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.     

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.     

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.     

Conditional Lethality of Deletions Which Include uvrB in Strains of Escherichia coli Lacking Deoxyribonucleic Acid Polymerase I

Deletions of the uvrB gene were not obtained in polA1 strains of Escherichia coli either by selecting for spontaneous deletions or by transduction from strains carrying such deletions. A strain forming a temperature-sensitive deoxyribonucleic acid polymerase I and carrying a deletion of the uvrB gene is inviable at the nonpermissive temperature.     

Escherichia coli DNA polymerase I. Construction of a polA plasmid for amplification and an improved purification scheme

Previous attempts to clone the Escherichia coli polA+ gene onto a high copy number plasmid were unsuccessful. The apparent lethality of unregulated overproduction of DNA polymerase I can be eliminated by cutting at a BglII site 100 nucleotides upstream from the ATG start codon of the polA gene. This permitted the construction of plasmid pMP5 which contains both the coding sequence for DNA polymerase I and the lambda pL promoter for conditional control of polA gene expression. BglII cutting only damages but does not eliminate the polA promoter activity; the BglII site thus lies within the polA promoter region. Leakiness of the damaged polA promoter results in overproduction of DNA polymerase I even under conditions where pL is fully repressed. This overproduction is inhibitory of cell growth, as reflected in both growth rate and in the frequency of appearance of mutant plasmids which are nonproducers of DNA polymerase I. Transformation of plasmid pMP5 into E. coli N4830 yields strain ATL100 which under inducing conditions provides 138-fold amplification of DNA polymerase I. Optimization of growth and expression conditions are presented together with an optimized rapid polymerase purification scheme. In addition to providing a convenient source for preparation of DNA polymerase I, this work serves as the basis for a future detailed molecular genetic analysis of the polA gene product.     

Construction and characterization of an Escherichia coli strain with a uncI mutation.

A strain of Escherichia coli with a mutation in the promoter proximal gene ( uncI ) of the unc operon has been constructed by using a new gene replacement method. The mutation is a deletion of a defined sequence of 196 base pairs. It was constructed by homologous integration and segregation of a ColE1-derived recombinant plasmid containing the mutation, in a temperature-sensitive polA strain. The mutant strain is phenotypically unc+ but has a reduced growth yield compared to a normal sibling strain.     

X-ray Sensitivity and Repair Capacity of a polA1 exrA Strain of Escherichia coli K-12

A polA1 exrA strain of Escherichia coli K-12 was constructed. It was found to be more sensitive to aerobic or anoxic X irradiation than were mutants containing either polA1 or exrA alone. The ability of polA1 exrA and related strains to repair X-ray-induced single-strand breaks in deoxyribonucleic acid DNA was examined. The polA1 strain was deficient in type II (buffer) repair but not in type III (growth medium-dependent) repair. The exrA strain was not deficient in type II repair but was deficient in type III repair (similar to rec strains). The double mutant polA1 exrA was deficient in both type II and type III repair. Thus, the increased X-ray sensitivity of the polA1 exrA double mutant was correlated with its decreased ability to repair X-ray-induced single-strand breaks in DNA. We have tested the hypothesis that polA rec double mutants are not viable because they lack the types II and III systems for the repair of DNA single-strand breaks. Since the polA1 exrA strain is viable and is deficient in both of these repair processes, this hypothesis seems not to be correct.     

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.     

Repair of hydrogen peroxide-induced single-strand breaks in Escherichia coli deoxyribonucleic acid.

Near-ultraviolet (300 to 400 nm) irradiation of L-tryptophan yielded H2O2 (a toxic photoproduct) that was selectively lethal for rec and polA1 Escherichia coli mutants. H2O2 treatment of cells resulted in the induction of single-strand deoxyribonucleic acid breaks. These breaks were repaired to only a small extent in polA1, recA recB, and recA mutants, but were efficiently repaired in wild-type strains. We conclude that H2O2 deoxyribonucleic acid lesions require both the polA+ and recA+ pathways for repair.     

Role of deoxyribonucleic acid polymerase III in the repair of single-strand breaks produced in Escherichia coli deoxyribonucleic acid by gamma radiation.

Cell survival, deoxyribonucleic acid (DNA) degradation, and the repair of DNA single-strand breaks were measured for Escherichia coli K-12 pol+, polA1, polC1026(ts), and polA1 polC1026(ts) cells after 137Cs gamma irradiation. The results indicate that DNA polymerase III is required for growth medium-dependent (type III) repair in polA+ or polA cells. In pol+ or polC cells, DNA polymerase I performs type II repair efficiently. The relative deficiencies of each of these strains in DNA repair generally correlate with their relative sensitivities to cell killing and with the extent of DNA degradation observed.     

Anaerobic incubation enhances the colony formation of a polA recB strain of Escherichia coli K-12.

Escherichia coli strain E247 (polA1 recB21) has reduced colony formation (even at the permissive temperature of 30 degrees C) because of a poor suppressor mutation (sup-126). The colony formation was enhanced in the absence of oxygen about 3-fold at 30 degrees C and 10(6)-fold at 43 degrees C, suggesting that a polA recB strain was inviable due to oxygen toxicity. Colony formation was also increased by incubation in an agar medium containing the reducing agent thioglycolate and incubation in the presence of chloroform-killed Saccharomyces cerevisiae pet+ cells, but not pet cells. Since the E247 strain viability was inversely dependent on the oxygen pressure and since the strain was more sensitive to superoxide radical than either the polA or the recB mutant, it seems likely that the polA and recB genes play a role in repairing DNA damage during respiration.     

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.     

Recombinant levels of Escherichia coli K-12 mutants deficient in various replication, recombination, or repair genes.

Escherichia coli strains containing mutations in lexA, rep, uvrA, uvrD, uvrE, lig, polA, dam, or xthA were constructed and tested for conjugation and transduction proficiencies and ability to form Lac+ recombinants in an assay system utilizing a nontandem duplication of two partially deleted lactose operons (lacMS286phi80dIIlacBK1). lexA and rep mutants were as deficient (20% of wild type) as recB and recC strains in their ability to produce Lac+ progeny. All the other strains exhibited increased frequencies of Lac+ recombinant formation, compared with wild type, ranging from 2- to 13-fold. Some strains showed markedly increased conjugation proficiency (dam uvrD) compared to wild type, while others appeared deficient (polA107). Some differences in transduction proficiency were also observed. Analysis of the Lac+ recombinants formed by the various mutants indicated that they were identical to the recombinants formed by a wild-type strain. The results indicate that genetic recombination in E. coli is a highly regulated process involving multiple gene products.     

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.     

Effect of repair deficiency and R plasmids on spontaneous and radiation-induced mutability in Pseudomonas aeruginosa.

The effect of R plasmids on spontaneous and radiation (ultraviolet and gamma)-induced mutability in Pseudomonas aeruginosa was studied in strains containing the radiation-sensitive markers polA3 or rec-2 and the revertable auxotrophic markers hisO27 and trpB1. In the absence of an R plasmid, the radiation-induced mutability was dependent on the recA+ genotype and independent of the polA+ genotype, whereas spontaneous mutability was similar in all genetic backgrounds. R plasmids pPL1, R2, and pMG15 increased the ultraviolet radiation survival and ultraviolet-induced mutability of wild-type and polA host cells but did not alter either effect in a recA mutant. These R plasmids also increased the gamma radiation survival and gamma-induced mutability of wild-type host cells bud pMG15 also enhanced the level of spontaneous mutagenesis in wild-type host cells but not in a polA or recA mutant. These data suggested that a common plasmid gene product(s) may participate in various recA-dependent, error-prone deoxyribonucleic acid repair pathways of P. aeruginosa. The properties of a mutant R plasmid, pPL2, originally selected because it lacked enhanced ultraviolet-induced mutability, supported this conclusion.     

Survival and the repair of single-stranded DNA breaks in gamma-irradiated Escherichia coli cells adapted to methylmethane sulfonate]

The survival and repair of single-strand breaks of DNA in gamma-ray-irradiated E. coli adapted to MMS (20 mkg/ml during 3 hours) have been investigated. It is shown that the survival of adapted bacteria of radioresistant strains B/r, H/r30, AB1157 and W3110 pol+ increases with DMF (dose modification factor) ranging within 1.4-1.8 and in radiosensitive strains Bs-1, AB1157 recA13 and AB1157 lexA3 with DMF ranging within 1.3-1.4, and does not change in strains with mutation in polA gene P3478 polA1 and 016 res-3. There is no increase in radioresistance during the adaptation to MMS under the action of the protein synthesis inhibitor chloramphenicol. The increase in radioresistance during the adaptation to MMS correlates with the acceleration of repair of gamma-ray-induced single-strand breaks in the radioresistant strains B/r and W3110 pol+ and with the appearance of the ability to repair some part of DNA single-strand breaks in the mutant Bs-1, which beyond the adaptation to MMS does not repair these damages. The incomplete reparability of DNA single-strand breaks in P3478 polA1 strain cells, both adapted and non-adapted to MMS, is equal.     

Modulation of mutagenesis involving precise excision of transposon Tn10

Precise excision of transposon Tn10 results in reversion of the Trp- phenotype to Trp+ in a trp-1014::Tn10 strain of Salmonella typhimurium, and also occurs at a markedly higher frequency in a strain carrying the temperature-sensitive polA7 allele. The frequency with which precise excision events occurs can be modified by the plating medium, results indicating that the great majority of mutants which arise on broth-supplemented or tryptophan-supplemented minimal media actually arise on the selective plating medium. Trp+ revertants (1000) arising from excision of Tn10 were purified by re-streaking for single colonies; none were found to retain the Tn10 encoded resistance to tetracycline. Yields of Trp+ revertants of the polA7 strain were consistently higher when glycerol rather than glucose was used as sole carbon source in the selective medium. Clean excision of Tn10 can also be increased by ultraviolet irradiation in (R) plasmid-free strains, and is further increased in strains carrying an N-group plasmid (R205, R46 or pKM101). Ultraviolet-induced precise excision of Tn10 also occurs at a much enhanced frequency in a strain with a deletion through the uvrB gene; in this case, however, the addition of plasmid pKM101 leads to a decrease in yields of ultraviolet-induced precise excision events.     

Role of the 5´ → 3´ exonuclease and Klenow fragment of <Emphasis Type="Italic">Escherichia coli</Emphasis> DNA polymerase I in base mismatch repair

We have previously demonstrated that the Escherichia coli strain mutS ΔpolA had a higher rate of transition and minus frameshift mutations than mutS or ΔpolA strains. We argued that DNA polymerase I (PolI) corrects transition mismatches. PolI, encoded by the polA gene, possesses Klenow and 5′ → 3′ exonuclease domains. In the present study, rates of mutation were found to be higher in Klenow-defective mutS strains and 5′ → 3′ exonuclease-defective mutS strains than mutS or polA strains. The Klenow-defective or 5′ → 3′ exonuclease-defective mutS strains showed a marked increase in transition mutations. Sites of transition mutations in mutS, Klenow-defective mutS and 5′ → 3′ exonuclease-defective mutS strains are different. Thus, it is suggested that, in addition to mutS function, both the Klenow and 5′ → 3′ exonuclease domains are involved in the decrease of transition mutations. Transition hot and warm spots in mutS ⁺ polA ⁺ strains were found to differ from those in mutS and mutS ΔpolA strains. We thus argue that all the spontaneous transition mutations in the wild-type strain do not arise from transition mismatches left unrepaired by the MutS system or MutS PolI system.     

Enzymatic production of deoxyribonucleic acid double-strand breaks after ultraviolet irradiation of Escherichia coli K-12.

We have observed the enzymatic production of deoxyribonucleic acid (DNA) doublestrand breaks in Escherichia coli K12 after ultraviolet irradiation. Doublestrand breaks appeared in wild-type, polA1, recB21, recA, and exrA strains after incubation in minimal medium. THE UVRA6 strain showed no evidence of double-strand breakage under the same conditions. Our data suggest that uvr+ cells, which are proficient in the incision step of excision repair, accumulate double-strand breaks in their DNA as a result of the excision repair process, i.e., arising from closely matched incisions, excision gaps, or incisions and gaps on opposite strands of the DNA twin helix. Furthermore, strains deficient in excision repair subsequent to the incision step (i.e., polA, rec, exrA) showed more double-strand breaks than the wild type strain. The results raise the possibility that a significant fraction of the lethal events in ultraviolet-irradiated, repair-proficient (uvr+) cell may be enzymatically-induced DNA double-strand breaks.