RESUMEN
The formation of araB-lacZ coding sequence fusions in Escherichia coli is a particular type of chromosomal rearrangement induced by Mucts62, a thermoinducible mutant of mutator phage Mu. Fusion formation is controlled by the host physiology. It only occurs after aerobic carbon starvation and requires the phage-encoded transposase pA, suggesting that these growth conditions trigger induction of the Mucts62 prophage. Here, we show that thermal induction of the prophage accelerated araB-lacZ fusion formation, confirming that derepression is a rate-limiting step in the fusion process. Nonetheless, starvation conditions remained essential to complete fusions, suggesting additional levels of physiological regulation. Using a transcriptional fusion indicator system in which the Mu early lytic promoter is fused to the reporter E. coli lacZ gene, we confirmed that the Mucts62 prophage was derepressed in stationary phase (S derepression) at low temperature. S derepression did not apply to prophages that expressed the Mu wild-type repressor. It depended upon the host ClpXP and Lon ATP-dependent proteases and the RpoS stationary phase-specific sigma factor, but not upon Crp. None of these four functions was required for thermal induction. Crp was required for fusion formation, but only when the Mucts62 prophage encoded the transposition/replication activating protein pB. Finally, we found that thermally induced cultures did not return to the repressed state when shifted back to low temperature and, hence, remained activated for accelerated fusion formation upon starvation. The maintenance of the derepressed state required the ClpXP and Lon host proteases and the prophage Ner-regulatory protein. These observations illustrate how the cts62 mutation in Mu repressor provides the prophage with a new way to respond to growth phase-specific regulatory signals and endows the host cell with a new potential for adaptation through the controlled use of the phage transposition machinery.
Asunto(s)
Bacteriófago mu/genética , Bacteriófago mu/fisiología , Proteínas de Escherichia coli , Escherichia coli/genética , Escherichia coli/virología , Proteasa La , Recombinación Genética , Proteasas ATP-Dependientes , Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/metabolismo , Bacteriófago mu/metabolismo , Proteínas Portadoras , Proteína Receptora de AMP Cíclico/metabolismo , Endopeptidasa Clp , Escherichia coli/metabolismo , Genes Bacterianos , Proteínas de Choque Térmico/metabolismo , Operón Lac , Lisogenia , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Serina Endopeptidasas/metabolismo , Factor sigma/metabolismo , Activación ViralRESUMEN
Formation of araB-lacZ coding-sequence fusions is a key adaptive mutation system. Eighty-four independent araB-lacZ fusions were sequenced. All fusions carried rearranged MuR linker sequences between the araB and lacZ domains indicating that they arose from the standard intermediate of the well-characterized Mu DNA rearrangement process, the strand transfer complex (STC). Five non-standard araB-lacZ fusions isolated after indirect sib selection had novel structures containing back-to-back inverted MuR linkers. The observation that different isolation procedures gave rise to standard and non-standard fusions indicates that cellular physiology can influence late steps in the multi-step biochemical sequence leading to araB-lacZ fusions. Each araB-lacZ fusion contained two novel of DNA junctions. The MuR-lacZ junctions showed 'hot-spotting' according to established rules for Mu target selection. The araB-MuR and MuR-MuR junctions all involved exchanges at regions of short sequence homology. More extensive homology between MuR and araB sequences indicates potential STC isomerization a resolvable four-way structure analogous to a Holliday junction. These results highlight the molecular complexity of araB-lacZ fusion formation, which may be thought of as a multi-step cell biology process rather than a unitary biochemical reaction.
Asunto(s)
Clonación Molecular , Escherichia coli/genética , Genes Bacterianos/genética , Operón Lac/genética , Selección Genética , beta-Galactosidasa/genética , Bacteriófago mu/genética , Secuencia de Bases , Análisis Mutacional de ADN , Genes Reguladores , Datos de Secuencia Molecular , Mutagénesis Insercional , Homología de Secuencia de Ácido NucleicoRESUMEN
The potency of 2-amino-3-methylimidazo(4,5-f)quinoline (IQ) adducts to induce -2, -1 and +1 frameshift mutations has been determined on specific target DNA sequences, namely short runs of alternating GpC sequences and short runs of guanines. The genetic control of the mutational processes has been analyzed using different Escherichia coli mutants, affected either in the control or in the mutagenesis pathway of the SOS system. We have shown that IQ adducts induce very efficiently both -1 and -2 frameshift mutations in E. coli. Both types of deletion mutations are induced in bacteria without the need of SOS induction, indicating that no LexA-controlled functions, in particular the UmuDC proteins, are required for mutation fixation. We have also shown that the frequency of IQ-induced -2 frameshift mutations in alternating GC sequences increases with the length of the repetition. The efficiency of IQ adducts to induce -1 and -2 frameshift mutations is similar to that of N-2-acetylaminofluorene (AAF) adducts. Both chemicals are potent carcinogens which form covalent adducts at the C8 position of guanines. We suggest that in both cases the adduct-induced DNA structure allows the replication complex to perform a mutagenic bypass of the lesion by a slippage mechanism. However, in contrast to AAF-induced frameshift mutagenesis, IQ-induced frameshift mutagenesis is SOS-independent.
Asunto(s)
Aductos de ADN , Mutación del Sistema de Lectura , Mutágenos/toxicidad , Quinolinas/toxicidad , Respuesta SOS en Genética , Repeticiones de Dinucleótido , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , MutagénesisAsunto(s)
2-Acetilaminofluoreno/farmacología , Carcinógenos/farmacología , Daño del ADN , Mutación del Sistema de Lectura , Mutagénesis , Conformación de Ácido Nucleico , Composición de Base , Secuencia de Bases , ADN/química , ADN/efectos de los fármacos , ADN Bacteriano/química , ADN Bacteriano/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Desnaturalización de Ácido Nucleico , Ácidos Nucleicos Heterodúplex , Eliminación de SecuenciaRESUMEN
dinP is an Escherichia coli gene recently identified at 5.5 min of the genetic map, whose product shows a similarity in amino acid sequence to the E. coli UmuC protein involved in DNA damage-induced mutagenesis. In this paper we show that the gene is identical to dinB, an SOS gene previously localized near the lac locus at 8 min, the function of which was shown to be required for mutagenesis of nonirradiated lambda phage infecting UV-preirradiated bacterial cells (termed lambdaUTM for lambda untargeted mutagenesis). A newly constructed dinP null mutant exhibited the same defect for lambdaUTM as observed previously with a dinB::Mu mutant, and the defect was complemented by plasmids carrying dinP as the only intact bacterial gene. Furthermore, merely increasing the dinP gene expression, without UV irradiation or any other DNA-damaging treatment, resulted in a strong enhancement of mutagenesis in F'lac plasmids; at most, 800-fold increase in the G6-to-G5 change. The enhanced mutagenesis did not depend on recA, uvrA, or umuDC. Thus, our results establish that E. coli has at least two distinct pathways for SOS-induced mutagenesis: one dependent on umuDC and the other on dinB/P.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Genes Bacterianos , Respuesta SOS en Genética/genética , Secuencia de Bases , Daño del ADN , ADN Bacteriano/genética , ADN Polimerasa Dirigida por ADN , Expresión Génica , Prueba de Complementación Genética , Mutagénesis , Plásmidos/genética , Regiones Promotoras GenéticasRESUMEN
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
Asunto(s)
Carcinógenos/toxicidad , Daño del ADN , Reparación del ADN , Mutágenos/toxicidad , Alquilantes/toxicidad , Animales , Cricetinae , Humanos , Masculino , Ratones , Ratas , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
2-Acetilaminofluoreno/toxicidad , Proteínas Bacterianas/fisiología , Proteínas de Escherichia coli , Escherichia coli/efectos de los fármacos , Mutación del Sistema de Lectura , Citosina , ADN Polimerasa Dirigida por ADN , Escherichia coli/genética , Escherichia coli/fisiología , Guanina , Rec A Recombinasas/metabolismoRESUMEN
The araB-lacZ fusion system has been a key case in the 'directed mutation' controversy. Fusions did not occur detectably during normal growth but formed readily after prolonged incubation on selective Ara-Lac medium. To distinguish the roles of starvation stress and selective substrates in coding sequence fusions, we applied sib selection and PCR technologies. Sib selection of the prefusion strain, MCS2, starved under aerobic conditions permitted us to isolate active fusion clones which had never been in contact with arabinose or lactose. Hence, a directive role for selective substrates is not essential. Aerobiosis was necessary for fusions to appear in glucose-starved cultures. The difference in fusion formation between normal and starved conditions is best explained by the response of a signal transduction network to physiological stimuli to activate Mu prophage joining of araB and lacZ sequences. PCR analysis revealed that direct plating on selective Ara-Lac agar yielded mostly a single class of 'standard' fusions, while sib selection yielded a broader spectrum of fusion structures. Standard fusions were found to occur within a narrow 9 bp window in lacZ. The high frequency of standard fusions in glucose-starved cultures suggested efficient and/or specific Mu action.
Asunto(s)
Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas Recombinantes de Fusión/genética , Recombinación Genética , Selección Genética , beta-Galactosidasa/genética , Secuencia de Bases , Evolución Biológica , Inducción Enzimática , Glucosa/deficiencia , Datos de Secuencia Molecular , Mutagénesis/genética , Reacción en Cadena de la Polimerasa , Transducción de SeñalRESUMEN
Bacteriophage Mu is a transposon and a temperate phage which has become a paradigm for the study of the molecular mechanism of transposition. As a prophage, Mu has also been used to study some aspects of the influence of the host cell growth phase on the regulation of transposition. Through the years several host proteins have been identified which play a key role in the replication of the Mu genome by successive rounds of replicative transposition as well as in the maintenance of the repressed prophage state. In this review we have attempted to summarize all these findings with the purpose of emphasizing the benefit the virus and the host cell can gain from those phage-host interactions.
Asunto(s)
Bacteriófago mu/genética , Replicación del ADN , Elementos Transponibles de ADN , Bacteriófago mu/crecimiento & desarrollo , Secuencia de Bases , Regulación Viral de la Expresión Génica , Genoma Viral , Modelos Genéticos , Datos de Secuencia Molecular , Nucleotidiltransferasas/metabolismo , Transposasas , Activación ViralRESUMEN
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.
Asunto(s)
Acetoxiacetilaminofluoreno/farmacología , Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Mutación del Sistema de Lectura , Genes Bacterianos , Respuesta SOS en Genética , Serina Endopeptidasas , Rayos Ultravioleta , Proteínas Bacterianas/metabolismo , Secuencia de Bases , ADN Polimerasa Dirigida por ADN , Escherichia coli/efectos de los fármacos , Escherichia coli/efectos de la radiación , Genes Bacterianos/efectos de los fármacos , Genotipo , Mutagénesis , Plásmidos , Rec A Recombinasas/genética , Mapeo RestrictivoRESUMEN
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.
Asunto(s)
Alelos , Escherichia coli/genética , Mutación del Sistema de Lectura , Rec A Recombinasas/genética , Reparación del ADN , Genes Bacterianos , Mutagénesis , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
The SOS hypothesis postulated that the mutator effect on undamaged DNA that generates phage-untargeted mutagenesis (UTM) results directly from the mechanism of targeted mutagenesis. RecA protein, which stimulates the cleavage of both the LexA repressor and UmuD protein, and the UmuDC gene products are required for UV-induced targeted mutagenesis. The use of phage lambda for analyzing UV-induced mutagenesis has permitted a distinction to be made between the mechanisms of targeted and untargeted mutagenesis, in that the two processes differ with respect to their genetic requirements for recA+ and umuDC+ genes. In this paper, we show that (i) proficiency for excision repair is required for UTM in double-stranded DNA phage but not in single-stranded DNA phage; (ii) the umuC function, which is not required for UTM of the double-stranded DNA phage lambda, is necessary for untargeted mutagenesis of the single-stranded DNA phages M13 and phi X174; (iii) for both single-stranded and double-stranded DNA phage, UV irradiation of the host increases the level of recA730-induced UTM. Our results are also consistent with the interpretation that the expression of untargeted mutagenesis in phage lambda and in M13 depends on the polymerase and to a lesser extent on the exonuclease 5'----3', activities of PolI. These results suggest that the involvement of the RecA and UmuDC proteins may be related to more than the presence of base damage in the DNA substrate.
Asunto(s)
Bacteriófagos/genética , Reparación del ADN , Mutación , Respuesta SOS en Genética , Proteínas Bacterianas/genética , Bacteriófago lambda/genética , Bacteriófago phi X 174/genética , ADN Polimerasa I/metabolismo , ADN de Cadena Simple/genética , ADN de Cadena Simple/efectos de la radiación , ADN Viral/genética , ADN Viral/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Exonucleasas/metabolismo , Genes Virales , Rec A Recombinasas/genética , Rayos UltravioletaRESUMEN
In Escherichia coli, induction of the SOS functions by UV irradiation or by mutation in the recA gene promotes an SOS mutator activity which generates mutations in undamaged DNA. Activation of RecA protein by the recA730 mutation increases the level of spontaneous mutation in the bacterial DNA. The number of recA730-induced mutations is greatly increased in mismatch repair deficient strains in which replication errors are not corrected. This suggests that the majority of recA730-induced mutations (90%) arise through correctable, i.e. non-targeted, replication errors. This recA730 mutator effect is suppressed by a mutation in the umuC gene. We also found that dam recA730 double mutants are unstable, segregating clones that have lost the dam or the recA mutations or that have acquired a new mutation, probably in one of the genes involved in mismatch repair. We suggest that the genetic instability of the dam recA730 mutants is provoked by the high level of replication errors induced by the recA730 mutation, generating killing by coincident mismatch repair on the two unmethylated DNA strands. The recA730 mutation increases spontaneous mutagenesis of phage lambda poorly. UV irradiation of recA730 host bacteria increases phage untargeted mutagenesis to the level observed in UV-irradiated recA+ strains. This UV-induced mutator effect in recA730 mutants is not suppressed by a umuC mutation. Therefore UV and the recA730 mutation seem to induce different SOS mutator activities, both generating untargeted mutations.
Asunto(s)
Reparación del ADN , Escherichia coli/genética , Genes Bacterianos , Respuesta SOS en Genética , Bacteriófago lambda/genética , Bacteriófago lambda/efectos de la radiación , Daño del ADN , Reparación del ADN/efectos de la radiación , Escherichia coli/efectos de la radiación , Mutación , Rec A Recombinasas/genética , Respuesta SOS en Genética/efectos de la radiación , Rayos UltravioletaRESUMEN
Circular heteroduplex DNAs of bacteriophage phi X174 have been constructed carrying either a G:T (Eam+/Eam3) or a G:A (Bam+/Bam16) mismatch and containing either two, one or no GATC sequences. Mismatches were efficiently repaired in wild-type Escherichia coli transfected with phi X174 heteroduplexes only when two unmethylated GATC sequences were present in phi X174 DNA. The requirements for GATC sequences in substrate DNA and for the E. coli MutH function in E. coli mismatch repair can be alleviated by the presence of a persistent nick (transfection with nicked heteroduplex DNA in ligase temperature-sensitive mutant at 40 degrees C). A persistent nick in the GATC sequence is as effective in stimulating mutL- and mutS-dependent mismatch repair as a nick distant from the GATC sequence and from the mismatch. These observations suggest that the MutH protein participates in methyl-directed mismatch repair by recognizing unmethylated DNA GATC sequences and/or stimulating the nicking of unmethylated strands.
Asunto(s)
Bacteriófago phi X 174/genética , Reparación del ADN , Escherichia coli/genética , Mutación , Secuencia de Bases , ADN Bacteriano/genética , ADN Viral/genética , Ácidos Nucleicos Heterodúplex/genéticaRESUMEN
N-2-Acetylaminofluorene (AAF), a well-known chemical carcinogen, when covalently linked to guanine residues constitutes a premutagenic lesion that is converted in vivo into frameshift mutations. In Escherichia coli, it is thought that -AAF adducts block the replication fork and that the mutagenic processing of the -AAF adducts is mediated by the SOS response. The construction in vitro of plasmids containing -AAF adducts in one strand only of a double-stranded DNA molecule enabled us to investigate the segregation of the strands and the mutagenicity of the lesions in vivo. The two DNA strands were "genetically labelled" by means of a single base-pair mismatch in the tetracycline-resistance gene, one strand carrying the wild-type allele and the other strand a mutant tetracycline-sensitive allele. The two strands contained either no -AAF adducts, -AAF adducts in one strand or -AAF adducts in both strands. When such constructions are used to transform bacterial cells the following are found. When no -AAF adducts are present on either strand of the DNA, a mixture of plasmids having information from both parent strands is found in 80% of the transformed bacterial clones. With -AAF adducts present in one strand only, in 90% of the transformants there is a consistent loss of the parent strand information that contained the -AAF adducts. In the constructions having -AAF adducts in both strands, the transformed bacteria carry either one or the other allele in a pure form. Our results suggest that when blocking lesions (-AAF adducts) are present in one strand only, they trigger the specific loss of that strand. The forward mutation frequency (i.e. the tetracycline-resistance gene inactivation frequency) was found to be more than ten times lower when the -AAF adducts are bound to one strand only compared with the situation where both strands carry the premutagenic lesions. Moreover, when the isolated mutants were sequenced, the mutations were found to consist of a mixture of true -AAF-induced mutations (i.e. -1 or -2 frameshift mutation at previously determined mutation hot spots) and of mutations that are not targeted at -AAF adducts. We suggest that these "background" mutants arose from the mutagenic processing of cryptic lesions present in our DNA. The low mutagenic efficiency of -AAF adducts, when present in one strand only of a duplex DNA, most probably results from the above-described loss of the damaged strand.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
2-Acetilaminofluoreno/farmacología , ADN Bacteriano/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Mutación , Ácidos Nucleicos Heterodúplex , Plásmidos , Respuesta SOS en GenéticaRESUMEN
The Escherichia coli mismatch repair system greatly improves DNA replication fidelity by repairing single mispaired and unpaired bases in newly synthesized DNA strands. Transient undermethylation of the GATC sequences makes the newly synthesized strands susceptible to mismatch repair enzymes. The role of unmethylated GATC sequences in mismatch repair was tested in transfection experiments with heteroduplex DNA of phage phi 174 without any GATC sequence or with two GATC sequences, containing in addition either a G:T mismatch (Eam+/Eam3) or a G:A mismatch (Bam+/Bam16). It appears that only DNA containing GATC sequences is subject to efficient mismatch repair dependent on E. coli mutH, mutL, mutS and mutU genes; however, also in the absence of GATC sequence some mut-dependent mismatch repair can be observed. These observations suggest that the mismatch repair enzymes recognize both the mismatch and the unmethylated GATC sequence in DNA over long distances. The presence of GATC sequence(s) in the substrate appears to be required for full mismatch repair activity and not only for its strand specificity according to the GATC methylation state.
Asunto(s)
Reparación del ADN , Replicación del ADN , Escherichia coli/genética , Bacteriófago phi X 174/genética , Secuencia de Bases , Enzimas de Restricción del ADN , ADN Bacteriano/genética , Ácidos Nucleicos Heterodúplex/genética , TransfecciónRESUMEN
Untargeted UV mutagenesis of bacteriophage lambda--i.e., the increased recovery of lambda mutants when unirradiated lambda infects UV-irradiated Escherichia coli--is thought to be mediated by a transient decrease in DNA replication fidelity, generating mutations in the newly synthesized strands. Using the bacteriophage lambda cI857----lambda c mutation system, we provide evidence that the RecA protein, shown previously to be required for this mutagenic pathway, is no longer needed when the LexA protein is inactivated by mutation. We suggest that the error-prone DNA replication responsible for UV-induced untargeted mutagenesis is turned on by the presence of replication-blocking lesions in the host cell DNA and that the RecA protein is required only to derepress the relevant din gene(s). This is in contrast to mutagenesis of irradiated bacteria or irradiated phage lambda, in which activated RecA protein has a second role in mutagenesis in addition to the cleavage of the LexA protein. Among the tested din genes, the dinB gene product (in addition to the uvrA and uvrB gene products) was found to be required for untargeted mutagenesis of bacteriophage lambda. To our knowledge, a phenotype associated with the dinB gene has not been reported previously.
Asunto(s)
Bacteriófago lambda/genética , Genes Bacterianos , Mutación/efectos de la radiación , Rec A Recombinasas/genética , Serina Endopeptidasas , Proteínas Bacterianas/genética , Replicación del ADN , Rayos UltravioletaRESUMEN
Bacteriophage lambda and phi X 174 DNAs, carrying sequenced mutations, have been used to construct in vitro defined species of heteroduplex DNA. Such heteroduplex DNAs were introduced by transfection, as single copies, into E. coli host cells. The progeny of individual heteroduplex molecules from each infective center was analyzed. The effect of the presence of GATC sequences (phi X 174 system) and of their methylation (lambda system) was tested. The following conclusions can be drawn: some mismatched base pairs trigger the process of mismatch repair, causing a localized strand-to-strand information transfer in heteroduplex DNA: transition mismatches G:T and A:C are efficiently repaired, whereas the six transversion mismatches are not always readily recognized and/or repaired. The recognition of transversion mismatches appears to depend on the neighbouring nucleotide sequence; single unpaired bases (frameshift mutation "mismatches") are recognized and repaired, some equally efficiently on both strands (longer and shorter), some more efficiently on the shorter (-1) strand; large non-homologies (about 800 bases) are not repaired by the Mut H, L, S, U system, but some other process repairs the non-homology with a relatively low efficiency; full methylation of GATC sequences inhibits mismatch repair on the methylated strand: this is the chemical basis of strand discrimination (old/new) in mismatch correction; unmethylated GATC sequences appear to improve mismatch repair of a G:T mismatch in phi X 174 DNA, but there may be some residual mismatch repair in GATC-free phi X 174, at least for some mismatches.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Bacteriófago lambda/genética , Bacteriófago phi X 174/genética , Composición de Base , ADN Bacteriano/genética , Escherichia coli/genética , Ácidos Nucleicos Heterodúplex/genética , Secuencia de Bases , Replicación del ADN , TransfecciónRESUMEN
This paper retraces the evolution of hypotheses concerning mechanisms of SOS induced mutagenesis. Moreover, it reports some recent data which support a new model for the mechanism of targeted and untargeted mutagenesis in E. coli. In summary, the SOS mutator effect, which is responsible for untargeted mutagenesis and perhaps for the misincorporation step in targeted mutagenesis, is believed to involve a fidelity function associated with DNA polymerase III and does not require the umuC gene product. umuC and umuD gene products are probably required specifically for elongation of DNA synthesis past blocking lesions, i.e. to allow mutagenic replication of damaged DNA.
Asunto(s)
Reparación del ADN , Escherichia coli/genética , Mutación , Bacteriófago lambda/genética , ADN Polimerasa III/fisiología , Replicación del ADN/efectos de los fármacos , ADN Bacteriano/genética , ADN Bacteriano/efectos de la radiación , ADN Viral/genética , ADN Viral/efectos de la radiación , Escherichia coli/efectos de los fármacos , Escherichia coli/efectos de la radiación , Genes Bacterianos , Mutágenos/farmacología , Fotoquímica , Dímeros de Pirimidina , Rec A Recombinasas/fisiología , Rayos UltravioletaRESUMEN
It has been proposed that the mutation fixation processes stimulated by SOS induction result from an induced infidelity of DNA replication (Radman 1974). The aim of this study was to determine if mutator mutations in the E. coli DNA polymerase III might affect UV-induced mutagenesis. Using a phage lambda mutation assay which can discriminate between targeted and untargeted mutations, we show that the polC74 mutator mutation (Sevastopoulos and Glaser 1977) primarily affects untargeted mutagenesis, which occurs in a recA1 genetic background and is amplified in the recA+ genetic background. The polC74 mutation also increases the UV-induced mutagenesis of the bacterial chromosome. These results suggest that DNA polymerase III is involved in the process of UV-induced mutagenesis in E. coli.