Replication of plasmid R6K in DNA polymerase deficient mutants of Escherichia coli.

The plasmid R6K has been introduced into a number of Escherichia coli polymerase deficient (pol) mutants. In polCts mutants transferred to the nonpermissive temperature to inactivate polymerase III, R6K replicates but the replication products have a density in dye-CsCl gradients intermediate between supercoiled and linear forms. This aberrant replication requires normal cellular levels of polymerase I since it does not occur in polA polCts mutants. Normal R6K replication and maintenance occur in a polA polB polC+ host, however, we cannot tell from our experiments wheather polymerase I or III replicates R6K in polA+ polC+ host. Polymerase II, the polB gene product, has no detectable role in R6K replication.     

Multiple pathways for repair of oxidative DNA damages caused by X rays and hydrogen peroxide in Escherichia coli.

The responses of Escherichia coli to X rays and hydrogen peroxide were examined in mutants which are deficient in one or more DNA repair genes. Mutant cells deficient in either exonuclease III (xthA) or endonuclease IV (nfo) had normal resistance to X rays, but an xthA-nfo double mutant showed a sensitivity increased over that of either parental strain. A DNA polymerase I mutant (polA) was more sensitive than the xthA-nfo mutant. Cells bearing mutations in all of the polA, xthA, and nfo genes were more sensitive to X rays than polA and xthA-nfo mutants. Similar repair responses were obtained by exposing these mutant cells to hydrogen peroxide, with the exception of the xthA mutant, which was hypersensitive to this agent. The DNA polymerase III mutant (polC(Ts)) was slightly more sensitive to the agents than the wild-type strain at the restrictive temperature. The sensitivity of the polC-xthA-nfo mutant to X rays and hydrogen peroxide was greater than that of polC but almost the same as that of the xthA-nfo mutant. From these results it appears that there are at least four repair pathways, the DNA polymerase I-, exonuclease III/endonuclease IV and DNA polymerase I-, exonuclease III/endonuclease IV and DNA polymerase III-, and exonuclease III/endonuclease IV-dependent pathways, for the repair of oxidative DNA damages in E. coli.     

Multiple pathways for repair of hydrogen peroxide-induced DNA damage in Escherichia coli.

The repair response of Escherichia coli to hydrogen peroxide has been examined in mutants which show increased sensitivity to this agent. Four mutants were found to show increased in vivo sensitivity to hydrogen peroxide compared with wild type. These mutants, in order of increasing sensitivity, were recA, polC, xthA, and polA. The polA mutants were the most sensitive, implying that DNA polymerase I is required for any repair of hydrogen peroxide damage. Measurement of repair synthesis after hydrogen peroxide treatment demonstrated normal levels for recA mutants, a small amount for xthA mutants, and none for polA mutants. This is consistent with exonuclease III being required for part of the repair synthesis seen, while DNA polymerase I is strictly required for all repair synthesis. Sedimentation analysis of cellular DNA after hydrogen peroxide treatment showed that reformation was absent in xthA, polA, and polC(Ts) strains but normal in a recA cell line. By use of a lambda phage carrying a recA-lacZ fusion, we found hydrogen peroxide does not induce the recA promoter. Our findings indicate two pathways of repair for hydrogen peroxide-induced DNA damage. One of these pathways would utilize exonuclease III, DNA polymerase III, and DNA polymerase I, while the other would be DNA polymerase I dependent. The RecA protein seems to have little or no direct function in either repair pathway.     

Bacillus subtilis DNA polymerase III is required for the replication of DNA of bacteriophages SPP-1 and phi 105.

The replication of the Bacillus subtilis bacteriophages SPP-1 and phi 105 is sensitive to 6-(p-hydroxyphenylazo)-uracil (HPUra), a selective inhibitor of replicative DNA synthesis of B. subtilis which acts specifically at the levels of a replication-specific polymerase, DNA polymerase III (pol III). The origin of the HPUra-sensitive polymerase required for phage replication was examined by comparison of the drug sensitivity of phage development in a normosensitive host with that in a host carrying azp-12, a polC mutation that specifies production of an HPUra-resistant pol III. azp-12 specified HPUra-resistant phage host pol III. The host polIII requirement for SPP-1 replication also was confirmed by the demonstration that phage development was temperature sensitive in a host mutant carrying the polC mutation mut-1 (ts). Examination of the pol III activity of crude and purified cell-free preparations derived from phage-infected cells did not indicate any detectable changes in the specific activity, purification behavior, or drug sensitivity of the enzyme.     

Short deoxyribonucleic acid repair patch length in Escherichia coli is determined by the processive mechanism of deoxyribonucleic acid polymerase I.

The lengths of ultraviolet irradiation-induced repair resynthesis patches were measured in repair-competent extracts of Escherichia coli. Extracts containing wild-type deoxyribonucleic acid (DNA) polymerase I introduced a patch 15 to 20 nucleotides in length during repair of ColE1 plasmid DNA; extracts containing the polA5 mutant form of DNA polymerase I introduced a patch only about 5 nucleotides in length in a similar reaction. The repair patch length in the presence of either DNA polymerase corresponded to the processivity of that polymerase (the average number of nucleotides added per enzyme-DNA binding event) as determined with purified enzymes and DNA treated with a nonspecific endonuclease. The base composition of the repair patch inserted by the wild-type DNA polymerase was similar to that of the bacterial genome, whereas the patch inserted by the mutant enzyme was skewed toward greater pyrimidine incorporation. This skewing is expected, considering the predominance of pyrimidine incorporation occurring at the ultraviolet lesion and the short patch made by the mutant enzyme. Since the defect in the polA5 DNA polymerase which causes premature dissociation from DNA is reflected exactly in the repair patch length, the processive mechanism of the polymerase must be a central determinant of patch length.     

Time Scale for Rejoining of Bacteriophage λ Deoxyribonucleic Acid Molecules in Superinfected pol+ and polA1 Strains of Escherichia coli After Exposure to 4 MeV Electrons

The time scale for rejoining of radiation-induced deoxyribonucleic acid (DNA) single-strand breaks was measured in the presence and absence of oxygen. The involvement of DNA polymerase I in this repair process was studied. Formation and rejoining of DNA strand breaks were measured in lambda DNA infecting lysogenic pol(+) and polA1 strains of Escherichia coli irradiated by 4 MeV electrons under identical conditions. Irradiation and transfer to alkaline detergent could be completed in less than 180 ms. The initial yields of DNA strand breaks were identical in pol(+) and polA1 host cells and four- to fivefold higher in the presence of oxygen than in nitrogen anoxia. Evidence for the existence of a very fast repair process, independent of DNA polymerase I, was not found, since no rejoining of radiation-induced DNA strand breaks was observed during incubation from 45 ms to 3 s. In pol(+) host cells most of the strand breaks produced in the presence of oxygen were rejoined within the first 30 to 40 s of incubation, whereas no rejoining could be detected within the same period of time in anoxic cells. Since no rejoining of broken lambda DNA molecules was observed in polA1 host cells, it is concluded that the synthetase activity of DNA polymerase I is involved in the rejoining of DNA breaks induced by radiation in the presence of oxygen.     

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.     

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.     

Cloning the polB gene of Escherichia coli and identification of its product

Using an in vivo mini-Mu cloning system, we have cloned the polB gene of Escherichia coli into the multicopy plasmid, pUC18. A chromosomal insert of 4.9 kilobases gave 30-40-fold overproduction of DNA polymerase II, and the cells containing the plasmid showed normal growth. The restriction pattern of the polB gene does not match that of either the polA gene or polC gene. Plasmid-directed protein synthesis demonstrates peptides of 99 and 82 kDa which are not expressed by derivative plasmids without DNA polymerase II activity. It appears from in situ gel assays and high performance liquid chromatography that 82- and 55-kDa proteins are derived from the 99-kDa protein by degradation, but all retain activity. DNA polymerase I or DNA polymerase III antibody does not inhibit the synthesis reaction of partially purified DNA polymerase II, but DNA polymerase II antibody does. By the criteria of restriction pattern of the polB gene, molecular weight of the protein, and antibody inhibition of reaction, DNA polymerase II can be demonstrated to be a distinct DNA polymerase.     

DNA polymerases of low-GC gram-positive eubacteria: identification of the replication-specific enzyme encoded by dnaE

dnaE, the gene encoding one of the two replication-specific DNA polymerases (Pols) of low-GC-content gram-positive bacteria (E. Dervyn et al., Science 294:1716-1719, 2001; R. Inoue et al., Mol. Genet. Genomics 266:564-571, 2001), was cloned from Bacillus subtilis, a model low-GC gram-positive organism. The gene was overexpressed in Escherichia coli. The purified recombinant product displayed inhibitor responses and physical, catalytic, and antigenic properties indistinguishable from those of the low-GC gram-positive-organism-specific enzyme previously named DNA Pol II after the polB-encoded DNA Pol II of E. coli. Whereas a polB-like gene is absent from low-GC gram-positive genomes and whereas the low-GC gram-positive DNA Pol II strongly conserves a dnaE-like, Pol III primary structure, it is proposed that it be renamed DNA polymerase III E (Pol III E) to accurately reflect its replicative function and its origin from dnaE. It is also proposed that DNA Pol III, the other replication-specific Pol of low-GC gram-positive organisms, be renamed DNA polymerase III C (Pol III C) to denote its origin from polC. By this revised nomenclature, the DNA Pols that are expressed constitutively in low-GC gram-positive bacteria would include DNA Pol I, the dispensable repair enzyme encoded by polA, and the two essential, replication-specific enzymes Pol III C and Pol III E, encoded, respectively, by polC and dnaE.     

DNA polymerase III requirement for repair of DNA damage caused by methyl methanesulfonate and hydrogen peroxide.

The pcbA1 mutation allows DNA replication dependent on DNA polymerase I at the restrictive temperature in polC(Ts) strains. Cells which carry pcbA1, a functional DNA polymerase I, and a temperature-sensitive DNA polymerase III gene were used to study the role of DNA polymerase III in DNA repair. At the restrictive temperature for DNA polymerase III, these strains were more sensitive to the alkylating agent methyl methanesulfonate (MMS) and hydrogen peroxide than normal cells. The same strains showed no increase in sensitivity to bleomycin, UV light, or psoralen at the restrictive temperature. The sensitivity of these strains to MMS and hydrogen peroxide was not due to the pcbAl allele, and normal sensitivity was restored by the introduction of a chromosomal or cloned DNA polymerase III gene, verifying that the sensitivity was due to loss of DNA polymerase III alpha-subunit activity. A functional DNA polymerase III is required for the reformation of high-molecular-weight DNA after treatment of cells with MMS or hydrogen peroxide, as demonstrated by alkaline sucrose sedimentation results. Thus, it appears that a functional DNA polymerase III is required for the optimal repair of DNA damage by MMS or hydrogen peroxide.     

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.     

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.     

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.     

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.     

In vitro host cell reactivation of alkylated bacteriophage T7 deoxyribonucleic acid by repair-deficient strains of Escherichia coli.

An in vitro system capable of packaging bacteriophage T7 deoxyribonucleic acid (DNA) into phage heads to form viable phage particles has been used to monitor the biological consequences of DNA dam aged by alkylating agents, and an in vitro DNA replication system has been used to examine the ability of alkylated T7 DNA to serve as template for DNA synthesis. The survival of phage resulting from in vitro packaging of DNA preexposed to various concentrations of methyl methane sulfonate or ethyl methane sulfonate closely paralleled the in vivo situation, in which intact phage were exposed to the alkylating agents. Host factors responsible for survival of alkylated T7 have been examined by using wild-type strains of EScherichia coli and mutants deficient in DNA polymerase I (polA) or 3-methyladenine-DNA glycosylase (tag). For both in vivo and in vitro situations, a deficiency in 3-methyladenine-DNA glycosylase dramatically reduced phage survival relative to that in the wild type, whereas a deficiency in DNA polymerase I had an intermediate effect. Furthermore, when the tag mutant was used as an indicator strain, phage survival was enhanced when alkylated DNA was packaged with extracts prepared from a wild-type strain in place of the tag mutant or by complementing a tag extract with an uninfected tag+ extract, indicating in vitro repair during packaging.     

The nature of DNA damage and its repair after treatment of bacteria with dioxidine

The nature of the lethal effect of antimicrobial drug dioxidin was studied. The treatment of bacterial cells by dioxidin results in an instant repression of DNA synthesis and formation of single strand gaps in DNA molecule. The repair of single strand gaps in polA+ cells involves the DNA polymerase I. The deficit of this enzyme leads to the increased degradation of DNA. The products of the recA, polA1, lexA, recB are relevant for bacterial resistance to dioxidin while the products of uvrA, uvrE and recF genes are not. On the basis of the obtained data dioxidin may be defined as a "gamma-type" agent due to the nature of dioxidin-induced lesions in DNA and their repair.     

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.