Biomedical subjects

R P Fuchs

Publications and source records attributed to R P Fuchs.

At least 73 records · Page 4Linked to original sources

PubMed1994

Use of single-turnover kinetics to study bulky adduct bypass by T7 DNA polymerase.

The mechanism by which T7 DNA polymerase (exo-) bypasses N-2-acetylaminofluorene (AAF) and N-2-aminofluorene (AF) adducts was studied by single-turnover kinetics. These adducts are known to be mutagenic in several cell types, and their bypass was studied in the framework of understanding how they promote mutations. Synthetic primer/templates were made from a template sequence containing a single guanine, to which the adducts were covalently attached, and one of three primers whose 3' ends were various distances from the adduct in the annealed substrates. Upon approaching the site of either adduct, the polymerase was found to add nucleotides as rapidly as to unmodified primer/templates, until just opposite the lesion. The incorporation rate of dCTP (at 100 microM) opposite AF-dG or AAF-dG was approximately 5 x 10(4)- and 4 x 10(6)-fold slower, respectively, than incorporation at the same position into an unmodified primer/template. The polymerase dissociated from the sites of the adducts at approximately the same rate that it dissociated from unmodified DNA. Correct nucleotide incorporation was favored both opposite and immediately after AF-dG. However, at both positions, dATP was the most rapidly misincorporated nucleotide. Misincorporation of dATP was more rapid than correct nucleotide incorporation both opposite and immediately after AAF-dG. These results are discussed in terms of the effects of AF and AAF adducts in vivo.

2-Acetylaminofluorene↗

PubMed1994

Endonuclease VII of phage T4 nicks N-2-acetylaminofluorene-induced DNA structures in vitro.

We have tested in vitro the activity of T4 endonuclease VII on three different double-stranded oligonucleotides bearing a single N-2-acetylaminofluorene (AAF) adduct covalently bound to each of the three guanine residues located within the NarI site (G1G2CG3CC), a strong frameshift mutation hot spot in E. coli. With the oligonucleotides modified at G2 and G3 a specific cleavage pattern with T4 endonuclease VII was observed in the complementary strand while no cleavage was found in the adduct-bearing strand. On the other hand, when G1 was modified, only a very faint cleavage band was observed (< 1%). These differences in nicking among the three AAF-modified DNA substrates are discussed in terms of the polymorphic nature in adduct-induced DNA structures as previously shown. This "non-physiological" activity of a DNA resolvase is discussed in terms of a potential role for such enzymes in the induction of frameshift mutations.

2-Acetylaminofluorene↗

PubMed1993

Greater susceptibility to mutations in lagging strand of DNA replication in Escherichia coli than in leading strand.

Models of DNA replication in Escherichia coli involve an asymmetric DNA polymerase complex that replicates concurrently the leading and the lagging strands of double-stranded DNA. The effect of asymmetry on mutagenesis was tested with pairs of plasmids containing the unidirectional ColE1 origin of replication and a single lesion located in the leading or lagging strand. The lesion used was the covalent adduct that the chemical carcinogen N-2-acetylaminofluorene (AAF) forms with the C-8 position of guanine. Whether SOS was induced or not, mutations arose at about a 20-fold higher frequency when the AAF adduct was located in the lagging strand than when in the leading strand.

2-Acetylaminofluorene↗

PubMed1993

DNA adduct-induced stabilization of slipped frameshift intermediates within repetitive sequences: implications for mutagenesis.

Chemical carcinogens such as the aromatic amide 2-acetylaminofluorene (AAF) are known to induce -1 frameshift mutation hotspots at repetitive sequences. This mutagenesis pathway was suggested to involve slipped intermediates formed during replication. To investigate the stability and structure of such intermediates we have constructed DNA duplexes containing single AAF adducts within a run of three guanine residues. The strand complementary to that bearing the AAF adducts contained either the wild-type sequence (homoduplexes) or lacked one cytosine directly opposite the run of guanines containing the AAF adduct and thus modeled the putative slipped mutagenic intermediates (SMIs). The melting temperature of AAF-modified homoduplexes or the unmodified SMI was reduced by approximately 10 degrees C relative to the unmodified homoduplex. Surprisingly, AAF adducts stabilized the SMIs as evidenced by an increase in melting temperature to a level approaching that of the unmodified homoduplex. The chemical probes hydroxylamine and bromoacetaldehyde were strongly reactive toward cytosine residues opposite the adduct in AAF-modified homoduplexes, indicating adduct-induced denaturation. In contrast, no cytosine reactivities were observed in the AAF-modified SMIs, suggesting that the two cytosines were paired with unmodified guanines. Use of diethyl pyrocarbonate to probe the guanine residues showed that all three guanines in the unmodified SMI adopted a transient single-stranded state which was delocalized along the repetitive sequence. However, when an AAF adduct was present, reduced diethyl pyrocarbonate reactivity at guanines adjacent to the adduct in AAF-modified SMIs reflected localization of the bulge to the adducted base. Our results suggest that AAF exerts a local denaturing and destabilizing effect within the homoduplex which is alleviated by the formation of a bulge. The stabilization by the AAF adduct of the SMIs may contribute to the dramatic increase in -1 frameshift mutation frequency induced by AAF adducts in repetitive sequences.

2-Acetylaminofluorene↗

PubMed1992

Structure of UvrABC excinuclease-UV-damaged DNA complexes studied by flow linear dichroism. DNA curved by UvrB and UvrC.

The interaction between UvrABC excinuclease from Escherichia coli and ultraviolet light-(UV) damaged DNA was studied by flow linear dichroism. The dichroism signal from DNA was drastically decreased in intensity upon incubation with UvrA and UvrB or whole enzyme in the presence of effector ATP. The change was specific for UV-damaged DNA, and a concluded suppressed DNA orientation suggests the wrapping of DNA around the protein. The incubation with the UvrC subunit alone also somewhat reduces the signal, however, in this case the change was smaller and not specific for UV-damaged DNA. The structural modification of DNA, promoted by the (UvrA2-UvrB) complex, probably facilitates or stabilizes the interaction of the UvrC subunit with DNA for the excision.

Adenosine Triphosphatases↗

PubMed1992

Carcinogen-induced frameshift mutagenesis in repetitive sequences.

We have constructed plasmids pS3G-1 and pSG4 that contain single acetylaminofluorene adducts within contiguous runs of three (5'-CCCG1G2G3-3') and four (5'-CG1GGG4T-3') guanine residues, respectively. In Escherichia coli, the frequency of induced -1 frameshift mutations was strongly dependent on the position of modification: pS3G-G3 was approximately 100-fold and 10-fold more mutagenic than pS3G-G1 and pS3G-G2, respectively; pSG4-G4 was approximately 600-fold more mutagenic than pSG4-G1. Mutagenesis was SOS-dependent and was markedly reduced in bacteria that were proficient in nucleotide excision repair as compared to a repair-deficient uvrA6 mutant. DNA sequencing showed that -1 frameshift events in pS3G-1 consisted of either targeted mutations (greater than 90% of induced mutations) within the guanine sequence or semitargeted mutations (greater than 10%) in the 5' flanking repetitive cytosine sequence. Semitargeted events, which were observed when acetylaminofluorene modification was at G1 and G2, show that a lesion can reduce the fidelity of replication at positions 5' to its location on the template strand. No semitargeted frameshifts were observed in plasmid pSG4, which lacks a repetitive sequence 5' to the adduct. Our results are consistent with a model for frameshift mutagenesis in which the acetylaminofluorene adduct (i) allows accurate incorporation of cytosine opposite the bulky lesion during DNA synthesis and (ii) impedes elongation of primer/template termini formed opposite the adduct or 5' to the adduct on the template strand, providing increased opportunity for the formation of slipped frameshift intermediates.

2-Acetylaminofluorene↗

PubMed1992

A umuDC-independent SOS pathway for frameshift mutagenesis.

The chemical carcinogen N-acetoxy-N-2-acetylaminofluorene induces mainly frameshift mutations, which occur within two types of sequences (mutation hot spots): -1 frameshift mutations within contiguous guanine sequences and -2 frameshift mutations within alternating GC sequences such as the NarI and BssHII restriction site sequences. We have investigated the genetic control of mutagenesis at these sequences by means of a reversion assay using plasmids pW17 and pX2, which contain specific targets for contiguous guanine and alternating GC sequences, respectively. Our results suggest that mutations at these hot spot sequences are generated by two different genetic pathways, both involving induction of SOS functions. The two pathways differ both in their LexA-controlled gene and RecA protein requirements. In the mutation pathway that acts at contiguous guanine sequences, the RecA protein participates together with the umuDC gene products. In contrast, RecA is not essential for mutagenesis at alternating GC sequences, except to cleave the LexA repressor. The LexA-regulated gene product(s), which participate in this latter mutational pathway, do not involve umuDC but another as yet uncharacterized inducible function. We also show that wild-type RecA and RecA430 proteins exert an antagonistic effect on mutagenesis at alternating GC sequences, which is not observed either in the presence of activated RecA (RecA*), RecA730 or RecA495 proteins, or in the complete absence of RecA as in recA99. It is concluded that the -1 mutation pathway presents the same genetic requirements as the pathway for UV light mutagenesis, while the -2 mutation pathway defines a distinct SOS pathway for frameshift mutagenesis.

Acetoxyacetylaminofluorene↗

PubMed1992

Workshop on DNA repair.

A workshop on DNA repair with emphasis on eukaryotic systems was held, under the auspices of the EC Concerted Action on DNA Repair and Cancer, at Noordwijkerhout (The Netherlands) 14-19 April 1991. The local organization of the meeting was done under the auspices of the Medical Genetic Centre South-West, The Netherlands (MGC), c/o Department of Radiation Genetics and Chemical Mutagenesis, University of Leiden (The Netherlands). Local organizers were: D. Bootsma (chairman), W. Ferro, J.H.J. Hoeijmakers, A.R. Lehmann, P.H.M. Lohman, L. Mullenders, and A.A. van Zeeland (secretarial assistance: Mrs. C. Escher-van Heerden and Mrs. R. Bontre). Over 190 scientists participated, and the format of the meeting followed that of the 1987 workshop on the 'Molecular Aspects of DNA Repair' (Friedberg et al., 1987). Plenary review talks in the mornings were followed, in the afternoon, by poster viewing in three or four parallel sessions. Groups of 15-20 posters were discussed in detail, and later on, in plenary sessions, chairpersons of the poster discussions reviewed the afternoons' posters. The principal themes of the meeting were the isolation and characterisation of repair genes and proteins, repair in specific sequences, consequences of defective DNA repair, and new methods for detecting DNA damage and repair. Remarkable progress has been made recently in all of these areas, and many exciting new results were presented. It is impossible to summarize all contributions to this (intensive) one-week meeting. Therefore, and for the sake of coherence, presentations that did not fit easily into any of the general themes of the meetings have not been included.

Animals↗

PubMed1992

Mutation spectra of the two guanine adducts of the carcinogen 4-nitroquinoline 1-oxide in Escherichia coli. Influence of neighbouring base sequence on mutagenesis.

When the chemical carcinogen 4-nitroquinoline 1-oxide binds to DNA in vitro, two major adducts are formed, both at guanine residues, but at different positions, i.e. the C8 or the N2 position. Well-defined adducts (either C8 or N2 guanine adducts) can be formed in vitro by reacting DNA with 4-actoxyaminoquinoline 1-oxide (Ac-4HAQO) under different reaction conditions. Forward mutations induced by each of both main 4NQO adducts in the tetracycline resistance gene of pBR322 were determined. In total, 30 independent 4NQO-induced mutations were characterized, showing mainly base-pair substitution mutations and some frameshift mutations. We have observed that the 5' neighbouring base influences the specificity of dGuo-N2-AQO induced base-pair substitutions mutagenesis; a similar effect does not occur with dGuo-C8-AQO. This study reveals the importance of the N2 guanine adduct in the mutagenesis induced by 4NQO in vivo.

4-Nitroquinoline-1-oxide↗

PubMed1991

Polymorphism in N-2-acetylaminofluorene induced DNA structure as revealed by DNase I footprinting.

In this paper, we have constructed double stranded helices (60-mers) containing a single N-2-acetylaminofluorene (-AAF) adduct covalently bound to one of the three guanine residues of the Narl site (G1G2CG3CC). This sequence was identified as a strong frameshift mutation hot spot for many carcinogens that bind to the C8 position of guanine. Using DNase I as a probe for DNA conformation we show i) that the average size of the helix deformation extends over 3 to 5 base pairs in both directions from the adduct site, and ii) that there is a strong polymorphism in the adduct induced DNA conformation. The present study supports the idea that adducts induce specific sequence dependent local conformational changes in DNA that are differentially recognized and processed by the enzymatic machineries that lead to repair or mutagenesis.

2-Acetylaminofluorene↗

PubMed1991

Strong sequence-dependent polymorphism in adduct-induced DNA structure: analysis of single N-2-acetylaminofluorene residues bound within the NarI mutation hot spot.

We have used a set of chemical probes to characterize and to compare the structural deformation of double-stranded oligomers bearing a single N-2-acetylaminofluorene (AAF) adduct covalently bound to each of the three guanine residues located within the frameshift mutation hot spot sequence -G1G2CG3CC-(NarI site). Two classes of chemical probes have been used, probes that sense the geometry of the helix, giving rise to cuts at every nucleotide (for example, 1,10-phenanthroline-copper), and probes that react with specific bases depending on their conformation (e.g., diethyl pyrocarbonate). For all probes that were tested, a distinct pattern of reactivity was observed according to the position of the adduct within the DNA sequence, revealing an important polymorphism in the adduct-induced DNA structure. With 1,10-phenanthroline-copper at least three base pairs 3' of the AAF-modified guanine were reactive on each strand, showing that the deformation of the DNA helix extends over a region of 4-6 bases pairs centered around the adduct and sensed by the probe in both strands. With the base-specific probes, reactivities were limited to the base complementary to the modified guanine and to adjacent bases. Within this sequence context, the three possible AAF adducts have previously been shown to exhibit strong differences in biological responses such as excision repair [Seeberg, E., & Fuchs, R. P. P. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 191-194] and mutagenesis [Burnouf, D., Koehl, P., & Fuchs, R. P. P. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4147-4151].(ABSTRACT TRUNCATED AT 250 WORDS)

2-Acetylaminofluorene↗

PubMed1991

Roles of recA mutant allele (recA495) in frameshift mutagenesis.

The chemical carcinogen N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF) induces frameshift mutations located within two types of specific sequences (mutation hot spots): i) contiguous guanine sequences and ii) alternating GC sequences. The genetic requirements of these frameshift events were investigated using specific reversion assays. AAF-induced -2 frameshift mutagenesis at alternating GC sequences is peculiar in that it requires a LexA- controlled function which is not UmuDC and occurs in the absence of RecA protein, provided the SOS regulon is derepressed. Moreover, the non-activated form of the RecA protein was shown to act as an inhibitor in this mutation pathway. As we were interested in elucidating this mutation pathway, we have developed a convenient spot reversion assay specific for the detection of this class of mutations. This assay allowed us to isolate E coli mutants affected either in repair or mutagenesis functions. One particular mutant, recA495, is very sensitive to UV and N-AcO-AAF, and is defective in recombination and UV mutagenesis. The RecA495 protein exhibits very low binding to both single- and double-stranded DNA. We show that when the SOS regulon is derepressed, the recA495 allele has two contrasting roles in frameshift mutagenesis: i) it prevents the induction of -1 frameshift mutations at repetitive sequences and ii) it is permissive for the induction of -2 frameshift mutations within alternating GC sequences.

Alleles↗

PubMed1990

DNA sequence analysis of mutations induced by N-2-acetylamino-7-iodofluorene in plasmid pBR322 in Escherichia coli.

The spectrum of mutations induced by N-2-acetylamino-7-iodofluorene (AAIF) was analyzed in a forward mutation system based on mutagenesis directed to a small restriction fragment in the tetracycline resistance gene of plasmid pBR322. AAIF was found to induce frameshift mutations and base-pair substitutions at approximately equal frequencies. The frameshift mutations were mostly deletions of single base-pairs, but -2 frameshifts and +1 frameshifts were also detected. With one exception, the substitutions were transversions initiated at a G.C base-pair. Both frameshift mutations and transversions occurred preferentially at sites of repetitive guanine residues. Although AAIF and the related aromatic amines N-2-acetylaminofluorene (AAF) and N-2-aminofluorene (AF) all bind to the C-8 position of guanine, they have different effects on DNA conformation, and these differences are reflected in their mutation spectra. Previous studies have provided evidence that AAF adducts can trigger a B to Z conformational change in alternating GC sequences or displacement of the guanine by the fluorene ring in other sequences; the principal result is two classes of frameshift mutations. AF, whose DNA interaction involves outside binding rather than insertion and denaturation, primarily induces base-pair substitutions. AAIF adducts are chemically similar to AAF adducts, but the iodo group apparently hinders insertion of the fluorene ring into DNA. Consistent with this model, the mutation spectrum of AAIF combines properties of the mutation spectra of both AAF and AF.

2-Acetylaminofluorene↗

PubMed1990

Kinetic studies of the modulation of ada promoter activity by upstream elements.

We have determined the kinetics of initiation of transcription of the wild-type ada promoter by abortive initiation assays. In the absence of activation, it is a weak promoter, with an association constant KB and an isomerization rate constant k2 comparable to those obtained under the same conditions for other positively regulated promoters (0.36 x 10(7) M-1 and 1.7 x 10(-2) s-1, respectively, at 37 degrees C and 50 mM NaCl, on a supercoiled template). As already observed for other promoters, these constants are modulated by varying the supercoiling of the plasmid. However, the strength of the promoter (given by the KB.k2 product) remains almost constant, because the maximum value of KB and k2 are obtained for different values of the superhelical density. The ada promoter has a stretch of seven adenosine residues (A7) in its upstream region. We have analysed the effect of this upstream sequence on the efficiency of initiation of the ada promoter by comparing the wild-type sequence with an up-mutant promoter characterized by the inversion of the central base pair in the sequence (A7) leading to the sequence (A3TA3). Although the mutation, which is located outside the promoter consensus regions, has no effect on the isomerization step, it affects the equilibrium constant KB that characterizes the association step. In the mutant promoters, the supercoiling of the plasmid modulates the isomerization and association constants in such a way that both KB and k2 are maximum for the same superhelical density (-0.05), leading to a 12-fold increase of the strength of the promoter, on a supercoiled template.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1990

Position of a single acetylaminofluorene adduct within a mutational hot spot is critical for the related mutagenic event.

2-Acetylaminofluorene, a potent rat liver carcinogen, which binds primarily to C8 of guanines, has been shown to induce mainly frameshift mutations in the bacteria Escherichia coli. Mutations occur at specific sequences, known as mutation hot spots, of which two types may be considered. First, repetitive sequences, where deletions of a single unit occur (GGGGG----GGGG). Second, the so-called NarI site, 5'GGCGCC3', where only -2-bp deletions are observed (G1G2CG3CC----GGCC). Mutagenesis within repetitive sequences is dependent on the UmuCD+ gene functions, whereas mutagenesis in the NarI site is not. These differences in the genetic requirements of mutagenesis at these hot spots suggest that two different pathways operate. In order to precisely determine the actual involvement of each of the three premutagenic lesions that may form in the NarI site in the course of the mutational process, we designed a single adduct mutagenesis experiment, and found that AAF binding to the G3 induced only a -2 frameshift mutation event. This result will be discussed in terms of local DNA conformation.

2-Acetylaminofluorene↗

PubMed1990

Induction of -2 frameshift mutations within alternating GC sequences by carcinogens that bind to the C8 position of guanine residues: development of a specific mutation assay.

Using a forward mutation assay we have previously found that N-2-acetylaminofluorene (AAF), a strong chemical carcinogen, induces a majority of frameshift mutations located at specific sequences called mutation hot spots. Among these hot spot sequences, the NarI sequence (GGCGCC), is specific for -2 frameshifts (GGCGCC)----GGCC). Interestingly, these frameshift mutations occur independently of a functional umuDC locus. Being interested in elucidating this mutation pathway we have developed a reversion assay that is specific for this class of mutations. The assay is based on the reversion of a +2 frameshift mutant of plasmid pBR322 from tetracycline sensitivity to tetracycline resistance. It is shown that only "true" reversion events lead to tetracycline resistance. The carcinogen AAF induces this reversion event at a frequency that is increased four- to fivefold over the background frequency. A series of chemical carcinogens which, like AAF, bind covalently to the C8 position of guanine, are compared for their efficiency to induce this specific mutation event. Large variations in the mutagenic efficiency of these chemicals are observed and discussed in terms of the anti/syn conformation of the carcinogen-modified guanine residue. Based on this test, we describe a convenient spot assay that this presently used in our laboratory to isolate Escherichia coli mutants affected in this mutation pathway.

2-Acetylaminofluorene↗

PubMed1990

Acetylaminofluorene bound to different guanines of the sequence -GGCGCC- is excised with different efficiencies by the UvrABC excision nuclease in a pattern not correlated to the potency of mutation induction.

DNA fragments were constructed in which acetylaminofluorene adducts were introduced specifically at each one of the three different guanines of the 6-base-pair sequence -GGCGCC-. Incubation of the DNA with the UvrABC excision nuclease in vitro revealed major differences in the efficiency of adduct excision depending on the site of modification. Oligonucleotide excision of adducts bound to the second guanine was only 15% as efficient as excision of adducts at the first guanine, whereas the excision efficiency for adducts bound to the third guanine was intermediary. However, recognition of DNA damage appeared to occur with nearly 100% efficiency at all three adduct positions, as judged from DNase I footprint analysis of the DNA/protein binding complexes. Hence, it appears that the structural elements for DNA damage recognition by the UvrABC enzyme are different from the signals for excision. Furthermore, the repair pattern observed is not inversely correlated with the potential of these adducts to induce mutations since mutation analysis of single-adduct DNA has shown that only adducts at the third guanine are strongly premutagenic. We conclude that the effectiveness of excision repair depends on the context of the DNA sequence and that ineffectively repaired adduct sites are not necessarily mutational hot spots.

2-Acetylaminofluorene↗

PubMed1990

Single d(ApG)/cis-diamminedichloroplatinum(II) adduct-induced mutagenesis in Escherichia coli.

The mutation spectrum induced by the widely used antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP) showed that cisDDP[d(ApG)] adducts, although they account for only 25% of the lesions formed, are approximately 5 times more mutagenic than the major GG adduct. We report the construction of vectors bearing a single cisDDP[d(ApG)] lesion and their use in mutagenesis experiments in Escherichia coli. The mutagenic processing of the lesion is found to depend strictly on induction of the SOS system of the bacterial host cells. In SOS-induced cells, mutation frequencies of 1-2% were detected. All these mutations are targeted to the 5' base of the adduct. Single A----T transversions are mainly observed (80%), whereas A----G transitions account for 10% of the total mutations. Tandem base-pair substitutions involving the adenine residue and the thymine residue immediately 5' to the adduct occur at a comparable frequency (10%). No selective loss of the strand bearing the platinum adduct was seen, suggesting that, in vivo, cisDDP[d(ApG)] adducts are not blocking lesions. The high mutation specificity of cisDDP[d(ApG)]-induced mutagenesis is discussed in relation to structural data.

Base Composition↗