Characterization of a new radiation-sensitive mutant, Escherichia coli K-12 radC102

A new radiation-sensitive mutant, radC , has been isolated. The radC gene is located at 81.0 min on the Escherichia coli K-12 linkage map. The radC mutation sensitized cells to uv radiation, but unlike most DNA repair mutations, sensitization to X rays was observed only for rich medium-grown cells. For cells grown in rich medium, the radC mutant was normal for gamma-radiation mutagenesis, but showed less uv-radiation mutagenesis than the wild-type strain; it showed normal amounts of X- and uv-radiation-induced DNA degradation, and it was approximately 60% deficient in recombination ability. The radC strain was normal for host cell reactivation of gamma-and uv-irradiated bacteriophage lambda; the radC mutation did not sensitize a recA strain, but did sensitize a radA and a polA strain to X and uv radiation and a uvrA strain to uv radiation. Therefore, we suggest that the radC gene product plays a role in the growth medium-dependent, recA gene-dependent repair of DNA single-strand breaks after X irradiation, and in postreplication repair after uv irradiation.     

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.     

Toxicity of organic acids for repair-deficient strains of Escherichia coli

The wild-type strain and four DNA repair-deficient strains (uvrA6, uvrB5, recA56, and polA1) of Escherichia coli K-12 were treated with acetic acid, lactic acid, and p-aminobenzoic acid at pH 3.5 during their stationary phase of growth. All three acids were highly toxic to the polymerase-deficient strain. The greater sensitivity of the strain carrying the polA1 gene than its isogenic pol+ derivatives suggested that damage caused by acidity requires polA+ gene products for repair.     

Genetic and phenotypic analyses indicating occurrence of the recN262 and radB101 mutations at the same locus in Escherichia coli.

The radB101 and recN262 mutations showed essentially identical phenotypes when compared in isogenic Escherichia coli strains for their effects on gamma and UV radiation survival and on conjugal recombination in a uvrA recB recC sbcB sbcC strain. Complementation tests involving attempts to reconstitute a radB+ recN+ strain by transductions between radB101 and recN262 donors and recipients, and tests involving plasmids carrying recN+ and recN::Tn1000 inserts, indicated that the radB and recN genes are identical. We suggest that the radB101 mutation now be referred to as recN2001.     

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 the radB gene in postreplication repair in UV-irradiated Escherichia coli uvrB

In UV-irradiated Escherichia coli, the radB101 mutation sensitized uvrB recF cells 4-fold and uvrB recB cells 1.2-fold, but did not sensitize uvrB recB recF cells. The radB mutation had very little effect (1.2-fold or less) on the repair of UV radiation-induced DNA daughter-strand gaps in uvrB cells, but it did cause about a 3-fold deficiency in the repair of the DNA double-strand breaks that arise in association with nonrepaired daughter-strand gaps in UV-irradiated uvrB recF cells. Thus, the radB gene does not appear to be involved in the recF-dependent or recF recB-independent processes for the repair of DNA daughter-strand gaps, but is involved in the recB-dependent postreplication repair of DNA double-strand breaks.     

Role of constitutive and inducible repair in radiation resistance of Escherichia coli

Radiation resistance of Escherichia coil cells depends on how efficiently DNA is recovered after damage, which is determined by the function of constitutive and inducible repair branches. The effects of additional mutations of the key genes of constitutive and inducible repair (recA, lexA, recB, polA, lig, gyr, recE, recO, recR, recJ, recQ, uvrD, helD, recN, and ruv) on radiation resistance were studied in E. coli K-12 strain AB 1157 and highly radiation-resistant isogenic strain Gam(r)444. An optimal balance ensuring a high gamma resistance of the Gam(r)444 radiation-resistant E. coli mutant was due to expression of the key SOS repair genes (recA, lexA, recN, and ruv) and activation of the presynaptic functions of the RecF homologous recombination pathway as a result of a possible mutation of the uvrD gene, which codes for repair helicase II.     

Conditional Lethality of recA and recB Derivatives of a Strain of Escherichia coli K-12 with a Temperature-Sensitive Deoxyribonucleic Acid Polymerase I

We have isolated a strain of Escherichia coli K-12 carrying a mutation, polA12, that results in the synthesis of a temperature-sensitive deoxyribonucleic acid (DNA) polymerase I. The double mutants polA12 recA56 and polA12 recB21, constructed at 30 C, are inviable at 42 C. About 90% of the cells of both double mutants die after 2 hr of incubation at 42 C. Both double mutants filament at 42 C and show a dependence on high cell density for growth at 30 C. In polA12 recB21 cells at 42 C, DNA and protein synthesis gradually stop in parallel. In polA12 recA56 cells, DNA synthesis continues for at least 1 hr at 42 C, and there is extensive DNA degradation. The results suggest that the primary lesion in these double mutants is not in DNA replication per se.     

Toxicity of organic acids for repair-deficient strains of Escherichia coli.

The wild-type strain and four DNA repair-deficient strains (uvrA6, uvrB5, recA56, and polA1) of Escherichia coli K-12 were treated with acetic acid, lactic acid, and p-aminobenzoic acid at pH 3.5 during their stationary phase of growth. All three acids were highly toxic to the polymerase-deficient strain. The greater sensitivity of the strain carrying the polA1 gene than its isogenic pol+ derivatives suggested that damage caused by acidity requires polA+ gene products for repair.     

New mutation (mmrA1) in Escherichia coli K-12 that affects minimal medium recovery and postreplication repair after UV irradiation

After UV irradiation, Escherichia coli uvrA mutant cells show higher survival on minimal than on rich growth medium, i.e., they show minimal-medium recovery. This effect of rich growth medium on survival is not observed in a uvrA mutant carrying an mmrA1 mutation, and the uvrA mmrA strain showed the same survival rate on minimal and rich growth media as the uvrA strain did on minimal medium plates. The mmrA1 mutation was isolated as a hidden mutation from a uvrA polA mutant strain and shown to map at 84.3 min on the E. coli K-12 linkage map. In contrast to the uvrA strain, the repair of DNA daughter strand gaps was not inhibited in the uvrA mmrA strain by rich growth medium after irradiation. However, the uvrA and uvrA mmrA strains were similar in their ability to repair DNA when compared in minimal medium. These data are consistent with the idea that the mmr gene product is not involved directly in the repair of UV radiation-induced DNA damage, but rather allows rich growth medium to inhibit a portion of postreplication repair.     

Conditional lethality of the recA441 and recA730 mutants of Escherichia coli deficient in DNA polymerase I

E. coli strains bearing the recA441 mutation and various mutations in the polA gene resulting in enzymatically well-defined deficiencies of DNA polymerase I have been constructed. It was found that the recA441 strains bearing either the polA1 or polA12 mutation causing deficiency of the polymerase activity of pol I are unable to grow at 42 degrees C on minimal medium supplemented with adenine, i.e., when the SOS response is continuously induced in strains bearing the recA441 mutation. Under these conditions the inhibition of DNA synthesis is followed in recA441 polA12 by DNA degradation and loss of cell viability. A similar lethal effect is observed with the recA730 polA12 mutant. The recA441 strain bearing the polA107 mutation resulting in the deficiency of the 5'-3' exonuclease activity of pol I shows normal growth under conditions of continuous SOS response. We postulate that constitutive expression of the SOS response leads to an altered requirement for the polymerase activity of pol I.     

Synergistic killing of Escherichia coli by near-UV radiation and hydrogen peroxide: distinction between recA-repairable and recA-nonrepairable damage.

Wild-type cells and six DNA repair-deficient mutants (lexA, recA, recB, recA, recB, polA1, and uvrA) of Escherichia coli K-12 were treated with near-ultraviolet radiation plus hydrogen peroxide (H2O2). At low H2O2 concentrations (6 X 10(-6) to 6 X 10(-4) M), synergistic killing occurred in all strains except those containing a mutation in recA. This RecA-repairable damage was absent from stationary-phase cells but increased in logarithmic cells as a function of growth rate. At higher H2O2 concentrations (above 6 X 10(-4) M) plus near-ultraviolet radiation, all strains, including those with a mutation in recA, were synergistically killed; thus, at high H2O2 concentrations, the damage was not RecA repairable.     

Frameshift mutagenesis by ultraviolet and gamma radiations in Salmonella: SOS inducibility and effects of DNA polymerase I

Ultraviolet (UV) and gamma-induced mutagenesis have been studied using a doubly auxotrophic strain of Salmonella typhimurium carrying the amber leuA150 mutation (which reverts by base-pair substitution) and the frameshift hisC3076 marker (which reverts by compensating frameshifts). In the initially constructed LT2 background, both markers were poorly revertible by UV and essentially non-revertible by gamma-radiation. A derivative of this strain carrying the mutation-enhancing plasmid pKM101 was however readily reverted by both UV and gamma, with either Leu+ (base substitution) or His+ (frameshift) revertants being observed on appropriate selective media. Photoreactivation experiments suggested that the lesions leading to formation of the two types of mutagenic event were similar if not identical. Support for this suggestion was obtained when it was found that yields of both types of UV-induced revertant were significantly increased in an excision-deficient background, while no revertants of either type were found in a recA background. Yields of gamma-induced revertants were not greatly altered in a uvrB background, but were also reduced to zero (for both markers) in the recA background. These results are consistent with what has previously been well-documented for UV and gamma-induced base-pair substitution mutagenesis, and serve to emphasize the similarities between base-pair substitution mutagenesis and frameshift mutagenesis by these agents. There are differences, however, since although UV-induced reversion of the leuA150 marker was little affected and gamma-induced reversion of leuA150 was somewhat reduced in the presence of a polA mutation (polA3), the yields of His+ frameshift revertants were significantly increased in the polA3 background following treatment with either UV or gamma. Thus while inducible DNA repair (SOS repair) appears to be involved in generating both types of mutational event following either UV- or gamma-irradiation, at some stage in the processing of premutational lesions the level (or type) of DNA polymerase I activity in the cell seems to have an important role in determining whether or not frameshifts or base-pair substitutions will be produced at a particular frequency.     

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.     

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.     

X-ray-induced mutations in Escherichia coli K-12 strains with altered DNA polymerase I activities

Spectra of ionizing radiation mutagenesis were determined by sequencing X-ray-induced endogenous tonB gene mutations in Escherichia coli polA strains. We used two polA alleles, the polA1 mutation, defective for Klenow domain, and the polA107 mutation, defective for flap domain. We demonstrated that irradiation of 75 and 50 Gy X-rays could induce 3.8- and 2.6-fold more of tonB mutation in polA1 and polA107 strains, respectively, than spontaneous level. The radiation induced spectrum of 51 tonB mutations in polA1 and 51 in polA107 indicated that minus frameshift, A:T-->T:A transversion and G:C-->T:A transversion were the types of mutations increased. Previously, we have reported essentially the same X-ray-induced tonB mutation spectra in the wild-type strain. These results indicate that (1) X-rays can induce minus frameshift, A:T-->T:A transversion and G:C-->T:A transversion in E. coli and (2) presence or absence of polymerase I (PolI) of E. coli does not have any effects on the process of X-ray mutagenesis.     

The effect of catalase on recovery of heat-injured DNA-repair mutants of Escherichia coli

The apparent sensitivity of Escherichia coli K12 to mild heat was increased by recA (def), recB and polA, but not by uvrA, uvrB or recF mutations. However, addition of catalase to the rich plating medium used to assess viability restored counts of heat-injured recA, recB and polA strains to wild-type levels. E. coli p3478 polA was sensitized by heat to a concentration of hydrogen peroxide similar to that measured in autoclaved recovery medium. The apparent heat sensitivity of DNA-repair mutants is thus due to heat-induced sensitivity to the low levels of peroxide present in rich recovery media. It is proposed that DNA damage in heated cells could occur indirectly by an oxidative mechanism. The increased peroxide sensitivity of heat-injured cells was not due to a decrease in total catalase activity but may be related specifically to inactivation of the inducible catalase/peroxidase (HPI).     

Comparative mutagenesis and interaction between near-ultraviolet (313- to 405-nm) and far-ultraviolet (254-nm) radiation in Escherichia coli strains with differing repair capabilities.

Comparative mutagenesis and possible synergistic interaction between broad-spectrum (313- to 405-nm) near-ultraviolet (black light bulb [BLB]) radiation and 254-nm radiation were studied in Escherichia coli strains WP2 (wild type), WP2s (uvrA), WP10 (recA), WP6 (polA), WP6s (polA uvrA), WP100 (uvrA recA), and WP5 (lexA). With BLB radiation, strains WP2s and WP6s demonstrated a high level of mutagenesis, whereas strains WP2, WP5, WP6, WP10, and WP100 did not demonstrate significant mutagenesis. In contrast, 254-nm radiation was mutagenic in strains WP2, WP2s, WP6, and WP6s, but strains WP5, WP10, and WP100 were not significantly mutated. The absence of mutagenesis by BLB radiation in lexA and recA strains WP10, WP5, and WP100 suggests that lex+ rec+ repair may play a major role in mutagenesis by both BLB and 254-nm radiation. The hypothesis that BLB radiation selectively inhibits rec+ lex+ repair was tested by sequential BLB-254-nm radiation. With strain WP2, a fluence of 30 J/m2 at 254 nm induced trp+ revertants at a frequency of 15 X 10(-6). However, when 10(5) J/m2 or more of BLB radiation preceded the 254-nm exposure, no trp+ revertants could be detected. A similar inhibition of 254-nm mutagenesis was observed with strain WP6 (polA). However, strains WP2s (uvrA) and wP6s (polA uvrA) showed enhanced 254-nm mutagenesis when a prior exposure to BLB radiation was given.     

Repair of near-ultraviolet (365 nm)-induced strand breaks in Escherichia coli DNA. The role of the polA and recA gene products.

The action of near-ultraviolet (UV-365 nm) radiation in cellular inactivation (biological measurements) and induction and repair of DNA strand breaks (physical measurements) were studied in a repair-proficient strain and in polA-, recA-, uvrA-, and polA uvrA-deficient strains of Escherichia coli K-12. The induction of breaks in the polA and polA uvrA strains was linear with dose (4.0 and 3.7 X 10(-5) breaks/2.5 X 10(9) daltons/Jm-2, respectively). However, in the recA-, uvrA-, and repair-proficient strains, there was an initial lag in break induction at low doses and then a linear induction of breaks at higher doses with rates of 4.6, 2.8, and 3.2 X 10(-5) breaks/2.5 X 10(9) daltons/Jm-2, respectively. We interpret these strain differences as indicating simultaneous induction and repair of breaks in polymerase 1 (polA)-proficient strains under the 0 degrees C, M9 buffer irradiation conditions that, for maximum efficiency, require both the polA and recA gene products. Strand-break rejoining also occurred at 30 degrees C in complete growth medium. We propose that at least three (and possibly four) distinct types of pathways can act to reduce the levels of 365-nm radiation-induced strand breaks. A quantitative comparison of the number of breaks remaining with the number of lethal events remaining after repair in complete medium at 30 degrees C showed that between one and three breaks remain per lethal event in the wild-type and recA strains, whereas in the polA strain one order of magnitude more breaks were induced.     

Streptococcus pneumoniae polA gene is expressed in Escherichia coli and can functionally substitute for the E. coli polA gene.

The Streptococcus pneumoniae polA+ gene was introduced into Escherichia coli on the recombinant plasmid pSM31, which is based on the pSC101 replicon. Extracts of E. coli polA5 mutants containing pSM31 showed DNA polymerase activity, indicating that the pneumococcal DNA polymerase I was expressed in the heterospecific host. Complete complementation of the E. coli polA5 mutation by the pneumococcal polA+ gene was detected in excision repair of DNA damage.