Improvement in the sensitivity of DNA polymerase I-deficient Escherichia coli for detecting mutagens and carcinogens.

The sensitivity of a polA strain to the antibacterial activity of mutagens and carcinogens may be increased by inserting one or both of the following characteristics, a lexA mutation or the R391 bacterial plasmid. The effects of the lexA mutation and the plasmid appear to be additive. The differential sensitivity of a polA lexA (R391) strain could be adapted as a preliminary screening test for mutagens and potential carcinogens.     

The effect of plasmid R391 and other IncJ plasmids on the survival of Escherichia coli after UV irradiation

The presence of the IncJ plasmids R391, R997, R705, R706, R748 and R749 was shown to sensitize Escherichia coli AB1157 and both its uvrA and lexA derivatives to UV irradiation. No alteration in post-irradiation survival was observed in a recA mutant containing these plasmids, compared with the non-plasmid-containing recA strain. Analysis of recombination frequency in Hfr crosses to recA+ cells containing plasmid R391 indicated a reduction in recombination frequency compared with that obtained in similar crosses to a non-plasmid-containing strain. This effect was not due to plasmid-encoded restriction or entry exclusion systems and therefore must be considered as a real block in recombination. When cells containing plasmid R391 were irradiated and allowed to photoreactivate, an increase in survival was observed which was comparable to that observed in the non-plasmid-containing derivative. This indicated that post-irradiation processing of UV-induced damage, or lack of such processing, by mechanisms other than photoreactivation was responsible for the UV sensitivity associated with plasmid R391.     

The Mechanism of Reca Pola Lethality: Suppression by Reca-Independent Recombination Repair Activated by the Lexa(def) Mutation in Escherichia Coli

The mechanism of recA polA lethality in Escherichia coli has been studied. Complementation tests have indicated that both the 5' -> 3' exonuclease and the polymerization activities of DNA polymerase I are essential for viability in the absence of RecA protein, whereas the viability and DNA replication of DNA polymerase I-defective cells depend on the recombinase activity of RecA. An alkaline sucrose gradient sedimentation analysis has indicated that RecA has only a minor role in Okazaki fragment processing. Double-strand break repair is proposed for the major role of RecA in the absence of DNA polymerase I. The lexA(Def)::Tn5 mutation has previously been shown to suppress the temperature-sensitive growth of recA200(Ts) polA25::spc mutants. The lexA(Def) mutation can alleviate impaired DNA synthesis in the recA200(Ts) polA25::spc mutant cells at the restrictive temperature. recF(+) is essential for this suppression pathway. recJ and recQ mutations have minor but significant adverse effects on the suppression. The recA200(Ts) allele in the recA200(Ts) polA25::spc lexA(Def) mutant can be replaced by δrecA, indicating that the lexA(Def)-induced suppression is RecA independent. lexA(Def) reduces the sensitivity of δrecA polA25::spc cells to UV damage by ~10(4)-fold. lexA(Def) also restores P1 transduction proficiency to the δrecA polA25::spc mutant to a level that is 7.3% of the recA(+) wild type. These results suggest that lexA(Def) activates a RecA-independent, RecF-dependent recombination repair pathway that suppresses the defect in DNA replication in recA polA double mutants.     

DNA polymerase I in constitutive stable DNA replication in Escherichia coli.

We examined the effects of mutations in the polA (encoding DNA polymerase I) and polB (DNA polymerase II) genes on inducible and constitutive stable DNA replication (iSDR and cSDR, respectively), the two alternative DNA replication systems of Escherichia coli. The polA25::miniTn10spc mutation severely inactivated cSDR, whereas polA1 mutants exhibited a significant extent of cSDR. cSDR required both the polymerase and 5'-->3' exonuclease activities of DNA polymerase I. A similar requirement for both activities was found in replication of the pBR322 plasmid in vivo. DNA polymerase II was required neither for cSDR nor for iSDR. In addition, we found that the lethal combination of an rnhA (RNase HI) and a polA mutation could be suppressed by the lexA(Def) mutation.     

Effect of host lex, recA, recF, and uvrD genotypes on the ultraviolet light-protecting and related properties of plasmid R46 in Escherichia coli.

The ability of plasmid R46 to reduce the lethal but enhance the mutagenic effect of ultraviolet (UV) irradiation was tested in sets of Escherichia coli K-12 derivatives, wild type or with different mutations affecting DNA repair capacity, but otherwise isogenic. UV protection and enhancement of UV mutagenic effect were obtained in uvrA6, uvrB5, uvrD3, and recF143 hosts, but not in a recA56 strain. The plasmid gave some UV protection in two lexA1 and two lexA101 strains and in one lexA102 host, but produced no such effect in another lexA102 host. The plasmid restored UV mutagenic effect in a lexB30 strain, the yield of induced mutants per survivor of irradiation (10 J/m2) being about the same for the lexB30(R46) and lex+(R46) strains; by contrast the plasmid, though it reduced the UV sensitivity of the lexB30 strain, did not make it as UV-resistant as the lex+ R-strain.     

Characterization of an Escherichia coli mutant (radB101) sensitive to gamma and uv radiation, and methyl methanesulfonate

After N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis of Escherichia coli K-12 (xthA14), and X-ray-sensitive mutant was isolated. This sensitivity is due to a mutation, radB101, which is located at 56.5 min on the E. coli K-12 linkage map. The radB101 mutation sensitized wildtype cells to gamma and uv radiation, and to methyl methanesulfonate. When known DNA repair-deficient mutants were ranked for their gamma-radiation sensitivity relative to their uv-radiation sensitivity, their order was (starting with the most selectively gamma-radiation-sensitive strain): recB21, radB101, wild type, polA1, recF143, lexA101, recA56, uvrD3, and uvrA6. The radB mutant was normal for gamma- and uv-radiation mutagenesis, it showed only a slight enhancement of gamma- and uv-radiation-induced DNA degradation, and it was approximately 60% deficient in recombination ability. The radB gene is suggested to play a role in the recA gene-dependent (Type III) repair of DNA single-strand breaks after gamma irradiation and in postreplication repair after uv irradiation for the following reasons; the radB strain was normal for the host-cell reactivation of gamma- and uv-irradiated bacteriophage lambda; the radB mutation did not sensitize a recA strain, but did sensitize a polA strain to gamma and uv radiation; the radB mutation sensitized a uvrB strain to uv radiation.     

Frameshift mutagenesis by nitracrine analogues in wild-type, uvrB, polA and recA strains of Salmonella typhimurium, with and without plasmid pKM101

The mutagenic potential of 9-[(3-dimethylaminopropyl)amino]-acridine and its 1-, 2-, 3- and 4-nitro derivatives was studied in several strains of Salmonella typhimurium carrying the frameshift marker hisC3076. The strains all carried deep rough (rfa) mutations, and were either wild-type with respect to DNA repair capacity or carried recA, uvrB, polA1 or polA3 (amber) mutations. Derivatives with and without plasmid pKM101 were also studied. The des-nitro compound resembled 9 aminoacridine and other simple intercalating compounds. Both toxicity and mutagenesis were apparently unaffected by the uvrB and recA mutations or by the presence of plasmid pKM101. However, mutagenicity was reduced by the polA1 mutation, and virtually eliminated by the polA3 mutation. The drug was substantially more toxic in the latter, slightly more toxic in the former, of these polA- strains. Plasmid pKM101 enhanced mutagenesis and protected from toxicity in both polA1- and polA3- strains, although it did not restore either of these parameters to the level in the wild-type strain. The 2-nitro compound was generally similar to the des-nitro compound, except that it was considerably more toxic and apparently non-mutagenic in the recA-bearing strain. By contrast, mutagenicity of the 3- and 4-nitro compounds was enhanced by the uvrB mutation and by the presence of the plasmid. These compounds were highly toxic but non-mutagenic in the recA- strain, and showed some increased toxicity in polA1- and polA3- strains. The 1-nitro compound has been previously found to cross-link DNA. Unlike well-characterised cross-linkers such as mitomycin C it was highly mutagenic in the uvrB- strain, and this mutagenesis was enhanced by plasmid pKM101, but eliminated by the recA mutation. At high doses, where the drug was completely toxic towards uvrB- or recA-carrying strains, it became mutagenic in the DNA-repair-proficient strains. This 'high-dose' mutagenesis was enhanced by plasmid pKM101, but reduced by the polA1 mutation and almost eliminated by the polA3 mutation. Although there are several possible interpretations of these data, they are compatible with the suggestion that the lesion induced by high doses (but not by low doses) of nitracrine is a cross-link, but that this is not the major mutagenic lesion.     

Role of DNA polymerase I in postreplication repair: a reexamination with Escherichia coli delta polA.

Using strains of Escherichia coli K-12 that are deleted for the polA gene, we have reexamined the role of DNA polymerase I (encoded by polA) in postreplication repair after UV irradiation. The polA deletion (in contrast to the polA1 mutation) made uvrA cells very sensitive to UV radiation; the UV radiation sensitivity of a uvrA delta polA strain was about the same as that of a uvrA recF strain, a strain known to be grossly deficient in postreplication repair. The delta polA mutation interacted synergistically with a recF mutation in UV radiation sensitization, suggesting that the polA gene functions in pathways of postreplication repair that are largely independent of the recF gene. When compared to a uvrA strain, a uvrA delta polA strain was deficient in the repair of DNA daughter strand gaps, but not as deficient as a uvrA recF strain. Introduction of the delta polA mutation into uvrA recF cells made them deficient in the repair of DNA double-strand breaks after UV irradiation. The UV radiation sensitivity of a uvrA polA546(Ts) strain (defective in the 5'----3' exonuclease of DNA polymerase I) determined at the restrictive temperature was very close to that of a uvrA delta polA strain. These results suggest a major role for the 5'----3' exonuclease activity of DNA polymerase I in postreplication repair, in the repair of both DNA daughter strand gaps and double-strand breaks.     

Influence of a uvrD mutation on survival and repair of X-irradiated Escherichia coli K-12 cells.

The presence of a uvrD mutation increased the X-ray sensitivities of E. coli wild-type and polA strains, but had no effect on the sensitivities of recA and recB strains, and little effect on a lexA strain. Incubation of irradiated cells in medium containing 2,4-dinitrophenol or chloramphenicol decreased the survival of wild-type and uvrD cells, but had no effect on the survival of recA, recB and lexA strains. Alkaline sucrose gradient sedimentation studies indicated that the uvrD strain is deficient in the growth-medium-dependent (Type III) repair of DNA single-strand breaks. These results indicate that the uvrD mutation inhibits certain rec+lex+-dependent repair processes, including the growth-medium-dependent (Type III) repair of X-ray-induced DNA single-strand breaks, but does not inhibit other rec+lex+-dependent processes that are sensitive to 2,4-dinitrophenol and chloramphenicol.     

Genetic Analysis of a Temperature-Resistant Revertant of the Conditional Lethal Escherichia coli Double Mutant polA12 uvrE502

Conditional lethality of the Escherichia coli polA12 uvrE502 double mutant may be overcome by a mutation that has been termed polA350. The polA350 mutation restored the polymerizing activity of deoxyribonucleic acid polymerase I at 42 C in the polA12 mutant and partially suppressed ultraviolet (UV) and methylmethane sulfonate sensitivities of the polA12. Mapping experiments have located polA350 between metE and polA12, very close to the latter. The strain carrying polA12 polA350 and recB21 was viable at 42 C. The effects of the recB21 and polA12 polA350 combination on the UV sensitivity were additive. The triple mutant polA12 polA350 uvrE502 was more UV sensitive than the single uvrE502 mutant.     

Characterization of polVR391: a Y-family polymerase encoded by rumA'B from the IncJ conjugative transposon, R391

Although best characterized for their ability to traverse a variety of DNA lesions, Y-family DNA polymerases can also give rise to elevated spontaneous mutation rates if they are allowed to replicate undamaged DNA. One such enzyme that promotes high levels of spontaneous mutagenesis in Escherichia coli is polV(R391), a polV-like Y-family polymerase encoded by rumA'B from the IncJ conjugative transposon R391. When expressed in a DeltaumuDC lexA(Def) recA730 strain, polV(R391) promotes higher levels of spontaneous mutagenesis than the related MucA'B (polR1) or UmuD'C (polV) polymerases respectively. Analysis of the spectrum of polV(R391)-dependent mutations in rpoB revealed a unique genetic fingerprint that is typified by an increase in C:G-->A:T and A:T-->T:A transversions at certain mutagenic hot spots. Biochemical characterization of polV(R391) highlights the exceptional ability of the enzyme to misincorporate T opposite C and T in sequence contexts corresponding to mutagenic hot spots. Purified polV(R391) can also bypass a T-T pyrimidine dimer efficiently and displays greater accuracy opposite the 3'T of the dimer than opposite an undamaged T. Our study therefore provides evidence for the molecular basis for the enhanced spontaneous mutator activity of RumA'B, as well as explains its ability to promote efficient and accurate bypass of T-T pyrimidine dimers in vivo.     

Spontaneous and UV-induced mutations in Escherichia coli K-12 strains with altered or absent DNA polymerase I.

The induction of mutations to valine resistance and to rifampin resistance occurs after UV irradiation in bacteria carrying a deletion through the polA gene (delta polA), showing that DNA polymerase I (PolI) is not an essential enzyme for this process. The PolI deletion strain showed a 7- to 10-fold-higher spontaneous mutation frequency than the wild type. The presence in the deletion strain of the 5'----3' exonuclease fragment on an F' episome caused an additional 10-fold increase in spontaneous mutation frequency, resulting in mutation frequencies on the order of 50- to 100-fold greater than wild type. The mutator effect associated with the 5'----3' exonuclease gene fragment together with much of the effect attributable to the polA deletion was blocked in bacteria carrying a umuC mutation. The mutator activity therefore appears to reflect constitutive SOS induction. Excision-proficient polA deletion strains exhibited increased sensitivity to the lethal effect of UV light which was only partially ameliorated by the presence of polA+ on an F' episome. The UV-induced mutation rate to rifampin resistance was marginally lower in delta polA bacteria than in bacteria carrying the polA+ allele. This effect is unlikely to be caused by the existence of a PolI-dependent mutagenic pathway and is probably an indirect effect caused by an alteration in the pattern of excision repair, since it did not occur in excision-deficient (uvrA) bacteria. An excision-deficient polA deletion strain possessed UV sensitivity similar to that of an isogenic strain carrying polA+ on an F' episome, showing that none of the functions of PolI are needed for postreplication repair in the absence of excision repair. Our data provide no evidence for a pathway of UV mutagenesis dependent on PolI, although it remains an open question whether PolI is able to participate when it is present.     

Frameshift mutagenesis by acridines in wild-type, uvrB and polA strains of Salmonella typhimurium with and without plasmid pKM101

A large range of acridines, including several anilinoacridines which are active as antitumour agents, have been studied for their ability to revert derivatives of Salmonella typhimurium strains carrying the frameshift marker hisC3076. The strains used all carried deep-rough (rfa) mutations, and were either wild-type with respect to DNA-repair capacity or carried uvrB, polA1 or polA3 (amber) mutations. Derivatives with and without the mutation-enhancing N group plasmid pKM101 were also used. 9-Aminoacridine and other acridines appeared similar to the anilinoacridines for the most part, in that frameshift mutagenesis and toxicity appeared to be unaffected by the uvrB mutation or by the presence of plasmid pKM101. Exceptions were ICR191, 3-NO2-acridine and 1- or 3-NO2-anilinoacridine derivatives in which mutagenesis was increased in uvrB strains and also when pKM101 was present. These compounds were slightly more toxic in the uvrB background, but less toxic when pKM101 was present in either the uvrB or wild-type backgrounds. Mutagenesis by most compounds was reduced by the polA1 mutation and virtually eliminated (except in the case of ICR191) by the polA3 mutation. Plasmid pKM101 occasionally enhanced mutagenesis in the polA1 strain, whereas in the polA3 it appeared to have no effect whatsoever. Again, there were no obvious differences in toxicity between Pol+ and Pol- strains.     

Isolation of recombinant plasmids and phage carrying the lexA gene of Escherichia coli K-12

The lexA gene of Escherichia coli K-12 was cloned from the plasmid pLC44-14 into pBR322. Plasmids carrying lexA+ were selected by their ability to complement a recessive tsl mutation, which is believed to be a mutation in lexA. The smallest lexA+ recombinant plasmid, pJL21, contained an EcoRI-PstI fragment 2.9 kilobases (kb) in length; two larger plasmids also contained this fragment, and genetic material to one or both sides of the EcoRI-PstI fragment. Plasmids homologous to pJL21, but carrying a dominant mutation, lexA3, or one of three recessive amber mutations in lexA, termed spr, were also isolated. To clone the EcoRI-PstI fragment onto a lambda vector, the PstI end was first converted to an EcoRI end by attachment of a 100-base pair PstI-EcoRI fragment isolated from the plasmid ColE1; the resultant EcoRI fragment was then cloned into the lambda vector lambda gt4. A restriction map of pLC44-14 was obtained for nine restriction enzymes. The orientation of this map was determined relative to the E. coli genetic map by complementation of the gene ubiA+ and by comparison with restriction enzyme digests of another plasmid, pLC11-9, which carries dnaB, a gene closely linked to lexA, but does not carry lexA.     

Revised methods for the Salmonella mutagenicity test

The methods for detecting carcinogens and mutagens with the Salmonella mutagenicity test were described previously (Ames et al., 1975b). The present paper is a revision of the methods. Two new tester strains, a frameshift strain (TA97) and a strain carrying an ochre mutation on a multicopy plasmid (TA102), are added to the standard tester set. TA97 replaces TA1537. TA1535 and TA1538 are removed from the recommended set but can be retained at the option of the investigator. TA98 and TA100 are retained. We discuss other special purpose strains and present some minor changes in procedure, principally in the growth, storage, and preservation of the tester strains. Two substitutions are made in diagnostic mutagens to eliminate MNNG and 9-aminoacridine. Some test modifications are discussed.     

Trimethoprim-induced DNA polymerase I deficiency in Escherichia coli K-12

Curing of the mini-ColE1 plasmid pML21 was observed among cells of Escherichia coli K-12 strain C600(pML21) grown under subinhibitory conditions in the presence of trimethoprim, a specific inhibitor of dihydrofolate reductase. Some of the cured colonies showed (i) a reduction in frequency of transformation with pML21 compared with those of isogenic strains not treated with trimethoprim, (ii) loss of viability after acquisition of a recA mutation, and (iii) UV sensitivity greater than that of the original isogenic strain. These colonies therefore had PolA- phenotypes. Moreover, they were found to be deficient in DNA polymerase I activity in the in vitro assays, indicating the occurrence of a polA mutation in them. Many of the colonies with PolA- phenotypes were also thyA deoC mutants, and these mutations, in addition to the polA mutations, appeared to be involved in the expression of the PolA- phenotypes.     

Mutagenic action of nitrous acid on prophage lambda

The lethal and mutagenic effects of nitrous acid (0,1 M NaNO2 in 0,1 M acetate buffer, pH 4.6) on prophage lambda cI857 ind- were studied in the wild-type cells of Escherichia coli and in 9 repair-deficient mutants: uvrA6, uvrA6 umuC36, uvrD3, uvrE502, polA1, recA13, lexA102, recF143 and xthA9. After treatment with HNO2, the prophage was heat-induced either immediately or after 90 min incubation in broth at 32 degrees C. The prophage survival after delayed induction was considerably higher than after immediate induction. The lethal action of HNO2 was highly expressed in uvrA- and uvrE- lysogens after delayed induction. The frequency of temperature-independent c mutants forming clear plaques at 32 degrees C reached 4% in the wild-type host after immediate induction, this value being 10-15% in uvrA, uvrA umuC, uvrD, uvrE, polA and xthA mutants, 0,8% in recF- lysogen and only 0,2-0,3% in recA and lexA mutants. Under these conditions, about 90% of c mutants are generated by recA+, lexA+-dependent repair mechanism (most probably, due to W-mutagenesis). After delayed induction, mutation frequency in the wild-type host declines considerably (down to 0,1%). Analogous phenomenon of mutation frequency decline was registered in uvrA, xthA, recF, polA, uvrE and uvrD lysogens. Under conditions of delayed induction, the frequency of HNO2-induced c mutations only slightly depends on the recA+ and lexA+ gene products and mutations are, apparently, fixed by replication.     

A multicopy phr-plasmid increases the ultraviolet resistance of a recA strain of Escherichia coli

It has been previously reported that the ultraviolet sensitivity of recA strains of Escherichia coli in the dark is suppressed by a plasmid pKY1 which carries the phr gene, suggesting that this is due to a novel effect of photoreactivating enzyme (PRE) of E. coli in the dark (Yamamoto et al., 1983a). In this work, we observed that an increase of UV-resistance by pKY1 in the dark is not apparent in strains with a mutation in either uvrA, uvrB, uvrC, lexA, recBC or recF. The sensitivity of recA lexA and recA recBC multiple mutants to UV is suppressed by the plasmid but that of recA uvrA, recA uvrB and recA uvrC is not. Host-cell reactivation of UV-irradiated lambda phage is slightly more efficient in the recA/pKY1 strain compared with the parental recA strain. On the other hand, the recA and recA/pKY1 strains do not differ significantly in the following properties: Hfr recombination, induction of lambda by UV, and mutagenesis. We suggest that dark repair of PRE is correlated with its capacity of excision repair.     

Mutational and recombinational events in carcinogen-modified plasmid DNA. Influence of host-cell repair genes

A plasmid containing the STR operon has been modified in vitro (i) by irradiation with UV light, (ii) by reaction with ethyl methanesulphonate (EMS), (iii) by reaction with N-acetoxy-2-acetylaminofluorene (AcO-AAF), (iv) by reaction with (+/-)trans-benzo[a]pyrene-7, 8-dihydrodiol-9,10-epoxide (BPDE), and (v) by heating at 70 degrees C to produce apurinic sites. Suitably modified plasmid DNA was then used to transform both repair-proficient and repair-deficient strains of Escherichia coli, and the mutation frequency in the plasmid-encoded rspL+ gene measured. The influence of host mutations in the uvrB+, recA+, umuC+ and lexA+, genes on the mutation frequency have been investigated. Transformation into a uvrB strain significantly decreased survival and increased the level of mutations observed for UV- and AcO-AAF-modified plasmid DNA, while only a small increase in mutation frequency was seen with EMS-modified DNA and no increase in mutation frequency with plasmid DNA containing apurinic sites. Mutagenesis in UV- and BPDE-modified DNA (and probably also DNA containing apurinic sites) was totally dependent on he recA+ gene product, while EMS and AcO-AAF induced mutagenesis was only partially independent on the recA+ gene. Transformation of UV- or BPDE-modified DNA into a umuC or lexA strain, on the other hand, showed no change in mutation frequency from that observed with wild-type strain. Pre-irradiation of the wild-type host with UV light before transformation led to a significant increase in mutation frequency for UV- and BPDE-modified plasmid DNA. These results are discussed in terms of mutational or recombinational pathways which may be available to act on modified plasmid DNA, and suggest that the majority of the mutational events measured in this system are due to recombination between homologous regions on the plasmid and chromosomal DNA.     

Analysis of pCU1 replication origins: dependence of oriS on the plasmid-encoded replication initiation protein RepA

The broad-host-range replicon of the plasmid pCU1 has three origins of vegetative replication called oriB, oriS, and oriV. In the multi-origin replicon, individual origins can distinguish among replication factors provided by the host. It has been found that during replication in Escherichia coli polA(-) host, oriS was the only active origin of a mutant pCU1 derivative bearing a mutation in the gene encoding replication initiation protein RepA. To further investigate the capacity of oriS to function in an E. coli polA(-) host we constructed a number of clones of the basic replicon of pCU1 containing oriS as the only replication origin. An oriS construct created with pUC18 could transform the polA(-) strain when RepA was supplied in trans. When the oriS region (between nucleotides 290 and 832) was ligated to an antibiotic resistance Omega fragment, the construct could be recovered as a plasmid from polA(+) strain if functional RepA was provided in trans. Our results therefore indicate that the basic replicon of pCU1, containing oriS as the sole origin, does require RepA to initiate plasmid replication in E. coli