fixation was expected to occur during gap-filling DNA synthesis. To test whether UVM is observable during normal replication by DNA polymerase III, we have examined mutagenesis at an epsilon C residue borne on M13 single-stranded DNA. By analyzing mutation frequency and specificity using a multiplex sequence assay, we now show that UVM is observable in UV-irradiated recA+, and in delta recA cells. These data indicate that UV irradiation induces a previously unrecognized mutagenic mechanism in Escherichia coli, and that this mechanism is manifested during gap-filling DNA synthesis as well as during normal DNA replication.

Base Sequence↗

PubMed1994

Base incorporation and extension at a site-specific ethenocytosine by Escherichia coli DNA polymerase I Klenow fragment.

Ethenocytosine (epsilon C) is a highly mutagenic exocyclic DNA lesion induced by carcinogens vinyl chloride and urethane. We have examined base incorporation and extension at a site-specific epsilon C residue by a quantitative gel electrophoretic assay using an exonuclease-deficient version of Escherichia coli DNA polymerase I (Klenow fragment) as the model enzyme. The data show that the KM for incorporation of adenine or thymine opposite epsilon C by is about 5 orders of magnitude higher than that for the incorporation of guanine opposite normal cytosine. The KM for base extension past epsilon C:A and epsilon C:T pairs is 1-2 orders of magnitude higher than that observed for a C:G pair. Although adenine misinsertion is favored over that of thymine, base extension occurs more readily when the base incorporated opposite epsilon C is thymine.

Alkylating Agents↗

PubMed1993

Quantitative multiplex sequence analysis of mutational hot spots. Frequency and specificity of mutations induced by a site-specific ethenocytosine in M13 viral DNA.

We describe an assay for determining the frequency and specificity of mutations occurring at hot spots within a population of DNA molecules. The procedure consists of (a) annealing the DNA population with a labeled oligonucleotide designed to prime DNA synthesis at the mutational hot spot; (b) DNA elongation in the presence of a single dideoxynucleoside triphosphate together with 1-3 deoxynucleoside triphosphates, and (c) quantitation of all limit elongation products by high-resolution gel electrophoresis followed by autoradiography and computing densitometry. Derivation of mutational frequency and specificity over a wide range of values is demonstrated for M13 viral DNA mixtures containing defined proportions of wild-type and mutant DNAs, as well as for M13 viral DNA populations obtained by transfection of DNA bearing a defined site-specific ethenocytosine lesion. The assay is shown to yield results similar to those obtained by laborious clone-by-clone sequencing of viral progeny. The method is not affected significantly by several tested variables and appears to be suitable for use as a quantitative assay for sequence microheterogeneity at defined positions within DNA populations. Application of the methodology demonstrates that ethenocytosine, an exocyclic DNA lesion induced by carcinogens such as vinyl chloride and urethane, is a highly efficient mutagenic lesion with a mutational specificity expected for noninstructive lesions.

Bacteriophage M13↗

PubMed1993

UV irradiation of Escherichia coli modulates mutagenesis at a site-specific ethenocytosine residue on M13 DNA. Evidence for an inducible recA-independent effect.

Mutagenic action of chemical and physical mutagens is mediated through DNA damage and subsequent misreplication at sites of unrepaired damage. Most DNA damage is noninstructive in the sense that the causative chemical modification either destroys the template information or renders it inaccessible to the DNA polymerase. Noninstructive adducts possess high genotoxicity because they stop DNA replication. Replication past noninstructive adducts is thought to depend on induced functions in addition to the regular replication machinery. In Escherichia coli, noninstructive DNA damage leads to induction of the SOS regulon, which in turn is thought to provide the inducible functions required for replicative bypass of the lesion. Because of the absence of accessible template instruction, base incorporation opposite noninstructive lesions is inherently error-prone and results in mutagenesis. Ethenocytosine (epsilon C), an exocyclic DNA lesion induced by carcinogens such as vinyl chloride and urethane, is a highly mutagenic, noninstructive lesion on the basis of its template characteristics in vivo and in vitro. However, mutagenesis at epsilon C does not require SOS functions, as evidenced by efficient mutagenesis in recA-deleted E. coli. Even though efficient mutagenesis in recA-deleted cells shows a lack of SOS dependence, the question remains whether SOS induction can modulate mutagenesis opposite epsilon C. To examine the possible contribution of SOS functions to mutagenesis at epsilon C, we constructed an M13 duplex circular DNA molecule containing an epsilon C residue at a unique site. The construct was transfected into nonirradiated or UV-irradiated E. coli.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage M13↗

PubMed1993

Mutagenic and recombinagenic effects of diethylstilbestrol quinone.

Estrogens are believed to be major contributors to many cancers of the human female genital tract, but the mechanism of their carcinogenic action is not well-understood. While a tumor-promoting role for estrogens is well-supported, whether they also act as tumor initiators has remained controversial. Here, we have sought to examine the mutagenic potential of diethylstilbestrol, a synthetic estrogen that is a powerful carcinogen in hamsters, and is suspected to be a human carcinogen. Phage M13 single-stranded DNA was treated in vitro with diethylstilbestrol quinone (DES Q: 1.25 mM) and transfected into Escherichia coli cells. DES Q treatment resulted in an apparent enhancement of mutagenesis in the LacZ(alpha) gene segment. DNA sequence analysis of LacZ(alpha) mutants obtained by transfection of DES Q-treated DNA revealed that the major effect of DES Q treatment has been a 6-fold elevation of recombination between the phage-borne LacZ(alpha) sequence and the LacZ delta M15 sequence on the E. coli fertility plasmid F. To confirm whether DES Q treatment is recombinagenic, we used an experimental system that allows the detection of recombination between a defective E. coli chromosomal LacY gene and a normal counterpart borne on a plasmid. Transfection of DES Q (0.06-12 mM) treated plasmid DNA showed significant enhancement (2-100-fold) in recombination, but not in mutagenesis. These results raise the possibility that estrogen quinones may induce recombinagenic DNA damage.

Bacteriophage M13↗

PubMed1991

Mechanisms of mutagenesis by exocyclic DNA adducts. Construction and in vitro template characteristics of an oligonucleotide bearing a single site-specific ethenocytosine.

By using a gene-targeted random DNA adduction approach, we have recently shown that chloroacetaldehyde, a metabolite of vinyl chloride, induces mutations predominantly at cytosines under conditions in which both ethenoadenine (epsilon A) and ethenocytosine (epsilon C) are formed. Although the observed mutational specificity of epsilon C suggested that it was a noninstructional lesion, the high efficiency of mutagenesis and an apparent lack of SOS dependence were reminiscent of mispairing lesions. To obtain more direct evidence showing that epsilon C has properties of a noninstructional mutagenic lesion, we have examined the in vitro template properties of a single epsilon C residue at a unique position in a synthetic oligonucleotide. The oligonucleotide was constructed by use of the following steps: (a) in vitro treatment of the pentameric oligodeoxyribonucleotide TTCTT with chloroacetaldehyde to convert the central cytosine to ethenocytosine; (b) purification and characterization of TT epsilon CTT; and (c) ligation of purified TT epsilon CTT to two decamers to create a 25 nt long oligodeoxyribonucleotide with a centrally located epsilon C residue. The template characteristics of epsilon C were examined by the annealing of end-labeled primers to the purified epsilon C-containing oligonucleotide and primer elongation by Escherichia coli DNA polymerase I in the presence of one or more nucleotide precursors. The elongation products were analyzed by high-resolution gel electrophoresis followed by autoradiography and quantitated by computing densitometry.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkylating Agents↗

PubMed1991

Mechanisms of mutagenesis by exocyclic DNA adducts. Transfection of M13 viral DNA bearing a site-specific adduct shows that ethenocytosine is a highly efficient RecA-independent mutagenic noninstructional lesion.

It is widely accepted that mutagenic DNA lesions fall into two categories: mispairing lesions hydrogen bond with an incorrect incoming base, generally do not stop replication, and possess high mutagenic efficiency without any requirement for induced functions; noninstructional lesions lack accessible template information, act as strong blocks to DNA replication (and are therefore toxic), and their mutagenic effects are SOS-dependent. Our recent results show that ethenocytosine (epsilon C), a noninstructional exocyclic DNA lesion induced by vinyl chloride, may have unusual mutagenic properties. To obtain more definitive experimental evidence for the observed effects, we have introduced a single epsilon C residue at a specific site of coliphage M13AB28 replicative form DNA by a "single-stranded linker-ligation" technique. The resulting DNA was purified and transfected into appropriate recA+ or recA- Escherichia coli host cells. The effect of epsilon C on survival was determined from transfection efficiency. Both the frequency and specificity of mutations induced by epsilon C were determined by direct sequence analysis of randomly picked progeny phage plaques. The results indicated that epsilon C has little effect on the survival of M13 DNA. Approximately 30% of the progeny phage obtained by transfecting epsilon C DNA had a base substitution mutation precisely at the lesion site. No such mutations were observed in progeny plaques obtained by transfecting the control DNA construct. All epsilon C-induced mutations were either C-to-T transitions or C-to-A transversions. Neither survival nor mutagenic efficiency was significantly affected in recA- host cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkylating Agents↗

PubMed1991

Genetic requirements for frameshift reversion induced by bulky DNA adducts in M13 DNA.

In order to analyze the genetic requirements and mechanisms of frameshift mutagenesis by activated aflatoxin B1 (AFB1), in vitro-modified phage M13 replicative form (RF) DNA was transfected into appropriate Escherichia coli cells and +1 or -1 frameshift revertants in the lacZ(alpha) gene were isolated. This analysis shows that both +1 and -1 frameshift mutagenesis by AFB1 is significantly reduced in a umuC- background. On the other hand, in the absence of RecA, +1 frameshift mutagenesis is partially reduced, but -1 frameshift mutagenesis is unaffected. DNA sequence analysis of +1 frameshifts induced by AFB1 in recA- cells suggests that the mutations occur at the same sites as in recA+ cells, but that there are significant differences in the specificity of the observed base changes. A model consistent with the observed effects in the absence of RecA suggests that an appreciable fraction of AFB1-adducted guanines can correctly template for a cytosine.

Animals↗

PubMed1991

Specificity of base substitutions induced by the acridine mutagen ICR-191: mispairing by guanine N7 adducts as a mutagenic mechanism.

As the most nucleophilic site in DNA, the guanine N7 atom is a major site of adduction by a large number of alkylating mutagens and carcinogens. Aflatoxin B1, a powerful mutagen, is believed to act through its reaction with this DNA site. On the basis of the specificity of base substitutions induced by various adduct forms of aflatoxin, we have proposed that bulky guanine N7 adducts elicit base substitutions by two mechanisms. The first mechanism is similar to that observed for a number of bulky noninstructive lesions, whereas the second mechanism invokes mispairing between N7-adducted guanine and thymine. A prediction of the mispairing hypothesis is that diverse bulky guanine N7 adducts (regardless of structural similarities with the aflatoxins) should induce predominantly G-to-A transitions. Accordingly, we have recently observed that base substitutions induced by the acridine half-mustard ICR-191 in the M13 double-stranded DNA transfection system are predominantly G:C-to-A:T transitions. Here, by transfecting ICR-191-treated M13 AB28 single-stranded DNA into Escherichia coli, we show that base substitutions are predominantly targeted to guanines. Since the N7-adducted-guanine:thymine mispairing is proposed to require N1 deprotonation promoted by the primary N7 lesion, guanine imidazole ring-opening should abolish this mispairing property, and thereby alter the specificity of mutagenesis. Here, we show that the incubation of ICR-191-treated RF DNA at pH 10.5 results in a significant reversal of the specificity of G:C-targeted substitutions such that G-to-T transversions predominated over G-to-A transitions.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminacrine↗

PubMed1990

Induction of G.C to A.T transitions by the acridine half-mustard ICR-191 supports a mispairing mechanism for mutagenesis by some bulky mutagens.

As the most nucleophilic atom in DNA, the guanine N7 atom is a major site of attack for a large number of chemical mutagens as well as chemotherapeutic agents. Paradoxically, while methylation of guanine N7 is believed to be largely nonmutagenic, aflatoxin B1, among the most potent mutagens, appears to exert its mutagenic activity through adduction at this site. On the basis of an analysis of the specificity of mutations induced by various adduct forms of aflatoxin B1, we have previously proposed mechanisms that can both resolve the paradox and account for the specificity of mutagenesis by aflatoxin B1. The hypothesized mechanisms specify how a bulky guanine N7 lesion can promote G.C to A.T transitions as well as frame-shift mutations. Since the proposed mechanisms are in principle lesion-independent, a simple test of the proposed mechanisms would be to examine the specificity of mutations induced by a structurally different bulky guanine N7 adduct. Toward this goal, M13 replicative form DNA was subjected to in vitro adduction with the acridine mutagen ICR-191 and transfected into Escherichia coli. Mutations in the LacZ(alpha) gene segments were scored and defined at the sequence level. The results show that ICR-191 adduction induces both base substitutions and frame shifts with near-equal efficiency. A clear majority of base substitutions were G.C to A.T transitions. On the other hand, unlike aflatoxin B1 which could induce both -1 and +1 frameshifts, ICR-191 appears to predominantly induce +1 frame shifts. This preference appears to arise by lesion-dependent mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminacrine↗

PubMed1990

Mechanisms of mutagenesis by the vinyl chloride metabolite chloroacetaldehyde. Effect of gene-targeted in vitro adduction of M13 DNA on DNA template activity in vivo and in vitro.

2-Chloroacetaldehyde (CAA), a metabolite of the carcinogenic industrial chemical vinyl chloride, reacts with single-stranded DNA to form the cyclic etheno lesions predominantly at adenine and cytosine. In both ethenoadenine and ethenocytosine, normal Watson-Crick hydrogen-bonding atoms are compromised. We have recently shown that CAA adduction leads to efficient mutagenesis in Escherichia coli predominantly at cytosines, and less efficiently at adenines. About 80% of the mutations at cytosines were C-to-T transitions, and the remainder were C-to-A transversions, a result similar to that of many noninstructional DNA lesions opposite which adenine residues are preferentially incorporated. It is widely believed that noninstructional lesions stop replication and depend on SOS functions for efficient mutagenesis. We have examined the effects of in vitro CAA adduction of the lacZ alpha gene of phage M13AB28 on in vivo mutagenesis in SOS-(UV)-induced E. coli. CAA adduction was specifically directed to a part of the lacZ sequence within M13 replicative form DNA by a simple experimental strategy, and the DNA was transfected into appropriate unirradiated or UV-irradiated cells. Mutant progeny were defined by DNA sequencing. In parallel in vitro experiments, the effects of CAA adduction on DNA replication by E. coli DNA polymerase I large (Klenow) fragment were examined. Our data do not suggest a strong SOS dependence for mutagenesis at cytosine lesions. While adenine lesions remain much less mutagenic than cytosine lesions, mutation frequency at adenines is increased by SOS. SOS induction does not significantly alter the specificity of base changes at cytosines or adenines.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaldehyde↗

PubMed1989

Mutagenesis by aflatoxin in M13 DNA: base-substitution mechanisms and the origin of strand bias.

The two goals of the experiments described here are: (a) to examine whether there is a strand bias in mutagenic processing of bulky lesions in M13 replicative form (RF) DNA, and (b) to examine the mutational mechanisms of metabolically activated aflatoxin. For these experiments, two types of nicked heteroduplex M13 RF DNA molecules (+WT/-am1 and +am1/-WT) in which either the minus (-) or the plus (+) strand carried a gene 1 amber nonsense codon, were constructed. Heteroduplex DNAs were modified in vitro with aflatoxin B1 activated by hamster liver S9 enzymes, and transfected into SOS(UV)-induced Escherichia coli (Supo/uvrA-/mucAB+). Forward mutations in the lacZ alpha-complementing gene segment were scored and sequenced. Results indicated that aflatoxin-induced mutation frequencies in the +WT/-am1 heteroduplex were significantly greater than those in the +am1/-WT heteroduplex, suggesting more efficient mutagenic processing of lesions in the plus strand. These results permit specific suggestions for improved mutation detection in the extensively used M13 forward mutagenesis system. Sequence analysis of point mutations from the +WT/-am1 experiments showed that most substitutions were targeted to plus-strand guanines. Both G-to-A transitions and G-to-T transversions were induced with equal efficiency. Since activated aflatoxin B1 is known to react almost exclusively with DNA guanines at the N7 position, these results suggest that bulky lesions at guanine N7 position may have the properties of mis-instructional as well as non-instructional lesions.

Aflatoxin B1↗

PubMed1989

Mechanisms of mutagenesis by chloroacetaldehyde.

A number of bifunctional chemical mutagens induce exocyclic DNA lesions. For example, 2-chloroacetaldehyde (CAA), a metabolite of vinyl chloride, readily reacts with single-stranded DNA to predominantly form etheno lesions. Here, we report on in vivo mutagenesis caused by CAA treatment of DNA in vitro. These experiments used partially duplex phage M13AB28 replicative form DNA in which a part of the lacZ gene sequence was held in single-stranded form to direct reaction with CAA. CAA-treated partial duplex DNA was transfected into Escherichia coli, and the induced base changes were defined by DNA sequencing. These experiments suggested that CAA treatment induced mutations at cytosines, much less efficiently at adenines, but not at guanines or thymines. Among mutations targeted to cytosine, 80% were C-to-T transitions and 20% were C-to-A transversions. Application of a post-labeling method detected dose-dependent formation of ethenoadenine and ethenocytosine in CAA treated DNA. These data indicate that ethenocytosine is a highly efficient mutagen with properties suggestive of a non-instructional DNA lesion in vivo. Paradoxically, ethenoadenines are efficiently bypassed by a mechanism which appears to be largely nonmutagenic.

Acetaldehyde↗

PubMed1988

Effects of SOS and MucAB functions on reactivation and mutagenesis of M13 replicative form DNA bearing bulky lesions.

We have previously determined the specificity of -1 frameshifts induced by aflatoxin-B1-2,3-dichloride (AFB1C12) in phage M13 double-strand replicative form (RF) DNA. The system consists of: (i) in vitro adduction of RF DNA of BK8, a lacZ + 1 frameshift derivative of phage M13mp8; (ii) transfection into unirradiated or UV-irradiated bacterial host cells; (iii) scoring and sequencing of revertants (i.e., -1 frameshifts). The previous data had shown that induction of SOS functions enhanced mutagenesis. However, this increase in mutagenesis is not accompanied by enhanced survival in a majority of the strains tested. Here, we present evidence to show that the lack of SOS reactivation is a specific property of the RF DNA system rather than a specific property of the lesion. A model mechanism based on the replicative strategy of transfected RF DNA can account for these observations. In addition, we have calculated individual Weigle mutagenesis factors at 8 major mutagen induced sites reported previously. Analysis of these data indicates that, within a restricted subset of possible mutational events (i.e., -1 frameshifts), Weigle mutagenesis is affected by both the DNA sequence environment of the mutation site as well as the repair phenotype of the cell.

Aflatoxin B1↗

PubMed1988

Mechanisms of mutagenesis by a bulky DNA lesion at the guanine N7 position.

In order to examine the mechanisms of mutagenesis by a bulky DNA lesion at the guanine N7 position, the replicative form DNA of phage M13AB28 (mp8 without the amber codons in phage genes) was modified in vitro with aflatoxin B1-2,3-dichloride and transfected into appropriate Escherichia coli cells. Forward mutations in the lacZ alpha-complementing gene segment were identified as light blue or colorless plaques on appropriate indicator plates, isolated, and defined by DNA sequencing. Transfection of modified DNA into uvrA-/mucAB+ cells without prior UV (SOS) induction increased mutation frequency eight-fold over untreated DNA, whereas this increase was 12-fold upon SOS induction. Transfection of modified DNA after conversion of the primary guanine-aflatoxin lesions to the stable imidazole ring-opened formamidopyrimidine-aflatoxin suggested that these lesions were nearly equally mutagenic. A majority of point mutations under all conditions affected G:C bp. Base substitutions were in the majority, but significant frameshift mutagenesis was also detected in SOS-induced cells. Both G-to-T transversions and G-to-A transitions were produced at equal efficiency and together accounted for virtually all of the base substitutions induced by the primary lesions. Point mutations occurred predominantly at predicted damage hotspots. The characteristics of base substitution and frameshift mutations, together with available information point to multiple mechanisms of mutagenesis by this class of mutagens. The data indicate that primary lesions have the properties of both a noninstructional and pseudo-instructional lesion. In addition, the sequence context appears to play a role in determining whether a frameshift or a base substitution is induced by this bulky lesion.

Aflatoxin B1↗

PubMed1987

Mechanisms of frameshift mutagenesis by aflatoxin B1-2,3-dichloride.

In order to characterize frameshift mutagenesis by aflatoxin B1-2,3-dichloride (AFB1Cl2), we have introduced a +1 (BK8) or a -1 (HS8) frameshift within the lacZ alpha gene segment contained in the phage M13mp8 to obtain lacZ alpha- derivatives. BK8 or HS8 replicative form DNA was modified with AFB1Cl2 in vitro, transfected into appropriate Escherichia coli hosts and lacZ alpha+ revertants scored and defined by DNA sequencing. The -1 frameshift (BK8) results suggest the following. (1) The E. coli recA gene is not absolutely required for AFB1Cl2-induced frameshift mutagenesis; however, in recA+ cells, ultraviolet light (SOS) induction enhances AFB1Cl2 mutagenesis, but such ultraviolet induction is not required. The plasmid pGW270 (mucAB+) significantly enhances the AFB1Cl2-induced frameshift mutagenesis. The uvrABC+ excision system plays a major role in the repair of AFB1Cl2-induced damage. (2) Sequence analysis reveals that AFB1Cl2 induces two classes of -1 frameshift mutations: the simple class in which the frameshift is due to the loss of one base-pair, and the complex class in which the loss of a base-pair is coupled to a vicinal base substitution. Both types of mutations occur predominantly at G.C runs, which are hotspots for AFB1Cl2 damage. The complex mutations appear to be concerted events targeted by a single AFB1Cl2 adduct. The frequency of these complex mutations is significantly enhanced by mucAB activity. In this system, recA activity is required for generation of significant levels of complex mutations. An analysis of the +1 frameshifts (HS8) reveals that AFB1Cl2 induces +1 frameshifts with an efficiency comparable to that for -1 frameshifts. Most +1 frameshifts occur by the addition of a base, and a third of the additions are complex mutations because they are accompanied by at least one base substitution. All simple additions occur at G.C runs; however, in a striking contrast to spontaneous insertions, a majority of the induced events introduce an A.T pair at these sites. Our data suggest a model for the generation of base substitution as well as simple and complex frameshift mutations induced by AFB1Cl2. To the extent determined, the frameshift specificity of aflatoxin B1 activated by metabolic enzymes is similar to that of AFB1Cl2.

Aflatoxin B1↗

PubMed1987

DNA replication-blocking properties of adducts formed by aflatoxin B1-2,3-dichloride and aflatoxin B1-2,3-oxide.

The carcinogen aflatoxin B1 (AFB1), upon activation to a hypothesized AFB1-2,3-oxide (AFB1-oxide), reacts with DNA guanines. Aflatoxin B1-2,3-dichloride (AFB1-Cl2) was originally synthesized as an electronic analog for the putative AFB1-oxide, which has never been isolated due to presumed reactivity. We have previously shown that AFB1-oxide reacts with base-paired DNA guanines in a sequence-specific manner, as revealed by an alkali-degradation analysis. On the basis of a replication-block analysis, we have shown that AFB1-Cl2 reacts with single-stranded DNA preferentially at inverted repeat sequences, which were suggested to be capable of forming intrastrand base-paired structures. Here, we present data to show the following. Both AFB1-oxide and AFB1-Cl2 react with guanines in double-stranded DNA to induce similar sequence-specific, alkali-labile sites. Reactivity with partial DNA duplexes as well as the use of single-strand specific chemical probes directly demonstrates that AFB1-Cl2, like AFB1-oxide, prefers base-paired guanines over non-base-paired guanines. DNA replication block patterns induced by AFB1-oxide are essentially similar to those induced by AFB1-Cl2. Unexpectedly, and unlike other tested DNA lesions, Mn2+ does not appear to affect the template blocking properties of the adduct formed by AFB1-Cl2 or AFB1-oxide. The sites for replication stoppage as well as the lack of a Mn2+ effect on adducted templates have implications for the mechanisms of mutagenesis by activated AFB1.

Aflatoxin B1↗