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R P Fuchs

Publications and source records attributed to R P Fuchs.

At least 19 recordsLinked to original sources
PubMed2001

Mechanism of DNA polymerase II-mediated frameshift mutagenesis.

Escherichia coli possesses three SOS-inducible DNA polymerases (Pol II, IV, and V) that were recently found to participate in translesion synthesis and mutagenesis. Involvement of these polymerases appears to depend on the nature of the lesion and its local sequence context, as illustrated by the bypass of a single N-2-acetylaminofluorene adduct within the NarI mutation hot spot. Indeed, error-free bypass requires Pol V (umuDC), whereas mutagenic (-2 frameshift) bypass depends on Pol II (polB). In this paper, we show that purified DNA Pol II is able in vitro to generate the -2 frameshift bypass product observed in vivo at the NarI sites. Although the Delta polB strain is completely defective in this mutation pathway, introduction of the polB gene on a low copy number plasmid restores the -2 frameshift pathway. In fact, modification of the relative copy number of polB versus umuDC genes results in a corresponding modification in the use of the frameshift versus error-free translesion pathways, suggesting a direct competition between Pol II and V for the bypass of the same lesion. Whether such a polymerase competition model for translesion synthesis will prove to be generally applicable remains to be confirmed.

2-Acetylaminofluorene↗
PubMed2001

DNA repair and sequence context affect (1)O(2)-induced mutagenesis in bacteria.

Electronic excited molecular oxygen (singlet oxygen, (1)O(2)) is known to damage DNA, yielding mutations. In this work, the mutagenicity induced by (1)O(2) in a defined sequence of DNA was investigated after replication in Escherichia coli mutants deficient for nucleotide and base excision DNA repair pathways. For this purpose a plasmid containing a (1)O(2)-damaged 14 base oligonucleotide was introduced into E.coli by transfection and mutations were screened by hybridization with an oligonucleotide with the original sequence. Mutagenesis was observed in all strains tested, but it was especially high in the BH20 (fpg), AYM57 (fpg mutY) and AYM84 (fpg mutY uvrC) strains. The frequency of mutants in the fpg mutY strain was higher than in the triple mutant fpg mutY uvrC, suggesting that activity of the UvrABC excinuclease can favor the mutagenesis of these lesions. Additionally, most of the mutations were G-->T and G-->C transversions, but this was dependent on the position of the guanine in the sequence and on repair deficiency in the host bacteria. Thus, the kind of repair and the mutagenesis associated with (1)O(2)-induced DNA damage are linked to the context of the damaged sequence.

Bacterial Proteins↗
PubMed2001

Induction of -2 frameshift mutations by 2-nitrofluorene, N-hydroxyacetylaminofluorene, and N-2-acetylaminofluorene in reversion assays in Escherichia coli strains differing in permeability and acetyltransferase activity.

The mutagenicity of 2-nitrofluorene (NF), N-hydroxyacetylaminofluorene (N-OH-AAF), and N-2-acetylaminofluorene (AAF) was measured in strains of Escherichia coli that contain a lacZ allele that reverts by -2 frameshift mutations from CG(5) to CG(4). Mutagenesis was compared in a strain having wild-type permeability and metabolism, a strain with increased permeability caused by a lipopolysaccharide-defective (LPS(d)) mutation, a strain with N- and O-acetyltransferase (NAT/OAT) activity conferred by the Salmonella nat gene on plasmid pYG219, and a strain carrying both an LPS(d) mutation and pYG219. The LPS(d) mutation facilitated the measurement of mutagenicity but was not absolutely required, in that lower levels of mutagenicity were detected in LPS(+) strains. The NAT/OAT activity conferred by pYG219 strongly potentiated the mutagenicity of NF and N-OH-AAF. Surprisingly, AAF was mutagenic in the NAT/OAT LPS(d) strain without an exogenous P450 metabolic activation system. Its activity may be ascribable to the detection of a directly mutagenic impurity by the highly sensitive strain or to a low level of metabolic activation by the bacteria under the assay conditions. The findings add to the evidence that the lacZ allele derived from E. coli strain CC109 is an effective indicator of -2 frameshift mutagenesis and that strains expressing high levels of NAT/OAT activity are highly sensitive in monitoring the mutagenicity of nitroarenes and aromatic amides.

2-Acetylaminofluorene↗
PubMed2001

DNA polymerases II and V mediate respectively mutagenic (-2 frameshift) and error-free bypass of a single N-2-acetylaminofluorene adduct.

The NarI sequence represents a strong mutation hot spot for -2 frameshift mutations induced by N-2-acetylaminofluorene (AAF), a strong chemical carcinogen. Only when bound to the third (underlined) guanine (5'-GGCGCC-->GGCC) can AAF trigger frameshift mutations, suggesting the involvement of a slipped replication intermediate with a two-nucleotide bulge. While base substitutions induced by UV light or abasic sites require DNA polymerase V (Pol V; umuDC), the AAF-induced -2 frameshift pathway requires DNA polymerase II, the polB gene product. Interestingly, error-free bypass of the G-AAF adduct requires Pol V. The genes encoding both Pol II and Pol V are induced by the SOS regulon, a co-ordinated cellular response to environmental stress. A given lesion, G-AAF, can thus be bypassed by two SOS-controlled DNA polymerases (II and V), generating mutagenic (-2 frameshifts) and error-free replication products respectively. Therefore both Pol II and Pol V can compete for the blocked replication intermediate in the vicinity of the lesion and engage in replication by transiently replacing the replicative DNA Pol III. Our data suggest that, in order to cope with the large diversity of existing DNA lesions, cells use a single or a combination of translesional DNA polymerases to achieve translesion synthesis.

2-Acetylaminofluorene↗
PubMed2001

Early detection of 2-amino-1-methyl-6-phenylimidazo (4,5-b)pyridine(PhIP)-induced mutations within the Apc gene of rat colon.

A large proportion of human cancers result from exposure of individuals to environmental or occupational carcinogens. The early detection of carcinogen-induced mutations is a prerequisite for the identification of individuals at risk for developing cancer. Short G-rich repetitive sequences have been previously identified as hot-spots for frameshift mutagenesis induced by a large variety of carcinogens belonging to several families of widespread environmental pollutants. In order to test if these sequences, when mutated, might serve as biomarkers for carcinogen exposure, we designed a sensitive PCR-based strategy that allows the detection of rare mutational events within a whole genome. 2-Amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP), the most abundant carcinogenic heterocyclic amine generated in cooked meat, induces mammary and colon carcinoma in F344 rats. About 25% of male rats exposed to 400 p.p.m. PhIP in the diet for >43 weeks present colon tumors with specific -1G mutations within 5'-GGGA-3' sequences of the APC: gene. Using our PCR assay we have assessed the occurrence of such specific events in rats exposed to PhIP for only 1, 2, 4 and 6 weeks. A specific amplification signal was already observed in the 1 week-treated population and increases in a treatment time-dependent manner. These data validate this approach for the early detection of mutations and demonstrate its usefulness for molecular epidemiology and early diagnosis.

Animals↗
PubMed2000

All three SOS-inducible DNA polymerases (Pol II, Pol IV and Pol V) are involved in induced mutagenesis.

Most organisms contain several members of a recently discovered class of DNA polymerases (umuC/dinB superfamily) potentially involved in replication of damaged DNA. In Escherichia coli, only Pol V (umuDC) was known to be essential for base substitution mutagenesis induced by UV light or abasic sites. Here we show that, depending upon the nature of the DNA damage and its sequence context, the two additional SOS-inducible DNA polymerases, Pol II (polB) and Pol IV (dinB), are also involved in error-free and mutagenic translesion synthesis (TLS). For example, bypass of N:-2-acetylaminofluorene (AAF) guanine adducts located within the NAR:I mutation hot spot requires Pol II for -2 frameshifts but Pol V for error-free TLS. On the other hand, error-free and -1 frameshift TLS at a benzo(a)pyrene adduct requires both Pol IV and Pol V. Therefore, in response to the vast diversity of existing DNA damage, the cell uses a pool of 'translesional' DNA polymerases in order to bypass the various DNA lesions.

2-Acetylaminofluorene↗
PubMed2000

Lesions in DNA: hurdles for polymerases.

Translesion synthesis (TLS) is one of the DNA damage tolerance strategies, which have evolved to enable organisms to replicate their genome despite the presence of unrepaired damage. The process of TLS has the propensity to produce mutations, a potential origin of cancer, and is therefore of medical interest. Significant progress in our understanding of TLS has come primarily from studies of the bacterium Escherichia coli, the budding yeast Saccharomyces cerevisiae and, more recently, human cells. Results from these analyses indicate that the fundamental mechanism of TLS and the proteins involved have been conserved throughout evolution from bacteria to humans.

DNA Damage↗
PubMed2000

The early detection of frameshift mutations induced by a food-borne carcinogen in rats: a new tool for molecular epidemiology.

The accumulation of genetic changes is considered as the main factor that determines the development of cancer. Recent progresses in genetics and molecular biology led to the discovery of many new molecular markers and to the development of techniques able to monitor these markers. As a consequence, molecular epidemiology has emerged as a powerful approach to study the ternary relationship between the environment, the behaviour and the genetic predisposition of each individual. Susceptibility to cancer is determined at different levels such as the genetic polymorphism of enzymes involved in the activation and detoxification of carcinogens, the polymorphism of genes that maintains the genome stability, like those involved in DNA repair or recombination processes, and finally the polymorphism in oncogenes or tumour suppressor genes. Consequently, the full assessment of each individual's genetic predisposition is a long and difficult task. As the accumulation of mutations in somatic cells integrates all these parameters, its measurement would facilitate the evaluation of the individual predisposition status, provided that a marker common to a large spectrum of carcinogens could be found. Our current studies on the molecular mechanisms of carcinogen-induced mutagenesis has revealed that G-rich repetitive sequences are mutational hot spots for several major classes of environmental genotoxins such as aromatic and heterocyclic amines, polycyclic hydrocarbons and oxidative agents. We thus consider the possibility that these sequences form a new class of biomarkers for carcinogen exposure. In order to validate this hypothesis, we designed a sensitive PCR-based assay able to detect specific mutations induced by a common food-borne carcinogen in the colon epithelium of rats exposed for a short period to this carcinogen. This assay is sensitive enough to allow early detection of induced mutations and therefore allows to differentiate between unexposed animal and those exposed for a period as short as 1 week.

2-Acetylaminofluorene↗
PubMed2000

The processing of a Benzo(a)pyrene adduct into a frameshift or a base substitution mutation requires a different set of genes in Escherichia coli.

Replication through a single DNA lesion may give rise to a panel of translesion synthesis (TLS) events, which comprise error-free TLS, base substitutions and frameshift mutations. In order to determine the genetic control of the various TLS events induced by a single lesion, we have chosen the major N2-dG adduct of (+)-anti-Benzo(a)pyrene diol epoxide [(+)-anti-BPDE] adduct located within a short run of guanines as a model lesion. Within this sequence context, in addition to the major event, i.e. error-free TLS, the adduct also induces base substitutions (mostly G --> T transversions) and -1 frameshift mutations. The pathway leading to G --> T base substitution mutagenesis appears to be SOS independent, suggesting that TLS is most probably performed by the replicative Pol III holoenzyme itself. In contrast, both error-free and frameshift TLS pathways are dependent upon SOS-encoded functions that belong to the pool of inducible DNA polymerases specialized in TLS (translesional DNA polymerases), namely umuDC (Pol V) and dinB (Pol IV). It is likely that, given the diversity of conformations that can be adopted by lesion-containing replication intermediates, cells use one or several translesional DNA polymerases to achieve TLS.

Bacterial Proteins↗
PubMed2000

The beta clamp targets DNA polymerase IV to DNA and strongly increases its processivity.

The recent discovery of a new family of ubiquitous DNA polymerases involved in translesion synthesis has shed new light onto the biochemical basis of mutagenesis. Among these polymerases, the dinB gene product (Pol IV) is involved in mutagenesis in Escherichia coli. We show here that the activity of native Pol IV is drastically modified upon interaction with the beta subunit, the processivity factor of DNA Pol III. In the absence of the beta subunit Pol IV is strictly distributive and no stable complex between Pol IV and DNA could be detected. In contrast, the beta clamp allows Pol IV to form a stable initiation complex (t 1/2 approximately 2.3 min), which leads to a dramatic increase in the processivity of PoI IV reaching an average of 300-400 nucleotides. In vivo, the beta processivity subunit may target DNA Pol IV to its substrate, generating synthesis tracks much longer than previously thought.

Bacterial Proteins↗
PubMed2000

Mechanisms of dinucleotide repeat instability in Escherichia coli.

The high level of polymorphism of microsatellites has been used for a variety of purposes such as positional cloning of genes associated with diseases, forensic medicine, and phylogenetic studies. The discovery that microsatellites are associated with human diseases, not only as markers of risk but also directly in disease pathogenesis, has triggered a renewed interest in understanding the mechanism of their instability. In this work we have investigated the role of DNA replication, long patch mismatch repair, and transcription on the genetic instability of all possible combinations of dinucleotide repeats in Escherichia coli. We show that the (GpC) and (ApT) self-complementary sequence repeats are the most unstable and that the mode of replication plays an important role in their instability. We also found that long patch mismatch repair is involved in avoiding both short deletion and expansion events and also in instabilities resulting from the processing of bulges of 6 to 8 bp for the (GpT/ApC)- and (ApG/CpT)- containing repeats. For each dinucleotide sequence repeat, we propose models for instability that involve the possible participation of unusual secondary structures.

DNA Replication↗
PubMed2000

Molecular approach in cancer epidemiology: early detection of carcinogen-induced mutations in a whole genome (Review).

Chronic exposure of organisms to endo- or exogenous genotoxic products results in the accumulation of mutations in the genome and eventually to the development of cancers. Early detection of these mutations would allow the identification of at risk individuals who present a high load of mutations either because of an occupational or environmental exposure, or because of less efficient DNA repair processes. However, highly specific and sensitive assays are required to allow the detection of point mutations in a whole genome. We review a long-term study on the mutagenesis induced in E.coli by an aromatic amide, the N-2-acetylaminofluorene. A major contribution of this work was to reveal the presence of specific mutation hot spot sequences. Taking advantage of this observation, we designed a specific, sensitive and semi-quantitative in vitro assay allowing the detection of carcinogen induced mutations. This assay has been validated in vivo and demonstrate the sensitivity of the technique in early detection of mutations and its usefullness in molecular epidemiology, early diagnostic and prognosis.

2-Acetylaminofluorene↗
PubMed1999

SOS mutagenesis results from up-regulation of translesion synthesis.

Irradiation of DNA with ultraviolet light generates a variety of photolesions. Among them, are cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts blocking lesions that interfere with DNA replication if left unrepaired. In addition to efficient pre-replicative excision repair mechanisms, cells have evolved damage tolerance pathways enabling them to replicate lesion-containing DNA molecules either by directly replicating through the damaged base (translesion synthesis, TLS) or by employing the locally undamaged complementary strand thus avoiding the lesion (damage avoidance pathways, DA). Using double-stranded vectors with a single T(6-4)T UV lesion and a strand segregation analysis (SSA), we have measured the relative utilization of the two tolerance pathways (TLS and DA) in Escherichia coli. During the SOS response the error-prone TLS pathway is strongly stimulated ( approximately 20-fold) at the expense of the error-free DA pathways. Thus, up-regulation of TLS may turn out to be a general property of the SOS response; a similar conclusion was previously reached with the frameshift-inducing N-2-acetylaminofluorene adduct. Therefore, as far as its contribution to damaged DNA replication is concerned, the SOS response appears to be an induced mutator state rather than a survival strategy. Depending on the base inserted opposite the lesion, TLS can be error-free or mutagenic. In a wild-type strain, both forms of TLS are increased to a similar extent during the SOS response. In contrast, in a DeltaumuDC strain induction of TLS is totally abolished, demonstrating that the UmuDC proteins usually thought to be specifically involved in mutagenesis facilitate the recovery of both error-free and mutagenic replication intermediates in vivo.

DNA↗
PubMed1999

Replication of damaged DNA: molecular defect in xeroderma pigmentosum variant cells.

Individuals with Xeroderma pigmentosum (XP) syndrome have a genetic predisposition to sunlight-induced skin cancer. Genetically different forms of XP have been identified by cell fusion. Cells of individuals expressing the classical form of XP (complementation groups A through G) are deficient in the nucleotide excision repair (NER) pathway. In contrast, the cells belonging to the variant class of XP (XPV) are NER-proficient and are only slightly more sensitive than normal cells to the killing action of UV light radiation. The XPV fibroblasts replicate damaged DNA generating abnormally short fragments either in vivo [A.R. Lehmann, The relationship between pyramidine dimers and replicating DNA in UV-irradiated human fibroblasts, Nucleic Acids Res. 7 (1979) 1901-1912; S.D. Park, J.E. Cleaver, Postreplication repair: question of its definition and possible alteration in Xeroderma pigmentosum cell strains, Proc. Natl. Acad. Sci. U.S.A. 76 (1979) 3927-3931.] or in vitro [S.M. Cordeiro, L.S. Zaritskaya, L.K. Price, W.K. Kaufmann, Replication fork bypass of a pyramidine dimer blocking leading strand DNA synthesis, J. Biol. Chem. 272 (1997) 13945-13954; D.L. Svoboda, L.P. Briley, J.M. Vos, Defective bypass replication of a leading strand cyclobutane thymine dimer in Xeroderma pigmentosum variant cell extracts, Cancer Res. 58 (1998) 2445-2448; I. Ensch-Simon, P.M. Burgers, J.S. Taylor, Bypass of a site-specific cis-syn thymine dimer in an SV40 vector during in vitro replication by HeLa and XPV cell-free extracts, Biochemistry 37 (1998) 8218-8226.], suggesting that in XPV cells, replication has an increased probability of being blocked at a lesion. Furthermore, extracts from XPV cells were found to be defective in translesion synthesis [A. Cordonnier, A.R. Lehmann, R.P.P. Fuchs, Impaired translesion synthesis in Xeroderma pigmentosum variant extracts, Mol. Cell. Biol. 19 (1999) 2206-2211.]. Recently, Masutani et al. [C. Masutani, M. Araki, A. Yamada, R. Kusomoto, T. Nogimori, T. Maekawa, S. Iwai, F. Hanaoka, Xeroderma pigmentosum variant (XP-V) correcting protein from HeLa cells has a thymine dimer bypass DNA polymerase activity, EMBO J. 18 (1999) 3491-3501.] have shown that the XPV defect can be corrected by a novel human DNA polymerase, homologue to the yeast DNA polymerase eta, which is able to replicate past cyclobutane pyrimidine dimers in DNA templates. This review focuses on our current understanding of translesion synthesis in mammalian cells whose defect, unexpectedly, is responsible for the hypermutability of XPV cells and for the XPV pathology.

Animals↗
PubMed1999

Sequence-dependent modulation of frameshift mutagenesis at NarI-derived mutation hot spots.

The NarI sequence is known to be the strongest mutation hot spot for induced frameshift mutagenesis. Indeed, a single N-2-acetylaminofluorene (AAF) adduct induces -2 frameshift mutations (5'-GGCGAAFCC--> 5'-GGCC) more than 10(7)-fold over background mutagenesis in Escherichia coli. The mechanism of induction of the frameshift mutation involves a two nucleotide primer-template misalignment event during replication of the adduct-containing sequence. The slipped mutagenic intermediate (SMI) that is thus formed is strongly stabilised by the AAF residue. In order to understand the origin of the extreme susceptibility of this sequence to frameshift mutagenesis, we analysed AAF-induced mutagenesis at sequences 5'-NaGCGAAFCNb-3' containing the core dinucleotide GCGC repeat present in the NarI sequence flanked by variable nucleotides Na and Nb. The nature of nucleotide Nb was found to strongly modulate the frequency of induced -2 frameshift mutagenesis (up to 30 to 50-fold), while little if any effect could be attributed to nucleotide Na. The induction of -2 frameshifts, regardless of nucleotides Na and Nb, was found to be SOS-inducible but umuDC-independent as previously found for the authentic NarI sequence. The NarI sequence (GGCGCC) and sequence TGCGCA (Na=T, Nb=A) were found to be equally "hot" for -2 frameshift mutation induction compared to the sequence AGCGCT where induced mutagenesis was 30 to 50-fold lower.The analysis of replication events using constructions containing a strand marker across from the adduct site allowed us to demonstrate that the large difference in -2 frameshift mutagenesis is due to an intrinsic difference in the propensity of these sequences to slip during replication. How the nature of the nucleotide flanking the adduct on its 3'-side (Nb) differentially stabilises the SMI will be discussed in the light of recent structural data and theoretical models.

2-Acetylaminofluorene↗
PubMed1999

The dinB gene encodes a novel E. coli DNA polymerase, DNA pol IV, involved in mutagenesis.

In Escherichia coli, the dinB gene is required for the SOS-induced lambda untargeted mutagenesis pathway and confers a mutator phenotype to the cell when the gene product is overexpressed. Here, we report that the purified DinB protein is a DNA polymerase. This novel E. coli DNA polymerase (pol IV) is shown to be strictly distributive, devoid of proofreading activity, and prone to elongate bulged (misaligned) primer/template structures. Site-directed mutagenesis experiments of dinB also demonstrate that the polymerase activity of DinB is required for its in vivo mutagenicity. Along with the sequence homologies previously found within the UmuC-like protein family, these results indicate that the uncovered DNA polymerase activity may be a common feature of all these homologous proteins.

Bacterial Proteins↗
PubMed1999

Sequence context modulation of translesion synthesis at a single N-2-acetylaminofluorene adduct located within a mutation hot spot.

Oligonucleotides containing a single N-(deoxyguanosin-8-yl)acetylaminofluorene lesion (dGuo-C8-AAF) at each guanine residue of the sequence (5'-G1G2G3) have been used as templates for in vitro primer extension reactions by several DNA polymerases [Escherichia coli DNA polymerase III holoenzyme, its alpha subunit, DNA polymerase I Klenow fragment proficient (exo+) or deficient (exo-) in its 3' --> 5' exonuclease activity, and Sequenase]. The dGuo-C8-AAF lesion appears to be a strong block for all DNA polymerases: exo+ DNA polymerases stop one nucleotide before encountering the lesion, while partial incorporation opposite the lesion is observed only with enzymes devoid of the exonuclease activity. The efficiency of incorporation across from the adduct depends on both the DNA polymerase and the position of the lesion. When polymerase I Klenow fragment exo- is used, translesion synthesis (TLS) is observed with efficiencies varying according to the position of the adduct (G2 > G1 > G3). Sequencing of the TLS products shows that error-free TLS is observed only when the AAF lesion is bound to G1, while all TLS events occurring at G2- or G3-AAF adducts are mutagenic. The major mutational event is a G deletion (27, 76, and 55% of the events for G1, G2, and G3, respectively), while two-G deletions occur to a lesser extent (17-30%). These results are discussed in view of the slippage model developed for frameshift mutagenesis occurring during translesion synthesis at replication blocking lesions.

2-Acetylaminofluorene↗