The SOS-function-inducing activity of chemical mutagens in Escherichia coli

The SOS-function-inducing activities of 42 chemical mutagens were investigated in Escherichia coli K12. The induction of the SOS function was assayed by monitoring the beta-galactosidase activity in the sulA::lacZ fusion strain PQ37 . To correct for the inhibitory effects of test chemicals on mRNA or protein synthesis, the level of the constitutive alkaline phosphatase was assayed in parallel. Most of the mutagens reported to be mutagenic to the Ames' Salmonella tester strains showed the SOS-function-inducing activity. The inducible SOS repair may be responsible for not only base-change mutations but also frameshift mutations. However, 9-aminoacridine, ethidium bromide and 4-nitro-o-phenylenediamine did not induce the SOS function, suggesting that the mutagenesis induced by these mutagens may occur independently of SOS repair. Present results support the SOS mutagenesis model that error-prone SOS repair plays an important role in mutagenesis induced by most chemical mutagens.     

60 years of SOS repair

This review integrates 60 years of research on SOS repair and SOS mutagenesis in prokaryotes and eukaryotes, from Jean Weigle’s experiment in 1953 (mutagenesis of lambda bacteriophage in UV-irradiated bacteria) to the latest achievements in studying SOS mutagenesis on all living organisms, i.e., Eukarya, Archaea and Bacteria. A key role in establishing of a biochemical basis for SOS mutagenesis belongs to the finding in 1998–1999 that specific error-prone DNA polymerases (PolV and others) catalyzed translesion synthesis on damaged DNA. This review focuses on recent studies that address new models for SOS-induced mutagenesis in Escherichia coli and Homo sapiens cells.     

Activated RecA protein may induce expression of a gene that is not controlled by the LexA repressor and whose function is required for mutagenesis and repair of UV-irradiated bacteriophage lambda.

The activated form of the RecA protein (RecA) is known to be involved in the reactivation and mutagenesis of UV-irradiated bacteriophage lambda and in the expression of the SOS response in Escherichia coli K-12. The expression of the SOS response requires cleavage of the LexA repressor by RecA and the subsequent expression of LexA-controlled genes. The evidence presented here suggests that RecA induces the expression of a gene(s) that is not under LexA control and that is also necessary for maximal repair and mutagenesis of damaged phage. This conclusion is based on the chloramphenicol sensitivity of RecA -dependent repair and mutagenesis of damaged bacteriophage lambda in lexA(Def) hosts.     

Mutagenic effect on Escherichia coli bacteria of 8-hydroxy-2'-deoxyguanosine--a DNA base damage product induced by oxygen radicals and ionizing radiation]

The effect of 8-oxo-2'-deoxyguanosine (8-oxo-dG) (8-hydroxydeoxyguanosine)--a DNA base damage product induced by oxygen radicals and irradiation on survival and mutagenesis in Escherichia coli strains C-600 and P-687 was investigated. Survival and mutagenesis curves, in dependence of 8-oxo-dG concentrations in the medium, ranging from 0.2 through 10 mM, were obtained. Bacterial survival at all 8-oxo-dG concentrations tested was shown to be no lesser than in the control. The mutagenic effect of 8-oxo-dG was tested by frequency of reversions in the absence of leucine and threonine. A non-linear dependence of mutagenesis on the concentration was observed. Linear increase in the amount of revertants took place at concentrations of 8-oxo-dG lower than 1 mM, and being kept constant at higher concentrations. Induction of SOS repair under the action of 8-oxo-dG in E. coli PQ37 strain was estimated according to alteration of activity of beta-galactosidase in the SOS chromotest. Weak induction of the SOS response was observed within the wide range of 8-oxo-dG concentration values, which points to a lack of genotoxicity and independence of mutagenesis on SOS repair.     

Temporal distinction between repair and mutagenesis of benzopyrene adducts after SOS induction in Escherichia coli

Plasmid DNA covalently modified with benzopyrene diol epoxide was introduced into Escherichia coli strains which differed in their capacity for repair and mutagenesis at various times after SOS induction. The uvrA+-dependent repair activity rose and fell before umuC+SOS-dependent mutagenesis was fully expressed.     

Bacterial SOS response: a food safety perspective

The SOS response is a conserved inducible pathway in bacteria that is involved in DNA repair and restart of stalled replication forks. Activation of the SOS response can result in stress resistance and mutagenesis. In food processing facilities and during food preservation, bacteria are exposed to stresses and stimuli that potentially activate the SOS response, resulting in resistant or adapted bacteria. This review places the bacterial SOS response in a food safety perspective by providing an overview of the known triggers of the SOS response mechanism and its impact on the survival of spoilage and pathogenic bacteria.     

The SOS system

In the bacterium Escherichia coli DNA damaging treatments such as ultraviolet or ionizing radiation induce a set of functions called collectively the SOS response, reviewed here. The regulation of the SOS response involves a repressor, the LexA protein, and an inducer, the RecA protein. After DNA damage an effector molecule is produced--possibly single stranded DNA--which activates the RecA protein to a form capable of catalysing proteolytic cleavage of LexA. The repressors of certain temperate prophages are cleaved under the same conditions, resulting in lysogenic induction. SOS functions are involved in DNA repair and mutagenesis, in cell division inhibition, in recovery of normal physiological conditions after the DNA damage is repaired, and possibly in cell death when DNA damage is too extensive. The SOS response also includes several chromosomal genes of unknown function, a number of plasmid encoded genes (bacteriocins, mutagenesis), and lysogenic induction of certain prophages. DNA damaging treatments seem to induce DNA repair and mutagenic activities and proviral development in many species, including mammalian cells. In general, substances which are genotoxic to higher eukaryotes induce the SOS response in bacteria. This correlation is the basis of the numerous bacterial tests for genotoxicity and carcinogenicity.     

Induction of the SOS response in Escherichia coli cells under osmotic stress and in the presence of N-methyl-N'-nitro-N-nitrosoguanidine

We investigated the dynamics of the SOS response induction and the frequency of reversions induced by the monofunctional alkylating compound N-methyl-N'-nitro-N-nitrosoguanidine in Escherichia coli cells exposed to osmotic stress for 1 h. During the stress treatment of the wild-type cultures adapted and not adapted to the alkylating agent, the maximum SOS response values and induced reversion frequencies were recorded twice. The SOS response values and induced reversion frequencies remained unchanged during the whole period after attaining the maximum values in adapted and nonadapted cells carrying a mutation in the excision repair gene. Presumably, the SOS mutagenesis mechanisms are turned on in the cells with an inactivated excision repair system earlier than in wild-type cells.     

The appearance of the UmuD'C protein complex in Escherichia coli switches repair from homologous recombination to SOS mutagenesis

The process of SOS mutagenesis in Escherichia coli requires (i) the replisome enzymes, (ii) RecA protein, and (iii) the formation of the UmuD'C protein complex which appears to help the replisome to resume DNA synthesis across a lesion. We found that the UmuD'C complex is an antagonist of RecA-mediated recombination. Homologous recombination in an Hfr x F^- cross decreased as a function of the UmuD'C cell concentration; this effect was challenged by increasing RecA concentration. Recombination of a u.v.-damaged F-lac with the lac gene of an F^- recipient was reduced by increasing the UmuD'C concentration while lac mutagenesis increased, showing an inverse relationship between recombination and SOS mutagenesis. We explain our data with the following model. The kinetics of appearance of the UmuD'C complex after DNA damage is slow, reaching a maximum after an hour. Within that period, excision and recombinational repair have had time to occur. When the UmuD'C concentration relative to the number of residual RecA filaments, not resolved by recombinational repair, becomes high enough, UmuD'C proteins provide a processive factor for the replisome to help replication bypass and repel the standing RecA filament. Thus, at a high enough concentration, the UmuD'C complex will switch repair from recombination to SOS mutagenesis.     

Induction of the SOS response in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells under osmotic stress and in the presence of N-methyl-N′-nitro-N-nitrosoguanidine

We investigated the dynamics of the SOS response induction and the frequency of reversions induced by the monofunctional alkylating compound N-methyl-N′-nitro-N-nitrosoguanidine in Escherichia coli cells exposed to osmotic stress for 1 h. During the stress treatment of the wild-type cultures adapted and not adapted to the alkylating agent, the maximum SOS response values and induced reversion frequencies were recorded twice. The SOS response values and induced reversion frequencies remained unchanged during the whole period after attaining the maximum values in adapted and nonadapted cells carrying a mutation in the excision repair gene. Presumably, the SOS mutagenesis mechanisms are turned on in the cells with an inactivated excision repair system earlier than in wild-type cells.     

Inducibility of error-prone DNA repair in yeast?

Whereas some experimental evidence suggests that mutagenesis in yeast after treatment with DNA-damaging agents involves inducible functions, a general-acting error-prone repair activity analogous to the SOS system of Escherichia coli has not yet been demonstrated. The current literature on the problem of inducibility of mutagenic repair in yeast is reviewed with emphasis on the differences in the experimental procedures applied.     

Overproduction of the beta subunit of DNA polymerase III holoenzyme reduces UV mutagenesis in Escherichia coli.

Overproduction of the beta subunit of DNA polymerase III holoenzyme caused a 5- to 10-fold reduction of UV mutagenesis along with a slight increase in sensitivity to UV light in Escherichia coli. The same effects were observed in excision-deficient cells, excluding the possibility that they were mediated via changes in excision repair. In contrast, overproduction of the alpha subunit of the polymerase did not influence either UV mutagenesis or UV sensitivity. The presence of the mutagenesis proteins MucA and MucB expressed from a plasmid alleviated the effect of overproduced beta on UV mutagenesis. We have previously suggested that DNA polymerase III holoenzyme can exist in two forms: beta-rich form unable to bypass UV lesions and a beta-poor form capable of bypassing UV lesions (O. Shavitt and Z. Livneh, J. Biol. Chem. 264:11275-11281, 1989). The beta-poor form may be related to an SOS form of DNA polymerase III designed to perform translesion polymerization under SOS conditions and thereby generate mutations. On the basis of this model, we propose that the overproduced beta subunit affects the relative abundance of the regular replicative beta-rich polymerase and the SOS bypass-proficient polymerase by sequestering the polymerase molecules to the beta-rich form and blocking the SOS form.     

SOS mutator effect in E. coli mutants deficient in mismatch correction.

We have used bacteriophage lambda to characterize the mutator effect of the SOS response induced by u.v. irradiation of Escherichia coli. Mutagenesis of unirradiated phages grown in irradiated or unirradiated bacteria was detected by measuring forward mutagenesis in the immunity genes or reversion mutagenesis of an amber codon in the R gene. Relative to the wild-type, the SOS mutator effect was higher in E. coli mismatch correction-deficient mutants (mutH, mutL and mutS) and lower in an adenine methylation-deficient mutant ( dam3 ). We conclude that a large proportion of SOS-induced 'untargeted' mutations are removed by the methyl-directed mismatch correction system, which acts on newly synthesized DNA strands. The lower SOS mutator effect observed in E. coli dam mutants may be due to a selective killing of mismatch-bearing chromosomes resulting from undirected mismatch repair. The SOS mutator effect on undamaged lambda DNA, induced by u.v. irradiation of the host, appears to result from decreased fidelity of DNA synthesis.     

Induction of SOS functions in Escherichia coli by lesions resulting from incorporation of 5-bromouracil into DNA

Lesions induced by 5-bromouracil (BU), after its incorporation into DNA, led to effective induction of prophage lambda and W reactivation (or BU reactivation). Prophage induction due to incorporated BU occurred only with the wild-type prophage, and not for the lambda c1857 mutant with a thermosensitive repressor. Antipain, a protease inhibitor, inhibited wild-type prophage induction 70-90%. This indicates that BU-induced lesions may induce the SOS repair system. The finding that such lesions provoke BU reactivation permits the inference that BU-induced mutagenesis also proceeds via involvement of the error-prone repair system, and not directly as a result of base-pairing errors. Genetic evidence suggests that induction of the SOS repair system as a result of incorporation of BU into DNA is linked to the subsequent appearance of uracil residues and apyrimidinic sites, resulting from dehalogenation of incorporated BU. Apyrimidinic sites appear to be more effective than uracil residues in induction of the SOS system.     

Antimutagenic effects of para-aminobenzoic acid in experiments with bacterial cells. Noninterference in the mutation process controlled by plasmid pKM101

The dependence of expression of PABA antimutagenic action in bacterial cells on the character of genetic control of the mutagenic process was studied. PABA antimutagenic activity was largely connected with the negative control of SOS repair which is controlled by bacterial cell genes, but not by pKM101 plasmid genes. These results are in agreement with the idea that the systems of repair and mutagenesis specified by cell genome and plasmids are not identical.     

Molecular mechanisms of mutagenesis caused by apurinic/apyrimidinic DNA sites

The subject of this review are molecular mechanisms and specificity of mutagenesis induced by apurinic/apyrimidinic (AP) sites representing a characteristic group of so called non-coding DNA lesions. The data available suggest that efficiency and specificity of AP sites-induced mutations depend, primarily, on genome structural organization. This is manifested in existence of DNA sequences highly prone to depurination/depyrimidination as well as in the ability of specific DNA regions to adopt potentially mutagenic conformations. The latter leads to mutations as consequence of AP sites' repair. Secondly, the AP sites-induced mutagenesis depends on functional state of genome, on the ability of replicative/repair cell apparatus to carry out some specific forms of mutagenic DNA repair, in particular, to bypass non-coding DNA lesions under conditions of SOS repair.     

A new model for SOS-induced mutagenesis: how RecA protein activates DNA polymerase V

In Escherichia coli, cell survival and genomic stability after UV radiation depends on repair mechanisms induced as part of the SOS response to DNA damage. The early phase of the SOS response is mostly dominated by accurate DNA repair, while the later phase is characterized with elevated mutation levels caused by error-prone DNA replication. SOS mutagenesis is largely the result of the action of DNA polymerase V (pol V), which has the ability to insert nucleotides opposite various DNA lesions in a process termed translesion DNA synthesis (TLS). Pol V is a low-fidelity polymerase that is composed of UmuD′₂C and is encoded by the umuDC operon. Pol V is strictly regulated in the cell so as to avoid genomic mutation overload. RecA nucleoprotein filaments (RecA*), formed by RecA binding to single-stranded DNA with ATP, are essential for pol V-catalyzed TLS both in vivo and in vitro. This review focuses on recent studies addressing the protein composition of active DNA polymerase V, and the role of RecA protein in activating this enzyme. Based on unforeseen properties of RecA*, we describe a new model for pol V-catalyzed SOS-induced mutagenesis.     

The σE stress response is required for stress‐induced mutation and amplification in Escherichia coli

Pathways of mutagenesis are induced in microbes under adverse conditions controlled by stress responses. Control of mutagenesis by stress responses may accelerate evolution specifically when cells are maladapted to their environments, i.e. are stressed. Stress‐induced mutagenesis in the Escherichia coli Lac assay occurs either by ‘point’ mutation or gene amplification. Point mutagenesis is associated with DNA double‐strand‐break (DSB) repair and requires DinB error‐prone DNA polymerase and the SOS DNA‐damage‐ and RpoS general‐stress responses. We report that the RpoE envelope‐protein‐stress response is also required. In a screen for mutagenesis‐defective mutants, we isolated a transposon insertion in the rpoE P2 promoter. The insertion prevents rpoE induction during stress, but leaves constitutive expression intact, and allows cell viability. rpoE insertion and suppressed null mutants display reduced point mutagenesis and maintenance of amplified DNA. Furthermore, σ^E acts independently of stress responses previously implicated: SOS/DinB and RpoS, and of σ³², which was postulated to affect mutagenesis. I‐SceI‐induced DSBs alleviated much of the rpoE phenotype, implying that σ^E promoted DSB formation. Thus, a third stress response and stress input regulate DSB‐repair‐associated stress‐induced mutagenesis. This provides the first report of mutagenesis promoted by σ^E, and implies that extracytoplasmic stressors may affect genome integrity and, potentially, the ability to evolve.