Do DNA repair systems affect N4-hydroxycytidine-induced mutagenesis?

It was tested whether mutations induced in E. coli by N4-hydroxycytidine (oh4Cyd): (i) undergo mutation frequency decline (MFD) when synthesis of protein is arrested, and (ii) are influenced by polA1, polA107 or xth mutations. It was also investigated whether oh4Cyd may provoke SOS response and prophage lambda induction. All these processes may involve the action of repair enzymes. It has been shown that none of these processes or repair enzymes affects oh4Cyd-induced mutagenesis.     

Duplication Mutation as an Sos Response in Escherichia Coli: Enhanced Duplication Formation by a Constitutively Activated Reca

The SOS response in Escherichia coli involves the induction of a multioperon regulatory system, which copes with the presence of DNA lesions that interfere with DNA replication. Induction depends on activation of the RecA protein to cleave the LexA repressor of SOS operons. In addition to inducible DNA repair, the SOS system produces a large increase in the frequency of point mutations. To examine the possibility that other types of mutations are induced as part of the SOS response, we have studied the production of tandem duplications. To avoid the complications of indirect effects of the DNA lesions, we have activated the SOS response by a constitutive mutation in the recA gene, recA730. The introduction of the recA730 mutation results in an increase in duplications in the range of tenfold or greater, as judged by two different criteria. Based on its genetic requirements, the pathway for induced duplication formation is distinct from the point mutation pathway and also differs from the major normal recombination pathway. The induction of pathways for both duplications and point mutations shows that the SOS system produces a broad mutagenic response. We have suggested previously that many types of mutations might be induced by severe environmental stress, thereby enhancing genetic variation in an endangered population.     

SOS-dependent replication past a single trans-syn T-T cyclobutane dimer gives a different mutation spectrum and increased error rate compared with replication past this lesion in uninduced cells.

We have transfected SOS-induced and uninduced cells of a uvrA6 strain of Escherichia coli with single-stranded M13mp7-based vectors that carried a single trans-syn T-T cyclobutane dimer at a unique site. Unlike constructs carrying the cis-syn isomer of this lesion, these vectors could be replicated with modest efficiency (14%) in the absence of SOS induction and therefore provided an opportunity to measure directly the influence of such induction on error rate and mutation spectrum. We found that translesion synthesis in the absence of SOS induction was remarkably accurate; only 4% of the replicated bacteriophage contained mutations, which were exclusively targeted single T deletions. In SOS-induced cells, error frequency increased to 11% and the resulting mutations included targeted substitutions and near-targeted single base additions, as well as the T deletions. Replication efficiency was 29% in these conditions. SOS induction therefore leads not only to an enhanced capacity to replicate damaged DNA but also to a marked change in mutation frequency and spectrum.     

UV-mutagenesis at a cloned target sequence: converted suppressor mutation is insensitive to mutation frequency decline regardless of the gene orientation

Premutational lesions produced by ultraviolet radiation in the Gln2 tRNA genes of E. coli B/r show differing sensitivities to a mutation avoidance phenomenon known as mutation frequency decline (MFD). A mutation event that changes the wild-type gene to an amber (UAG) suppressor is normally sensitive to MFD. Mutation of this amber suppressor to an ochre (UAA) suppressor is not sensitive to MFD. These two mutation events occur in the same anticodon region of the DNA. The dissimilarity of MFD sensitivity between these two mutations may result because the respective premutational photoproducts for the two are located in opposite strands of duplex DNA. To examine the effect of strand position of the premutational lesions on MFD, recombinant lambda phage were constructed that contained the amber suppressor as a mutation target in the two possible orientations. Comparison of MFD in bacterial lysogens containing either of the two types of recombinant prophage indicated that reversing the orientation of the target sequence relative to adjacent bacterial DNA had no effect on MFD. Since rotational inversion of the target sequence did not alter the sensitivity to MFD of mutation occurring at the cloned target gene, the antimutation process inherent to MFD can not be attributed to an asymmetrical interaction between the template strands and the DNA-replication complex.     

Involvement of DNA lesions and SOS functions in 5-bromouracil-induced mutagenesis

Mutagenesis resulting from incorporation of 5-bromouracil (BU) in the DNA of E. coli K12 proceeds largely (approximately 80%) via misrepair of the lesions resulting from incorporation of the analogue. The premutational lesions are due principally to dehalogenation of incorporated BU residues, leading to formation of uracil residues, and removal of these by uracil-DNA glycosylase with formation of apyrimidinic sites. In the xthA mutant, defective in AP endonuclease, there is a several-fold increase in the frequency of BU-induced mutations, underlining the importance of AP sites in BU-induced mutagenesis. Premutational lesions undergo mutation frequency decline (MFD), which is subject to delay in the xthA mutant, pointing to some role of AP endonuclease in MFD, and further supporting involvement of AP sites in BU-induced mutagenesis. Efficient BU mutagenesis is dependent on the functions of the genes recA and umuC and non-mutated lexA protein.     

Separation of the SOS-dependent and SOS-independent components of alkylating-agent mutagenesis.

Escherichia coli plasmids containing the rpsL+ gene (Strs phenotype) as the target for mutation were treated in vitro with N-methyl-N-nitrosourea. Following fixation of mutations in E. coli MM294A cells (recA+ Strs), an unselected population of mutant and wild-type plasmids was isolated and transferred into a second host, E. coli 6451 (recA Strr). Strains carrying plasmid-encoded forward mutations were then selected as Strr isolates, while rpsL+ plasmids conferred the dominant Strs phenotype in the second host. Mutation induction and reduced survival of N-methyl-N-nitrosourea-treated plasmids were shown to be dose dependent. Because this system permitted analysis and manipulation of the levels of certain methylated bases produced in vitro by N-methyl-N-nitrosourea, it afforded the opportunity to assess directly the relative roles of these bases and of SOS functions in mutagenesis. The methylated plasmid DNA gave a mutation frequency of 6 X 10(-5) (a 40-fold increase over background) in physiologically normal cells. When the same methylated plasmid was repaired in vitro by using purified O6-methylguanine DNA methyltransferase (to correct O6-methylguanine and O4-methylthymine), no mutations were detected above background levels. In contrast, when the methylated plasmid DNA was introduced into host cells induced by UV light for the SOS functions, rpsL mutagenesis was enhanced eightfold over the level seen without SOS induction. This enhancement of mutagenesis by SOS was unaffected by prior treatment of the DNA with O6-methylguanine DNA methyltransferase. These results demonstrate a predominant mutagenic role for alkylation lesions other than O6-methylguanine or O4-methylthymine when SOS functions are induced. The mutation spectrum of N-methyl-N-nitrosourea under conditions of induced SOS functions revealed a majority of mutagenic events at A . T base pairs.     

Modification of UV-induced mutation frequency and cell survival of Escherichia coli B/r WP2 trpE65 by treatment before irradiation

The UV radiation survival curve of exponentially growing cultures of Escherichia coli B/r WP2 trpE65 was modified by pretreatment for short incubation periods (up to 20 min) with chloramphenicol such that an extended exponential section of intermediate slope appeared between the shoulder and the final exponential slope. Surges of mutation to tryptophan independence occurred with each increase in slope of the survival curve. These surges were separated by extended sections of little mutation. Nalidixic acid prevented both the changes in survival and mutation. Mutation curves obtained with overnight cultures had three extended sections of little mutation alternating with sections of high mutation. Reincubation for 60 min in fresh medium reduced or eliminated the low-response sections. These reappeared after 80 to 90 min, when DNA had doubled in the culture and before the initial synchronous cell divisions had occurred. Nalidixic acid prevented this reappearance.     

Mutagenic DNA repair in escherichia coli. V. Mutation frequency decline and error-free post-replication repair in an excision-proficient strain.

Mutation frequency decline (MFD) is an irreversible loss of newly-induced suppressor mutations occurring in excision-proficient Escherichia coli during a short period of incubation in minimal medium before plating on broth- or Casamino acids-enriched selective agar. It is known that MFD of UV-induced mutations may occur before DNA containing pre-mutagenic lesions is replicated, but we conclude that MFD can also occur after the damaged DNA has been replicated on the basis of the following evidence. (1) Mutation fixation in rich medium (i.e., loss of susceptibility to mutation frequency decline) with ethyl methanesulphonate mutagenesis begins immediately, whereas with UV it is delayed for 20--30 min. (2) The delay in mutation fixation after UV can be explained neither by inhibition of DNA replication nor by a delay in the appearance of error-prone repair activity in the irradiated population. (3) MFD at later times after UV irradiation is more rapid and is less strongly inhibited by caffeine than is MFD immediately after irradiation. (4) Excision is virtually complete 20 min after 3 J m-2 UV but at that time virtually all mutations are still susceptible to MFD. We have presented evidence elsewhere that in bacteria there is an alternative error-free excision-dependent type of post-replication repair of potentially mutagenic daughter strand gaps. We suggest that this process is inhibited at tRNA loci in the presence of nutrient broth or Casamino acids, possibly because of a broth-dependent change in the structure of the single-stranded region including the tRNA locus.     

SOS and UVM pathways have lesion-specific additive and competing effects on mutation fixation at replication-blocking DNA lesions

Escherichia coli cells have multiple mutagenic pathways that are induced in response to environmental and physiological stimuli. Unlike the well-investigated classical SOS response, little is known about newly recognized pathways such as the UVM (UV modulation of mutagenesis) response. In this study, we compared the contributions of the SOS and UVM pathways on mutation fixation at two representative noninstructive DNA lesions: 3,N4-ethenocytosine (epsilonC) and abasic (AP) sites. Because both SOS and UVM responses are induced by DNA damage, and defined UVM-defective E. coli strains are not yet available, we first constructed strains in which expression of the SOS mutagenesis proteins UmuD' and UmuC (and also RecA in some cases) is uncoupled from DNA damage by being placed under the control of a heterologous lac-derived promoter. M13 single-stranded viral DNA bearing site-specific lesions was transfected into cells induced for the SOS or UVM pathway. Survival effects were determined from transfection efficiency, and mutation fixation at the lesion was analyzed by a quantitative multiplex sequence analysis procedure. Our results suggest that induction of the SOS pathway can independently elevate mutagenesis at both lesions, whereas the UVM pathway significantly elevates mutagenesis at epsilonC in an SOS-independent fashion and at AP sites in an SOS-dependent fashion. Although mutagenesis at epsilonC appears to be elevated by the induction of either the SOS or the UVM pathway, the mutational specificity profiles for epsilonC under SOS and UVM pathways are distinct. Interestingly, when both pathways are active, the UVM effect appears to predominate over the SOS effect on mutagenesis at epsilonC, but the total mutation frequency is significantly increased over that observed when each pathway is individually induced. These observations suggest that the UVM response affects mutagenesis not only at class 2 noninstructive lesions (epsilonC) but also at classical SOS-dependent (class 1) lesions such as AP sites. Our results add new layers of complexity to inducible mutagenic phenomena: DNA damage activates multiple pathways that have lesion-specific additive as well as suppressive effects on mutation fixation, and some of these pathways are not directly regulated by the SOS genetic network.     

Induction of presumed somatic gene mutations in mice by 2-naphthylamine

Plasmid pKM101, whose mucA and B genes endow cells with enhanced mutation frequency and enhanced resistance to far-ultraviolet radiation (FUV) (254 nm), had no influence on these properties when cells were damaged by near-ultraviolet radiation (NUV) (300-400 nm). Thus, NUV lesions did not lead to induction of SOS repair and subsequent expression of mucA and B genes on plasmid pKM101. Further, when cells were pre-irradiated with NUV and subsequently irradiated with FUV, there was a blockage of SOS repair, including the repair normally controlled by genes on pKM101.     

Chloramphenicol-promoted increase in resistance to UV damage in Escherichia coli B/r WP2 trpE65: development of the capacity for successful repair of otherwise mutagenic or lethal lesions in DNA

The ultraviolet radiation survival curve of exponentially growing cultures of Escherichia coli B/r WP2 trpE65 was modified by a short period (20 min) of chloramphenicol treatment before UV exposure, which produced an extended exponential section of intermediate slope between the shoulder and the final exponential slope. More prolonged incubation with chloramphenicol (up to 90 min) resulted in little further extension of the intermediate exponential slope, but caused a progressive expansion of the shoulder region. With each period of chloramphenicol pretreatment, a major surge of mutation to tryptophan independence always occurred after that UV fluence promoting the transition from the shoulder to the intermediate exponential slope of the survival curve, and another major surge occurred after that fluence promoting the transition from the intermediate exponential slope to the final exponential slope. A minor surge of mutation occurred after low fluences. The 3 surges in mutation and the increased slopes of the survival curve are ascribed to UV-inactivation of 3 qualitatively different DNA-repair systems, each with differentially increased resistances to UV caused by pretreatment by chloramphenicol.     

Differential repair of premutational UV-lesions at tRNA genes in E. coli.

Summary Ultraviolet radiation produces bacterial revertants that frequently are the result of suppressor mutation. When irradiated cells are incubated under conditions unfavorable for protein synthesis there may be a large decrease in the frequency of observed mutants (mutation frequency decline, or MFD). MFD occurs only in excision-proficient strains and is inhibited by inhibitors of pyrimidine dimer excision. It has therefore been interpreted as enhanced excision of some premutational lesions. Potential de novo UAG suppressor mutation is very susceptible to MFD. Potential conversion mutation, the conversion of a UAG to a UAA suppressor, is at least ten times less susceptible to MFD. A base pair transition at a GC target in a particular tRNA gene is suggested for both de novo suppressor mutation and for conversion mutation. We interpret these results as indicating differential repair of premutational UV photoproducts at two closely spaced sites in the same tRNA gene. The significant difference between these two types of mutation may be the orientation of this target base pair in double helical DNA. The C would be in the transcribed strand of DNA when a nucleic acid alteration produces de novo suppressor mutation. The C would be in the nontranscribed strand, two base pairs removed, when a mutagenic alteration produces suppressor conversion. A model involving facilitated incision by hybridization of the transcribed strand of DNA to its cognate tRNA, under conditions promoting MFD, is described to explain this differential repair.     

RecA protein of Escherichia coli has a third essential role in SOS mutator activity.

The DNA damage-inducible SOS response of Escherichia coli includes an error-prone translesion DNA replication activity responsible for SOS mutagenesis. In certain recA mutant strains, in which the SOS response is expressed constitutively, SOS mutagenesis is manifested as a mutator activity. Like UV mutagenesis, SOS mutator activity requires the products of the umuDC operon and depends on RecA protein for at least two essential activities: facilitating cleavage of LexA repressor to derepress SOS genes and processing UmuD protein to produce a fragment (UmuD') that is active in mutagenesis. To determine whether RecA has an additional role in SOS mutator activity, spontaneous mutability (tryptophan dependence to independence) was measured in a family of nine lexA-defective strains, each having a different recA allele, transformed or not with a plasmid that overproduces either UmuD' alone or both UmuD' and UmuC. The magnitude of SOS mutator activity in these strains, which require neither of the two known roles of RecA protein, was strongly dependent on the particular recA allele that was present. We conclude that UmuD'C does not determine the mutation rate independently of RecA and that RecA has a third essential role in SOS mutator activity.     

Study of MFD in Bacillus subtilis.

The frequency of UV-induced extragenic suppressor reversions to leucine independence in B. subtilis carrying a leu8 mutation decreased when irradiated cells were temporarily incubated in medium deprived of nitrogen sources. This mutation frequency decline (MFD) was inhibited by acriflavine and was poorly expressed in a uvr1 mutant. Consequently, MFD may be considered as the manifestation of an anti-mutagenic activity of excision repair. MFD was decelerated and even vanished in cells subjected to prolonged starvation of nitrogen sources before irradiation. MFD was accelerated in bacteria that were first irradiated and incubated in nutritional medium for at least 30 min. The stimulation of MFD by UV exposure was observed only in the uvr+ strain and depended on protein synthesis after irradiation. It is assumed that different rates of MFD in cells of various pre-radiation histories reflect different levels of the excision-repair activity inherent in these cells.     

Ultraviolet photoproducts at the ochre suppressor mutation site in the glnU gene of Escherichia coli: relevance to "mutation frequency decline"

Summary Ochre suppressor mutations induced by UV in the Escherichia coli glnU tRNA gene are CG to TA transitions at the first letter of the anticodon-encoding triplet, CAA. Premutational UV photoproducts at this site have long been known to exhibit an excision repair anomaly (mutation frequency decline or MFD), whereby post-irradiation inhibition of protein synthesis enhances their excision and reduces suppressor mutation yields ten-fold. We sought to clarify the basis of this unique repair response by determining the spectrum of UV photoproducts on both strands of a 36 by region of glnU which includes the anticodon-encoding triplet. We found that four different photolesions are produced within the 3 by sequence corresponding to the tRNA anticodon: (i) on the transcribed strand, TC (6–4) photoproducts and TC cyclobutane dimers are formed in equal numbers at the site of the C to T transition, indicating that this site is a hotspot for the usually less frequent (6–4) photoproduct; (ii) on the nontranscribed strand, TT dimers are found opposite the second and third letters of the anticodon-encoding triplet, adjacent to the mutation site; and (iii) on the nontranscribed strand, an alkali-sensitive lesion other than a (6–4) photoproduct is formed, apparently at the G in the mutation site. We suggest that mutation frequency decline may reflect excision repair activity at closely spaced UV lesions on opposite strands, resulting in double-strand breaks and the death of potential mutants.     

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.     

Carcinogen-induced mutation spectrum in wild-type, uvrA and umuC strains of Escherichia coli. Strain specificity and mutation-prone sequences

Forward mutations induced by the ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF) in the tetracycline resistance gene carried on plasmid pBR322 are shown to be dependent upon the induction of the host SOS functions in wild-type and umuC Escherichia coli cells. The mutation frequency in the umuC strain is equal to about 40% of the mutation frequency observed in the umu+ background. In the excision-repair-deficient uvrA mutant strain the mutagenic response is the same as in SOS-induced wild-type cells whether or not the uvrA bacteria are SOS-induced. Equal mutation frequencies are obtained in both the wild-type and the uvrA strains for equal modification levels although the survival of AAF-modified plasmid DNA is greatly reduced in the uvrA strain as compared to the wild-type strain. Sequence analysis of the mutations reveals that more than 90% of the N-Aco-AAF-induced mutations are frameshift mutations. Two types of mutational hotspots are observed occurring either at repetitive sequences or at non-repetitive sequences. Both types of mutants appear at similar locations and frequencies in both the wild-type and the uvrA strains. On the other hand, only the non-repetitive sequence mutants are obtained in the umuC background. These non-repetitive sequence mutants preferentially occur within the sequence 5' G-G-C-G-C-C 3' (the NarI restriction enzyme recognition sequence). The analysis of the -AAF binding spectrum to the same DNA fragment shows that there is no direct correlation between the modification spectrum and the mutation spectrum. We suggest that certain sequences are "mutation-prone" in the sense that only these sequences can be efficiently mutated as the result of an active processing mediated by specific proteins. When a sequence is said to be mutation-prone it probably corresponds to a particular structure that is induced within this sequence as a result of the binding to the DNA of the mutagen. This sequence-specific conformational change is the substrate for the protein(s) that fixes the mutation. The mutagenic processing pathway(s) is part of the cellular response to DNA-damaging agents (the so-called SOS response). Two pathways for frameshift mutagenesis are suggested by the data: an umuC-dependent pathway, which is involved in the mutagenic processing of lesions within repetitive sequences; an umuC-independent pathway responsible for the fixation of mutations within specific non-repetitive sequences.     

Mechanisms of mutagenesis by the vinyl chloride metabolite chloroacetaldehyde. Effect of gene-targeted in vitro adduction of M13 DNA on DNA template activity in vivo and in vitro

2-Chloroacetaldehyde (CAA), a metabolite of the carcinogenic industrial chemical vinyl chloride, reacts with single-stranded DNA to form the cyclic etheno lesions predominantly at adenine and cytosine. In both ethenoadenine and ethenocytosine, normal Watson-Crick hydrogen-bonding atoms are compromised. We have recently shown that CAA adduction leads to efficient mutagenesis in Escherichia coli predominantly at cytosines, and less efficiently at adenines. About 80% of the mutations at cytosines were C-to-T transitions, and the remainder were C-to-A transversions, a result similar to that of many noninstructional DNA lesions opposite which adenine residues are preferentially incorporated. It is widely believed that noninstructional lesions stop replication and depend on SOS functions for efficient mutagenesis. We have examined the effects of in vitro CAA adduction of the lacZ alpha gene of phage M13AB28 on in vivo mutagenesis in SOS-(UV)-induced E. coli. CAA adduction was specifically directed to a part of the lacZ sequence within M13 replicative form DNA by a simple experimental strategy, and the DNA was transfected into appropriate unirradiated or UV-irradiated cells. Mutant progeny were defined by DNA sequencing. In parallel in vitro experiments, the effects of CAA adduction on DNA replication by E. coli DNA polymerase I large (Klenow) fragment were examined. Our data do not suggest a strong SOS dependence for mutagenesis at cytosine lesions. While adenine lesions remain much less mutagenic than cytosine lesions, mutation frequency at adenines is increased by SOS. SOS induction does not significantly alter the specificity of base changes at cytosines or adenines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of SOS-induced Pol II, Pol IV, and Pol V DNA polymerases on UV-induced mutagenesis and MFD repair in Escherichia coli cells

Irradiation of organisms with UV light produces genotoxic and mutagenic lesions in DNA. Replication through these lesions (translesion DNA synthesis, TSL) in Escherichia coli requires polymerase V (Pol V) and polymerase III (Pol III) holoenzyme. However, some evidence indicates that in the absence of Pol V, and with Pol III inactivated in its proofreading activity by the mutD5 mutation, efficient TSL takes place. The aim of this work was to estimate the involvement of SOS-inducible DNA polymerases, Pol II, Pol IV and Pol V, in UV mutagenesis and in mutation frequency decline (MFD), a mechanism of repair of UV-induced damage to DNA under conditions of arrested protein synthesis. Using the argE3-->Arg(+) reversion to prototrophy system in E. coli AB1157, we found that the umuDC-encoded Pol V is the only SOS-inducible polymerase required for UV mutagenesis, since in its absence the level of Arg(+) revertants is extremely low and independent of Pol II and/or Pol IV. The low level of UV-induced Arg(+) revertants observed in the AB1157mutD5DumuDC strain indicates that under conditions of disturbed proofreading activity of Pol III and lack of Pol V, UV-induced lesions are bypassed without inducing mutations. The presented results also indicate that Pol V may provide substrates for MFD repair; moreover, we suggest that only those DNA lesions which result from umuDC-directed UV mutagenesis are subject to MFD repair.