Biomedical subjects

R P Fuchs

Publications and source records attributed to R P Fuchs.

At least 55 records · Page 3Linked to original sources

PubMed1996

Cellular strategies for accommodating replication-hindering adducts in DNA: control by the SOS response in Escherichia coli.

The replication of double-stranded plasmids containing a single adduct was analyzed in vivo by means of a sequence heterology that marks the two DNA strands. The single adduct was located within the sequence heterology, making it possible to distinguish trans-lesion synthesis (TLS) events from damage avoidance events in which replication did not proceed through the lesion. When the SOS system of the host bacteria is not induced, the C8-guanine adduct formed by the carcinogen N-2-acetylaminofluorene (AAF) yields less than 1% of TLS events, showing that replication does not readily proceed through the lesion. In contrast, the deacetylated adduct N-(deoxyguanosin-8-yl)-2-aminofluorene yields approximately 70% of TLS events under both SOS-induced and uninduced conditions. These results for TLS in vivo are in good agreement with the observation that AAF blocks DNA replication in vitro, whereas aminofluorene does so only weakly. Induction of the SOS response causes an increase in TLS events through the AAF adduct (approximately 13%). The increase in TLS is accompanied by a proportional increase in the frequency of AAF-induced frameshift mutations. However, the polymerase frameshift error rate per TLS event was essentially constant throughout the SOS response. In an SOS-induced delta umuD/C strain, both US events and mutagenesis are totally abolished even though there is no decrease in plasmid survival. Error-free replication evidently proceeds efficiently by means of the damage avoidance pathway. We conclude that SOS mutagenesis results from increased TLS rather than from an increased frameshift error rate of the polymerase.

2-Acetylaminofluorene↗

PubMed1996

DNA damage and repair in mutagenesis and carcinogenesis: implications of structure-activity relationships for cross-species extrapolation.

Previous studies on structure-activity relationships (SARs) between types of DNA modifications and tumour incidence revealed linear positive relationships between the log TD50 estimates and s-values for a series of mostly monofunctional alkylating agents. The overall objective of this STEP project was to further elucidate the mechanistic principles underlying these correlations, because detailed knowledge on mechanisms underlying the formation of genotoxic damage is an absolute necessity for establishing guidance values for exposures to genotoxic agents. The analysis included: (1) the re-calculation and further extension of TD50 values in mmol/kg body weight for chemicals carcinogenic in rodents. This part further included the checking up data for Swain-Scott s-values and the use of the covalent binding index (CBI); (2) the elaboration of genetic toxicity including an analysis of induced mutation spectra in specific genes at the DNA level, i.e., the vermilion gene of Drosophila, a plasmid system (pX2 assay) and the HPRT gene in cultured mammalian cells (CHO-9); and (3) the measurement of specific DNA alkylation adducts in animal models (mouse, rat, hamster) and mammalian cells in culture. The analysis of mechanisms controlling the expression of mammalian DNA repair genes (alkyltransferases, glycosylases) as a function of the cell type, differentiation stage, and cellular microenvironment in mammalian cells. The 3 classes of genotoxic carcinogens selected for the project were: (1) chemicals forming monoalkyl adducts upon interaction with DNA; (2) genotoxins capable of forming DNA etheno-adducts; and (3) N-substituted aryl compounds forming covalent adducts at the C8 position of guanine in DNA. In general, clear SARs and AARs (activity-activity relationships) between physiochemical parameters (s-values, O6/N7-alkylguanine ratios, CBI), carcinogenic potency in rodents and several descriptors of genotoxic activity in germ cells (mouse, Drosophila) became apparent when the following descriptors were used: TD50 estimates (lifetime doses expressed in mg/kg b.wt. or mmol/kg b.wt.) from cancer bioassays in rodents; the degree of germ-cell specificity, i.e., the ability of a genotoxic agent to induce mutations in practically all cell stages of the male germ-cell cycle of Drosophila (this project) and the mouse (literature search), as opposed to a more specific response in postmeiotic stages of both species; the Mexr-/Mexr+ hypermutability ratio, determined in a repair assay utilizing Drosophila germ cells; mutation spectra induced at single loci (the 7 loci used in the specific-locus test of the mouse (published data), and the vermilion gene of Drosophila); and doubling doses (DD) in mg/kg (mmol/kg) for specific locus test results on mice. By and large, the TD50 values, the inverse of which can be considered as measures of carcinogenic potency, were shown to be predictable from knowledge of the in vivo doses associated with the absorbed amounts of the investigated alkylators and with the second-order constant, kc, reaction at a critical nucleophilic strength, nc. For alkylating agents kc can be expressed as the second-order rate constant for hydrolysis, kH2O, and the substrate constant s:kH2OTD50 is a function of a certain accumulated degree of alkylation, here given as the (average) daily increment, ac, for 2 years exposure of the rodents. The TD*50 in mmol/kg x day) could then be written: [formula: see text] This expression would be valid for monofunctional alkylators provided the reactive species are uncharged. This is the case for most SN2 reagents. Although it appears possible to predict carcinogenic potency from measured in vivo doses and from detailed knowledge of reaction-kinetic parameter values, it is at present not possible to quantify the uncertainty of such predictions. One main reason for this is the complication due to uneven distribution in the body, with effects on the dose in target tissues. The estimation can be impro

Alkylating Agents↗

PubMed1996

Effects of aminofluorene and acetylaminofluorene DNA adducts on transcriptional elongation by RNA polymerase II.

A prominent model for the mechanism of transcription-coupled DNA repair proposes that an arrested RNA polymerase directs the nucleotide excision repair complex to the transcription-blocking lesion. The specific role for RNA polymerase II in this mechanism can be examined by comparing the extent of polymerase arrest with the extent of transcription-coupled repair for a specific DNA lesion. Previously we reported that a cyclobutane pyrimidine dimer that is repaired preferentially in transcribed genes is a strong block to transcript elongation by RNA pol II (Donahue, B.A., Yin, S., Taylor, J.-S., Reines, D., and Hanawalt, P. C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8502-8506). Here we report the extent of RNA polymerase II arrest by the C-8 guanine DNA adduct formed by N-2-aminofluorene, a lesion that does not appear to be preferentially repaired. Templates for an in vitro transcription assay were constructed with either an N-2-aminofluorene adduct or the helix-distorting N-2-acetylaminofluorene adduct situated at a specific site downstream from the major late promoter of adenovirus. Consistent with the model for transcription-coupled repair, an aminofluorene adduct located on the transcribed strand was a weak pause site for RNA polymerase II. An acetylaminofluorene adduct located on the transcribed strand was an absolute block to transcriptional elongation. Either adduct located on the nontranscribed strand enhanced polymerase arrest at a nearby sequence-specific pause site.

2-Acetylaminofluorene↗

PubMed1996

Binding and incision activities of UvrABC excinuclease on slipped DNA intermediates that generate frameshift mutations.

Previous in vivo studies involving sequence 5'-CCCG1G2G3-3' (SmaI site) have demonstrated that adducts of N-2-acetylaminofluorene (AAF) to any of the three guanine residues of the SmaI sequence induce, with different efficiencies, two classes of -1 frameshift events, namely -G and -C mutations, referred to as targeted and semitargeted mutations, respectively. It has been proposed that both events occur during replication as a consequence of slippage events involving slipped mutagenic intermediates (SMIs). In order to evaluate the potential role of the UvrABC excinuclease in frameshift mutagenesis, we have studied the interaction of this enzyme with DNA molecules mimicking SMIs in vitro. In all of our constructions, when present, the AAF adduct was located on the third guanine residue of the SmaI site (5'-CCCG1G2G3-3'). This strand was referred to as the top strand, the complementary strand being the bottom strand. Double-stranded heteroduplexes mimicking the targeted and semitargeted SMIs contained a deletion of a C and a G within the SmaI sequence in the bottom strand and were designated deltaC/3 and deltaG/3 when modified with the AAF on the third guanine residue in the top strand or deltaC/O and deltaG/O when unmodified. The modified homoduplex was designated SmaI/3. deltaC/O and deltaG/O were weakly recognized by UvrA2B, but not incised. All three AAF-modified substrates were recognized with similar efficiency and much more efficiently than unmodified heteroduplexes. With AAF-monomodified substrates, dissociation of UvrA2 from the UvrA2B-DNA complex occurred more readily in heteroduplexes than in the homoduplex. SmaI/3 and deltaC/3 were incised with equal efficiency, while deltaG/3 was less incised. The position of the AAF lesion dictated the position of the incised phosphodiester bonds, suggesting that the presence of a bulge can modulate the yield but not the incision pattern of AAF-modified substrates. The finding that UvrABC excinuclease acts on substrates that mimic SMIs suggests that the nucleotide excision repair pathway may help in fixing frameshift mutations before the following round of replication.

2-Acetylaminofluorene↗

PubMed1996

Mutagenicity of acridines in a reversion assay based on tetracycline resistance in plasmid pBR322 in Escherichia coli.

The mutagenicity of a series of acridine compounds was studied in an assay based on the reversion of mutations in the tetracycline-resistance gene (tet) of plasmid pBR322 in Escherichia coli. Mutations that restore the tetracycline-resistant phenotype were detected in tetracycline-sensitive strains carrying mutant plasmids. Mutations that revert by +2, +1, -1 and -2 frameshift mutations and by base-pair substitutions were used to analyze the mutagenicity of two simple acridines, two acridine mustards, and a nitroacridine. The simple acridines (9-aminoacridine and quinacrine) effectively induced -1 frameshifts and weakly induced +1 frameshifts. The acridine mustards (quinacrine mustard and ICR-191) were more potent inducers of -1 and +1 frameshifts than the simple acridines. Reactive acridines, including both the mustards and the nitroacridine Entozon, were effective inducers of -2 frameshifts but the simple acridines were not. The two classes of reactive acridines differed from one another, in that the mustards were better inducers of +1 frameshifts than Entozon, whereas Entozon was a particularly potent inducer of -2 frameshifts. None of the compounds induced +2 frameshifts, and the induction of base-pair substitutions was negligible. These results confirm and extend studies showing that adduct-forming acridines are stronger frameshift mutagens than simple intercalating acridines and that the acridines differ from one another not only in overall mutagenic potency but also in the prevalence of different classes of frameshift mutations.

Acridines↗

PubMed1996

NMR data show that the carcinogen N-2-acetylaminofluorene stabilises an intermediate of -2 frameshift mutagenesis in a region of high mutation frequency.

The heteroduplex, D(ACCGGCGCCACA) . d(TGTGG-CCGGT), containing two bulged bases, a cytosine and the guanine G7, either unmodified or modified with the carcinogen N-2-acetylaminofluorene, have been studied by NMR as models of slipped-mutagenic intermediates (SMI). The melting temperature of the modified heteroduplex is strongly increased compared with that of the unmodified heteroduplex. NMR studies have shown that all the bases of the unmodified heteroduplex are stacked within the helix, without any disruption of the sequential connectivities. The two strands are in a B-like conformation. Nevertheless, exchangeable-proton studies have revealed that base pairing is very weak, or even lacking, over two base pairs apart from the bulge. Concerning the modified heteroduplex, no B-like connectivity is observed in the G5-C9 segment. Moreover, the cytosine C8 is rejected outside the helix, whereas the N-2-acetylaminofluorene moiety is inserted within the helix. The G5.C18, C6.G17 and C9.G16 bases are remarkably stable when the temperature is increased, in agreement with the high melting temperature. Some small unassigned peaks reveal the presence of the minor conformation in equilibrium. The strong stabilisation of the N-2-acetylaminofluorene-modified heteroduplex compared with the unmodified duplex is in agreement with the high N-2-acetylaminofluorene-induced mutation frequency compared with the spontaneous frequency and with the hypothesis of mutagenesis occurring during replication.

2-Acetylaminofluorene↗

PubMed1995

Sequence determinants for -2 frameshift mutagenesis at NarI-derived hot spots.

The recognition sequence of the NarI restriction enzyme is known to be a strong hot spot for -2 frameshift mutations (G1G2CG3CC-->GGCC) induced by the chemical carcinogen N-2-acetylaminofluorene (AAF). In an attempt to define a "consensus sequence" for this mutation hot spot, we have investigated the role of the bases flanking the central dinucleotide GpC repeat in the NarI sequence (NaGCGCNb) on the mutation frequency induced by the carcinogen. Construction and random modification with AAF of the 16 plasmids resulting from the replacement of Na and Nb by A,T,G and C, respectively, have been undertaken. All 16 sequences tested are found to be -2 frameshift mutation hot spots. Indeed, a level of modification of approximately five AAF adducts per plasmid molecule induces a mutation frequency ranging between 500 and 5000-fold above background. The mutations observed are mainly (90%) deletion of a dinucleotide CpG in the targeted sequence NaGCGCNb. Previous studies on NarI mutagenesis (G1G2CG3CC-->GGCC) have shown that only AAF adducts at G3 in the template for lagging strand synthesis induce -2 frameshift mutagenesis at a high level. When the mutation data obtained in this work are analyzed as originating essentially from adducts to guanines in this strand, we find that it is the nucleotide Nb located 3' to the central dinucleotide GpC repeat that strongly modulates the mutation frequency, while the nucleotide Na located on the 5' side has little effect. Our present model of frameshift mutagenesis at NarI sites involves a template-primer misalignment step with a two-nucleotide slipped mutagenic intermediate. In the context of this model, the modulation of mutagenesis by nucleotide Nb located two nucleotides 3' from the putative adduct site in the template strand can be explained in view of the fact that the replication complex encounters this nucleotide before it encounters the adduct.

2-Acetylaminofluorene↗

PubMed1995

Frequency and fidelity of translesion synthesis of site-specific N-2-acetylaminofluorene adducts during DNA replication in a human cell extract.

We have previously analyzed the effects of site-specific N-2-acetylaminofluorene (AAF) adducts on the efficiency and frameshift fidelity of SV40-based DNA replication in a human cell extract (Thomas, D. C., Veaute, X., Kunkel, T. A., and Fuchs, R. P. P. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 7752-7756). Here we use two sets of substrates to examine the probability of replication termination and error-free and error-prone bypass of AAF adducts. The substrates contained site-specific adducts at one of three guanines in a NarI sequence (5'-GGCGCC-3') placed within the lacZ alpha reporter gene and located on the template for either leading or lagging strand replication. The presence of the adduct at any position strongly reduces the efficiency of a single round of replication in a HeLa cell extract. Product analysis reveals preferential replication of the undamaged strand and termination of replication of the damaged strand occurring one nucleotide before incorporation opposite either a leading or lagging strand adduct. Products resistant to restriction endonuclease cleavage at the adducted site were generated in amounts consistent with 16-48% lesion bypass during replication. Most of this bypass was error-free. However, two-nucleotide deletion errors were detected in the replication products of DNA containing an AAF adduct in either the leading or lagging strand, but only when present at the third guanine position. Collectively, the data suggest that the replication apparatus in a HeLa cell extract generates a template-primer slippage error at an AAF adduct once for every 30-100 bypass events.

2-Acetylaminofluorene↗

PubMed1995

N-2-aminofluorene and N-2 acetylaminofluorene adducts: the local sequence context of an adduct and its chemical structure determine its replication properties.

The strong rat liver carcinogen, N-2-acetylaminofluorene, forms mainly two types of guanine adducts at the C-8 position, the acetylaminofluorene adduct (dGuo-C8-AAF) and the aminofluorene adduct (dGuo-C8-AF). We have constructed different oligonucleotides bearing a single AF lesion at each of the guanine residues of the NarI mutagenesis hot spot (G1G2CG3CC) and analysed the structural distortion induced by this DNA adduct according to the sequence context. At position G1 and G2, the deformation induced by the AF adduct is smaller than the deformation induced by the corresponding acetylated form of this adduct (i.e. the AAF adduct at the G1 and G2), whereas both AF and AAF adducts induce a similar structural change when bound to G3. Single-stranded oligonucleotides modified with AF adducts were used in primer extension replication assays using purified DNA polymerases (PolIII holoenzyme, Klenow fragment (exo+ and exo-), Sequenase 2.0) and the data compared to the AAF containing substrates. Translesion synthesis (complete bypass) is found with all tested polymerases when AF adducts are bound to G1 or G2 while little or no bypass is seen when the AF adduct is bound to G3. On the other hand, irrespective of its position within the NarI sequence, AAF adducts completely block DNA synthesis. The results described in this paper show that the sole knowledge of the chemical structure of an adduct neither determines uniquely the conformational change it induces at the DNA level nor its replication properties. Indeed, although AF adducts are in most cases non-distorting adducts and as a consequence non-replication blocking lesions (as exemplified by adducts at G1 or G2), some AF adducts (as at position G3) behave almost as AAF adducts in terms of the structural distortion induced and its replication blocking property. These findings stress the strong modulation by the local sequence context of the structural and biological consequences of a given adduct.

2-Acetylaminofluorene↗

PubMed1995

Genetic instability within monotonous runs of CpG sequences in Escherichia coli.

Genetic information can be altered by base substitutions, frameshift mutations, and addition or deletion of nucleotides. Deletions represent an important class of genetic aberration occurring at DNA sequences where it is often possible to predict the existence of intermediates of mutation. Instability within tracts of repetitive sequence have recently been associated with several genetic disorders, including the so-called triplet repeat diseases and certain forms of colorectal cancers. In Escherichia coli, (GpC)n repetitive sequences have been shown to be deletion prone, but the precise mechanism of this mutagenic pathway is still unknown. We show here that interrupting the monotony of the (GpC)n run with an ApT or a GpT dinucleotide decreases the rate of deletions within these sequences. On the other hand, introducing purine-pyrimidine alternating sequences beside the GpC insert results in an increased rate of deletion. Two pathways can be envisioned: (1) (GpC)n tracts can be seen as potential Z-forming DNA sequences, and this unusual DNA structure can be processed by an unknown cellular mechanism to give rise to the observed deletions and (2) (GpC)n monotonous runs can be considered as a succession of direct or palindromic repeats, allowing formation of DNA structures that are known to participate to frameshift mutagenesis. The results presented in this article are discussed in the light of these two alternative pathways.

Base Sequence↗

PubMed1994

Human and E.coli excinucleases are affected differently by the sequence context of acetylaminofluorene-guanine adduct.

Synthetic DNA substrates containing an acetylaminofluorene (AAF) adduct at each of the three guanine in the G1G2CG3CC sequence were constructed and tested as substrates for reconstituted E.coli (A)BC excinuclease and human excinuclease in HeLa cell-free extract (CFE). The (A)BC excinulcease repaired the three substrates with relative efficiencies of G1:G2:G3 of 100:18:66 in agreement with an earlier report [Seeberg, E., and Fuchs, R.P.P. (1990) Proc. Natl Acad. Sci. USA 87, 191-194]. The same lesions were repaired by the human excinuclease with the strikingly different efficiencies of G1:G2:G3 as 38:100:68. These results reveal that the human excinuclease is affected by the sequence context of the lesion in a different manner than its prokaryotic counterpart.

Base Sequence↗

PubMed1994

NMR evidence of the stabilisation by the carcinogen N-2-acetylaminofluorene of a frameshift mutagenesis intermediate.

Two heteroduplexes d(C1A2C3T4C5G6C7A8C9A10C11)-d (G12T13G14T15G16G17A18G19T20G21) containing a bulged guanine either unmodified or modified with the carcinogen N-2-acetylaminofluorene (AAF) have been studied by nuclear magnetic resonance (NMR) as models of slipped mutagenic intermediates (SMI). Conformational equilibria are observed in both the unmodified and the AAF-modified heteroduplexes. The major conformation of the unmodified duplex is one where the extra guanine is stacked in the helix and the major conformation of the AAF-modified heteroduplex is one where the AAF is external to the helix. Unusual sugar proton chemical shifts of C5- and G6-AAF indicate that the AAF ring is pointing out in the 5' direction. A strong increase in the modified heteroduplex melting temperature (+15 degrees C) is observed. Moreover, in contrast to the unmodified heteroduplex, which shows extensive melting in the vicinity of the bulged guanine, the base pairs around the bulge in the AAF-modified heteroduplex remain paired at temperatures up to 30 degrees C. This exceptional stability of the site around the bulged modified guanine is suggested to be responsible for the high rate of -1 mutation induced by AAF at repetitive sequences.

2-Acetylaminofluorene↗

PubMed1994

MucAB but not UmuDC proteins enhance -2 frameshift mutagenesis induced by N-2-acetylaminofluorene at alternating GC sequences.

N-2-acetylaminofluorene has been shown efficiently to induce both -1 and -2 frameshift mutations in Escherichia coli as well as in mammalian cells. In E. coli, the genetic characteristics of -1 and -2 frameshift mutations were found to be distinct. The -1 frameshift mutation pathway occurs at monotonous runs of G residues (i.e. GGG-->GG). This pathway exhibits the same genetic requirements as UV light-induced base substitution mutagenesis. Indeed, optimal mutagenesis requires the expression of both UmuDC and the activated form of RecA. The -2 frameshift mutation pathway operates at short alternating GpC sequences, such as the NarI sequence (i.e. GGCGCC-->GGCC). In contrast to the -1 frameshift mutation pathway, optimal induction does not require the UmuDC and RecA proteins. This pathway involves a LexA-repressed function tentatively called Npf (for NarI processing factor). In this paper, we show that MucAB efficiently stimulates the -2 frameshift mutation pathway. However, unlike the Npf pathway, MucAB-mediated stimulation requires expression of the RecA protein.

2-Acetylaminofluorene↗

PubMed1994

Mutational spectrum induced in Saccharomyces cerevisiae by the carcinogen N-2-acetylaminofluorene.

The spectrum of mutations induced by the carcinogen N-2-acetylaminofluorene (AAF) was analysed in Saccharomyces cerevisiae using a forward mutation assay, namely the inactivation of the URA3 gene. The URA3 gene, carried on a yeast/bacterial shuttle vector, was randomly modified in vitro using N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF) as a model reactive metabolite of the carcinogen AAF. The binding spectrum of AAF to the URA3 gene was determined and found to be essentially random, as all guanine residues reacted about equally well with N-AcO-AAF. Independent Ura- mutants were selected in vivo after transformation of the modified plasmid into a ura3 delta yeast strain. Plasmid survival decreased as a function of AAF modification, leading to one lethal hit (37% relative survival) for an average of approximately 50 AAF adducts per plasmid molecule. At this level of modification the mutation frequency was equal to approximately 70 x 10(-4), i.e. approximately 50-fold above the background mutation frequency. UV irradiation of the yeast cells did not further stimulate the mutagenic response, indicating the lack of an SOS-like mutagenic response in yeast. Sequence analysis of the URA3 mutants revealed approximately 48% frameshifts, approximately 44% base substitutions and approximately 8% complex events. While most base substitutions (74%) were found to be targeted at G residues where AAF is known to form covalent C8 adducts, frameshift mutations were observed at GC base pairs in only approximately 24% of cases.(ABSTRACT TRUNCATED AT 250 WORDS)

2-Acetylaminofluorene↗

PubMed1994

Flow linear dichroism and electron microscopic analysis of protein-DNA complexes of a mutant UvrB protein that binds to but cannot kink DNA.

(A)BC excinuclease of Escherichia coli is the enzymatic activity resulting from sequential and partially overlapping actions of UvrA, UvrB, and UvrC protein. UvrA is a molecular matchmaker which promotes the formation of a stable UvrB-damaged DNA complex in which the DNA is kinked by about 130 degrees. The UvrB-DNA complex is then recognized by UvrC and two incisions are made in the DNA by the joint actions of UvrC and UvrB. A mutant of UvrB (D478A) can be loaded onto the DNA but it does not interact with UvrC to cause a nick 3' to the lesion. Based on the lack of a DNase-I-hypersensitive site in the footprint of the mutant, it was proposed that the lack of incision was due to the inability of the mutant UvrB to kink the DNA. In the current study we have investigated the interaction of the mutant UvrB with DNA using two biophysical methods, flow linear dichroism and electron microscopy. Both methods reveal that the mutant UvrB is unable to bend DNA.

Adenosine Triphosphatases↗

PubMed1994

Mutagenic replication in human cell extracts of DNA containing site-specific N-2-acetylaminofluorene adducts.

We have analyzed the effects of site-specific N-2-acetylaminofluorene (AAF) adducts on the efficiency and frameshift fidelity of bidirectional replication of double-stranded DNA in a human cell extract. Plasmid vectors were constructed containing the simian virus 40 origin of replication and single AAF adducts at one of three guanines in the Nar I sequence GGCGCC in a lacZ reporter gene. The presence of an AAF adduct diminishes replication efficiency in HeLa cell extracts by 70-80%. Replication product analyses reveal unique termination sites with each damaged vector, suggesting that when the replication fork encounters an AAF adduct, it often stops before incorporation opposite the adduct. We also observed a higher proportion of products representing replication of the undamaged strand compared to the damaged strand. This suggests that the undamaged strand is replicated more readily, either by uncoupling the first fork to encounter the lesion or by replication using the fork arriving from the other direction. Also included among replication products are covalently closed monomer-length molecules resistant to cleavage at the AAF-modified Nar I site. This resistance is characteristic of substrates containing the AAF adduct, suggesting that translesion bypass had occurred. Transformation of Escherichia coli cells with the replicated damaged DNA yielded lacZ alpha revertant frequencies significantly above values obtained with undamaged DNA or with damaged DNA not replicated in vitro. This increase was only seen with the substrate modified at the third guanine position. Analysis of mutant DNA demonstrated the loss of a GC dinucleotide at the Nar I sequence. Generation of this position-dependent AAF-induced frameshift error in a human replication system is consistent with previous observations in E. coli suggesting that, after incorporation of dCMP opposite modified guanine in the third position, realignment of the template-primer occurs to form an intermediate with two unpaired nucleotides in the template strand.

2-Acetylaminofluorene↗

PubMed1994

DNA sequence determinants of carcinogen-induced frameshift mutagenesis.

In order to examine the mechanisms of induced frameshift mutagenesis, we constructed double-stranded DNA plasmids which contain single N-2-acetylaminofluorene (AAF) adducts at specified positions within a run of contiguous guanine residues. The length of the homopolymeric run and the nature of the bases flanking the contiguous sequence were systematically varied. Monomodified plasmids were introduced into SOS-induced Escherichia coli, and -1 frameshift mutations were scored by means of a phenotypic assay. A strong positional effect of the DNA adduct within the contiguous sequence was observed irregardless of the nature of the flanking bases: the AAF-induced mutation frequency was 20-200-fold higher at the 3'-end of the contiguous sequence than at the 5'-end. In addition, for a given number of guanine residues flanking the GAAF adduct on its 5'-side, differences (up to 5-fold) in the induced mutation frequency were observed as a function of the base 3' to the adduct (CGGGAAFAT approximately CGGGAAFGT > CGGGAAFCT > CGGGAAFTT). These results are discussed, within the frame of an incorporation slippage model, in terms of differences in stability and occurrence of the slipped mutagenic intermediates.

2-Acetylaminofluorene↗

PubMed1994

Effect of single DNA lesions on in vitro replication with DNA polymerase III holoenzyme. Comparison with other polymerases.

In the present work, we have studied in vitro replication of N-2-acetylaminofluorene (AAF) or cis-diamminedichloroplatinum II (cis-DDP) single modified DNA templates. We used the holoenzyme (pol III HE) or the alpha subunit of DNA polymerase III, which is involved in SOS mutagenesis, and other DNA polymerases in order to compare enzymes having different biological roles and properties. Single-stranded oligonucleotides (63-mer) bearing a single AAF adduct at one of the different guanine residues of the NarI sequence (-G1G2CG3CC-) have been used in primer extension assays. Site-specifically platinated 5'd(ApG) or 5'd(GpG) oligonucleotides were constructed and similarly used in primer extension assays. In all cases, irrespective of both the chemical nature of the lesion (i.e. AAF or cis-DDP) and its local sequence context (i.e. the 3 different sites for AAF adducts within the NarI site) replication by pol III HE and pol I Klenow fragment (pol I Kf) stops one base prior to the adduct site. Removal of the 3'-->5' proofreading activity alone was not sufficient to trigger bypass of DNA lesions. Indeed, when proofreading activity of pol I is inactivated by a point mutation (pol I Kf (exo-)), the major replication product corresponds to the position opposite the adduct site showing that incorporation across from the AAF adduct is possible. These results suggest that a polymerase with proofreading activity is actually found to stop one nucleotide before the adduct not because it is unable to insert a nucleotide opposite the adduct but most likely because elongation past the adduct is strongly impaired, giving thus an increased time frame for the proofreading exonuclease to remove the base inserted across from the adduct. These results are discussed in terms of their implications for error-free and error-prone bypass in vivo.

2-Acetylaminofluorene↗