N-Acetoxy-N-acetyl-2-aminofluorene-induced mutagenesis in the lacI gene of Escherichia coli

Schaaper, Roel M.; Koffel-Schwartz, Nicole; Fuchs, Robert P. P.

doi:10.1093/carcin/11.7.1087

Cited by 68 publications

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“…Note that the fully extended product on the AP template (lane 23) was one nucleotide shorter than the 34AP template (34-mer). Probably, a single nucleotide deletion occurred at the AP site by the misinsertion strand slippage mechanism (31)(32)(33)(34)(35). In the present case, incorporation of dAMP opposite the AP site (see below) was followed by misalignment of the primer terminus to 5Ј-T in the template (3Ј-TXT-5Ј, where X is the AP site) and extension of the misaligned terminus.…”

Section: Resultsmentioning

confidence: 76%

Effects of a Guanine-derived Formamidopyrimidine Lesion on DNA Replication

Asagoshi

Terato

Ohyama

et al. 2002

Journal of Biological Chemistry

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2,6-Diamino-4-hydroxy-5-formamidopyrimidine derived from guanine (FapyG) is a major DNA lesion formed by reactive oxygen species. In this study, a defined oligonucleotide template containing a 5-N-methylated analog of FapyG (mFapyG) was prepared, and its effect on DNA replication was quantitatively assessed in vitro. The results were further compared with those obtained for 7,8-dihydro-8-oxoguanine and an apurinic/ apyrimidinic site embedded in the same sequence context. mFapyG constituted a fairly strong but not absolute block to DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment with and without an associated 3-5 exonuclease activity, thereby permitting translesion synthesis with a limited efficiency. The efficiency of translesion synthesis was G > 7,8-dihydro-8-oxoguanine > mFapyG > apurinic/apyrimidinic site. Analysis of the nucleotide insertion (f ins ‫؍‬ V max /K m for insertion) and extension (f ext ‫؍‬ V max /K m for extension) efficiencies for mFapyG revealed that the extension step constituted a major kinetic barrier to DNA synthesis. When mFapyG was bypassed, dCMP, a cognate nucleotide, was preferentially inserted opposite the lesion (dCMP (relative f ins ‫؍‬ 1) > > dTMP (2.4 ؋ 10 ؊4 ) Ϸ dAMP (8.1 ؋ 10 ؊5 ) > dGMP (4.5 ؋ 10 ؊7 )), and the primer terminus containing a mFapyG:C pair was most efficiently extended (mFapyG:C (relative f ext ‫؍‬ 1) > mFapyG:T (4.6 ؋ 10 ؊3 ) > > mFapyG:A and mFapyG:G (extension not observed)). Thus, mFapyG is a potentially lethal but not premutagenic lesion.Reactive oxygen species formed by aerobic metabolism, phagocytic blood cells upon inflammation, ionizing radiation, and photosensitized reactions generate structurally diverse oxidative damage to DNA that stores vital genetic information of cells (1-3). Base lesions thus formed are generally restored by the base excision repair pathway involving multiple enzymes such as N-glycosylase/AP 1 lyase, AP endonuclease, DNA polymerase, and DNA ligase (4, 5). However, if left unrepaired, they result in mutations and/or cell death. It has also been implied that oxidative DNA damage is involved in carcinogenesis and various degenerative diseases (6, 7). 7,8-Dihydro-8-oxoguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine derived from guanine (FapyG) have been identified as major products in the reaction of DNA with reactive oxygen species (Fig. 1A) (1, 2). When formed in DNA, 8-oxoG in a template directs incorporation of non-cognate dAMP as well as cognate dCMP during translesion synthesis by DNA polymerases, thereby inducing GC-to-TA transversions (8, 9). Similarly, when formed in a cellular nucleotide pool, a 2Ј-deoxyribonucleotide form of 8-oxoG can be incorporated opposite adenine as well as cytosine in a DNA template, inducing AT-to-CG transversions (10 -12). Mispair formation between 8-oxoG and adenine leading to mutation involves an unusual syn-conformer of 8-oxoG that can form two hydrogen bonds between O-6 and N-7-H in 8-oxoG and N-6 -H and N-1 in adenine without introducing significan...

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Section: Resultsmentioning

confidence: 76%

Effects of a Guanine-derived Formamidopyrimidine Lesion on DNA Replication

Asagoshi

Terato

Ohyama

et al. 2002

Journal of Biological Chemistry

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“…This is consistent with a model wherein the replication apparatus first incorporates cytosine opposite the modified guanine (Lambert et al, 1992). The template-primer then slips such that the two terminal bases in the primer (3Ј-CpG-5Ј) hydrogen-bond with a repeated downstream complementary 5Ј-GpC-3Ј dinucleotide in the template, thus forming a misaligned intermediate (Schaaper et al, 1990). Continued synthesis from this intermediate leads to a two-base deletion error.…”

Section: Discussionmentioning

confidence: 99%

“…Studies in Escherichia coli have revealed that AAF, which binds primarily at the C-8 position of guanine, is a strong mutagen for Ϫ1 and Ϫ2 frameshift mutations (Fuchs et al, 1981;Koffel-Schwartz et al, 1984;Schaaper et al, 1990), particularly within repetitive sequences that permit slippage to form misaligned intermediates (Burnouf et al, 1989;Lambert et al, 1992). Mutagenesis within these sequences is strongly dependent on adduct location.…”

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Frequency and Fidelity of Translesion Synthesis of Site-specific N-2-Acetylaminofluorene Adducts during DNA Replication in a Human Cell Extract

Thomas

Veaute

Fuchs

et al. 1995

Journal of Biological Chemistry

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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␣ 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.DNA replication in eukaryotic cells requires multiple proteins that function in an asymmetric manner to coordinately synthesize the leading and lagging strands (Kornberg and Baker, 1992). Given the asymmetry of replication of duplex DNA, unrepaired DNA adducts may have different potentials for blocking replication or promoting replication infidelity depending on whether the adduct is located on the leading or lagging strand. To examine this, we have been using the SV40 origin-dependent replication system (for review, see Stillman (1994), and references therein) as a model for human chromosomal replication. With this system, replication of undamaged DNA in mammalian cell extracts is highly accurate (Roberts and Kunkel, 1988;Hauser et al., 1988;Thomas et al., 1991). However, replication of DNA containing randomly placed cyclobutane pyrimidine dimers (CPDs) 1 is highly mutagenic Carty et al., 1993). The assay system was adapted to determine whether, as a result of the asymmetry of replication, CPDinduced errors arise at different rates during leading and lagging strand synthesis. Overall average error rates were equal for the leading and lagging strand replication machinery, with strand-specific differences in fidelity observed at some nucleotide positions .For several years, we have studied the effects of an encounter between the replication fork and the major adduct of a known carcinogen, N-2-acetylaminofluorene (AAF) located at ...

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“…Frameshift mutations are efficiently induced by a large variety of chemical carcinogens that present an aromatic moiety. Most frameshift mutations occur within short mononucleotide or dinucleotide repeats and can be explained by slippage events during TLS (14,(17)(18)(19). Indeed, adducts formed by aromatic amines strongly stabilize misaligned primer͞template replication intermediates [slipped mutagenic in-termediates (SMI), (20)(21)(22)].…”

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confidence: 99%

SOS factors involved in translesion synthesis

Napolitano

Lambert

Fuchs

1997

Proc. Natl. Acad. Sci. U.S.A.

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Mutations are permanent DNA sequence changes that can be induced when replication occurs on a damaged DNA template. In Escherichia coli, the process of translesion synthesis past a lesion that hinders replication requires the induction of SOS-controlled gene products, among which are those of the umuDC operon. To study translesion synthesis in vivo, we have constructed singlestranded vectors containing single 2-acetylaminof luorene adducts located within ؊1 and ؊2 frameshift mutation hot spots formed by short repetitive sequences. These adducts strongly hinder DNA replication as only 2-5% of the molecules give rise to progeny under non-SOS-induced conditions. Induction of the SOS response lead to a 10-fold increase in survival. Adducts present within repetitive sequences trigger the formation of misaligned primer͞template replication intermediates which, upon elongation, will result in the fixation of frameshift errors (mutagenic translesion synthesis). Surprisingly we find that elongation from the nonslipped intermediate depends upon functional umuDC ؉ gene products, whereas elongation from the slipped intermediate is umuDC ؉ independent but requires another, as yet biochemically uncharacterized, SOS function. These data are discussed in terms of the different steps involved during translesion synthesis through a replication-blocking lesion.Organisms have developed various strategies to protect their genomes from the damaging effects of endogenous and exogenous agents. Despite having robust excision repair systems that remove DNA lesions before replication, cells also possess efficient strategies for tolerating lesions persisting at the replication fork during DNA synthesis (1). Two basic strategies of lesion tolerance can be distinguished: (i) translesion synthesis (TLS) is a process during which the replication machinery reads through the lesion with an associated risk of fixing a mutation. If the lesion does not impede DNA synthesis, an unmodified replication complex can achieve TLS. However, a replication complex modified by accessory proteins encoded by genes associated with the SOS regulon is required for TLS of lesions that hinder the progression of replication; and (ii) damage avoidance designates a general strategy that facilitates replication of damaged DNA templates without the need for the polymerase to read through the lesion. This strategy takes advantage of the information contained in the complementary strand. Two models of damage avoidance have been proposed (1-3): (i) postreplication recombinational repair, in which the DNA polymerase, blocked at a lesion site, dissociates from the DNA and reinitiates replication downstream from the lesion, leaving a gap that is repaired by a recombination mechanism involving the sister chromatid; and (ii) polymerase strand switching, in which the DNA polymerase switches temporarily from the damaged parental template to the undamaged newly synthesized strand of the sister chromatid before returning to the parental template downstream from the lesion. B...

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N-Acetoxy-N-acetyl-2-aminofluorene-induced mutagenesis in the lacI gene of Escherichia coli

Cited by 68 publications

References 0 publications

Effects of a Guanine-derived Formamidopyrimidine Lesion on DNA Replication

Effects of a Guanine-derived Formamidopyrimidine Lesion on DNA Replication

Frequency and Fidelity of Translesion Synthesis of Site-specific N-2-Acetylaminofluorene Adducts during DNA Replication in a Human Cell Extract

SOS factors involved in translesion synthesis

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