Dithiothreitol pretreatment and inducible repair in UV-irradiated Escherichia coli K12 cells

The UV radiation survival of several Escherichia coli K12 strains was measured after pretreatment of the cells with dithiothreitol (DTT). In DNA repair-competent cells (AB1157), UV survival was enhanced (ER = 1.2) after pretreating cells for 1.0 h using 10 mmol dm-3 DTT and then incubating the cells for 1.5 h in buffer before UV irradiation. Similar experiments using the excision repair mutant, AB1886uvrA6, or the recombination repair and SOS-deficient mutant, AB2462recA, strains did not show enhanced UV survival. None of the E. coli strains tested were protected against UV killing by simultaneous treatment with DTT (10 mmol dm-3). These results, and the fact that incubation in chloramphenicol removed the wild-type response in DTT-pretreated, UV-irradiated cells, suggest that the observed UV radioprotection was a result of inducible enzymatic repair processes such as recA-dependent repair. The proposed stimulus for inducible repair in these cells is DNA damage caused by intracellular hydroxyl radicals arising from thiol oxidation. The involvement of oxygen radicals in the induction pathway is supported by results that showed superoxide dismutase and catalase could inhibit a portion (one-third) of the inducible repair.     

Radioadaptive enhancement of the repair of UV-induced postreplication gaps in Escherichia coli cells deficient in DNA repair

In UV-irradiated E. coli WP2 uvrA, deficient in excision repair of DNA with pyrimidine dimers, gamma-irradiation in low doses (radioadaptation) before UV-irradiation leads to the intensification of postreplication repair of DNA. This process in WP2 uvrA polA and uvrA lexA mutants is less than in WP2 uvrA cells, but in WP2 uvrA recA both postreplication repair and its radioadaptive intensification are absent. In E. coli AB1157 excising pyrimidine dimers the radioadaptive intensification of postreplication repair of DNA is expressed almost to the same extent as in WP2 uvrA. In GW2100 umuC mutant, deficient in DNA polymerase V, postreplication repair of DNA is expressed, but its radioadaptive intensification is absent, while in AB2463 recA13 both postreplication repair of DNA and radioadaptive intensification of postreplication repair of DNA are absent. The above data suggest that DNA polymerase I and LexA protein are needed for radioadaptive intensification of postreplication repair of DNA in uvrA strain, and DNA polymerase V is needed for radioadaptive intensification in E. coli AB1157, and that RecA protein is required for postreplication repair and radioadaptive intensification of postreplication repair of DNA.     

The RecF pathway of homologous recombination can mediate the initiation of DNA damage-inducible replication of the Escherichia coli chromosome.

DNA damage-inducible DNA replication in SOS-induced Escherichia coli cells, termed inducible stable DNA replication (iSDR), has previously been shown to require either the RecBCD or the RecE pathway of homologous recombination for initiation. Here, we demonstrate that recB recC sbcC quadruple mutant cells are capable of iSDR induction and that a mutation in the recJ gene abolishes the inducibility. These results indicate that the RecF pathway of homologous recombination can also catalyze iSDR initiation.     

Mutagenic characteristics of formaldehyde on bacterial systems

The mutagenic characteristics of formaldehyde on bacteria were examined. All the tester strains of Escherichia coli deficient in DNA-repair enzymes tested in the present study were significantly more sensitive to the killing effect of formaldehyde than the corresponding wild-type strain. Among the E. coli B strains, H/r30R (wild-type) and Hs30R (uvrA) were mutable, whereas NG30 (recA) and O16 (polA) were not. There is no appreciable difference in mutation frequency of E. coli B between the wild-type and the uvrA strains in a dose range below 4 mM. However, the mutation frequency of the wild-type strain started to decrease in a higher concentration range, whereas that of the uvrA strain continued to increase linearly. This was confirmed with the E. coli B/r tester strains. The decrease in mutation frequency may be produced by prolongation of the lag period before entering the S-phase so as to give the cells a greater chance for DNA repair through the excision mechanism. In fact, it was evidenced that formaldehyde retarded to a remarkable extent the initiation of DNA synthesis of the cells at the higher dose range used for mutation assay. Some discrepancies found between the results obtained in this study and those previously reported by Nishioka (1973) were pointed out.     

Increased UV sensitivity of Escherichia coli cells after introduction of foreign photolyase genes

High-expression plasmids for photolyase (phr) genes from the bacteria Escherichia coli, Anacystis nidulans, Streptomyces griseus and Halobacterium halobium and the yeast Saccharomyces cerevisiae were constructed and introduced into E. coli phr recA cells. As previously reported, al introduced phr genes provided the host cells with photoreactivation-repair activity and the introduced E. coli phr gene rendered the host cells more UV-resistant in the dark. E. coli cells harboring foreign phr genes, however, were found to be more sensitive to UV light in the dark than cells containing the vector plasmid only. These differences in UV sensitivity in the dark disappeared when the host cells had an additional mutation, uvrA, suggesting that the foreign photolyases inhibited the E. coli excision-repair system.     

Genotoxicity assay of oil dispersants in bacteria (mutation, differential lethality, SOS DNA-repair) and yeast (mitotic crossing-over)

5 oil dispersants and a sample of paraffin were devoid of mutagenic activity in the Ames reversion test, with and without S9 mix, using 7 his- S. typhimurium strains (TA1535, TA1537, TA1538, TA97, TA98, TA100, TA102). However, 3 dispersants produced direct DNA damage in E. coli WP2, which was not repairable in repair-deficient strains (WP2uvrA, CM871, TM1080), as shown by two different DNA-repair test procedures. The uvrA excision-repair system was in all cases the most important mechanism involved in repairing the DNA damage produced by oil dispersants, while the combination of uvrA with other genetic defects (polA, recA, lexA) decreased the efficiency of the system. The observed genotoxic effects were considerably lowered in the presence of S9 mix containing liver S9 fractions from Aroclor-treated rats. The sample of oil dispersant yielding the most pronounced DNA damage in repair-deficient E. coli failed to induce gene sfiA in E. coli (strain PQ37), using the SOS chromotest, or mitotic crossing-over in Saccharomyces cerevisiae (strain D5). The direct toxicity of the oil dispersant to both bacterial and yeast cells was markedly decreased in the presence of rat-liver preparations. These two short-term tests were effective in detecting the genotoxicity of both direct-acting compounds (such as 4-nitroquinoline N-oxide and methyl methanesulfonate) and procarcinogens (such as cyclophosphamide, 2-aminoanthracene and 2-aminofluorene). Moreover, the SOS chromotest was successfully applied to discriminate the activity of chromium compounds as related to their valence (i.e. Cr(VI) genotoxic and Cr(III) inactive). Combination of oil dispersants with Cr(VI) compounds did not affect the direct mutagenicity to S. typhimurium (TA102) of a soluble salt (sodium dichromate) nor did it result in any release of a water-soluble salt (lead chromate), as also confirmed by analytical methods. On the other hand, exposure to sunlight tended to decrease, to a slow rate, the direct genotoxicity of an oil dispersant in the bacterial DNA-repair test.     

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.     

Construction of a plasmid containing functional Escherichia coli uvrA, B, and C genes in a configuration potentially suitable for mammalian expression

A plasmid, pUVABC-2, was constructed that encodes functional uvrA, B, and C genes of Escherichia coli. This plasmid also contains the gpt and ampr genes for positive selection in either bacterial or mammalian systems. Each of the uvrA, B, C, and gpt genes is located between SV40 initiation and termination signals and retains the original bacterial promoters. This recombinant vector conferred a wild-type UV resistance phenotype to uvrA-, B-, and C- strains of E. coli. The results indicate that each of the uvr genes contained in pUVABC-2 function in E. coli. The plasmid is a potential biological probe for DNA repair in mammalian cells.     

The expression of the Escherichia coli uvrA gene in human cells

Cells cultured from xeroderma pigmentosum (XP) patients are defective in excision repair of damaged DNA specifically at the incision step. In Escherichia coli this step is mediated by the UvrA, UvrB and UvrC gene products. Our goal is to express each of these genes in XP cells, singly or in combination, and to determine the most suitable conditions for generating faithful E. coli Uvr protein copies in functional concentrations and properly localized for the eventual repair of damaged chromosomal DNA or DNA which is introduced exogenously. The E. coli gpt gene in pSV2gpt is used as a selection marker for uvr gene transfection into XP cells. The uvr genes were cloned into composite pBR322, SV40 and gpt vectors in which each E. coli gene is flanked by individual SV40 regulatory elements. SV40-transformed XP-A cells were transfected with pSV2uvrASV2gpt, gpt+ colonies were selected, and cell lines established. Several lines were examined in detail. Cell lines 714 and 1511 contain uvrA together with flanking SV40 regulatory elements integrated intact in genomic DNA and express UvrA protein as well as a 95,000-dalton UvrA-related protein. The expression of uvrA was found to be 50-100-fold lower than the expression of gpt. Attempts were made to assay the mammalian UvrA protein for functionality, but endogenous activities interfered with assays for each of the UvrA protein's three activities. The peptide maps derived from partial proteolysis of the "mammalian" UvrA protein are identical to the E. coli UvrA protein. The sub-cellular location of UvrA protein in uvrA+ XP cells was investigated by fractionation of cell extracts in which an indirect immunofluorescence method revealed its location as being largely extra-nuclear. Two uvrA+ cell lines were examined for their UV-resistant phenotype and not unexpectedly were found not to be reverted to a state of repair proficiency.     

Genetic rearrangements in the tyrB-uvrA region of the enterobacterial chromosome: a potential cause for different class B acid phosphatase regulation in Salmonella enterica and Escherichia coli

Unlike in Escherichia coli, in Salmonella enterica production of class B acid phosphatase (AphA) was detectable also in cells growing in the presence of glucose. Characterization of the aphA locus from a S. enterica ser. typhi strain showed that the aphA determinant is very similar to the E. coli homolog, and that its chromosomal location between the highly conserved tyrB and uvrA genes is retained. However, the aphA flanking regions were found to be markedly different in the two species, either between tyrB and aphA or between aphA and uvrA. The differences in the aphA 5'-flanking region, which in S. enterica is considerably shorter than in E. coli (183 vs. 1121 bp) and includes potential promoter sequences not present in E. coli, could be responsible for the different regulation of class B acid phosphatase observed in the two species.     

Haemophilus influenzae UvrA: overexpression, purification, and in cell complementation

UvrA protein is a major component of ABC endonuclease complex involved in nucleotide excision repair (NER) mechanism. Although NER system is best characterized in Escherichia coli, not much information is available in Haemophilus influenzae. However, based on amino acid homology, uvrA ORF has been identified on H. influenzae genome [gene identification No. HI0249, Science 269 (1995) 496]. H. influenzae Rd uvrA ORF was cloned and overexpressed in E. coli. The expressed UvrA protein was purified using a two-step column chromatography protocol to a single band of expected molecular weight (104 kDa) and characterized for its ATPase and DNA binding activity. In addition, when H. influenzae uvrA was introduced in E. coli uvrA mutant strain AB1886, its UV resistance was restored to near wild type level.     

Post-replication repair and recombination in uvrA umuC strains of Escherichia coli are enhanced by vanillin, an antimutagenic compound

Effects of vanillin on UV killing of umuC mutant strains of E. coli were investigated in order to analyze the antimutagenic role of vanillin in mutagenesis. UV-irradiated uvrA umuC cells showed higher survival when plated on medium containing vanillin rather than medium without vanillin. This increased survival associated with exposure to vanillin was observed more clearly in uvrA umuC lexA(Ind-) and uvrA umuC recF strains. However, the effect was inhibited by additional recB recC mutations and completely blocked by an additional recA mutation. As far as tested the increased survival of UV-treated cells by vanillin was dependent on a capacity for genetic recombination. The effect of vanillin on recombination frequency between 2 plasmid DNA, pATH4 (Cmr Tcs) and pBMX7 (Apr Tcs), in a uvrA umuC background was investigated. A significantly higher frequency of plasmid recombination was observed when vanillin was present in the culture medium. These findings suggest that the antimutagenic effect of vanillin is the result of enhancement of a recA-dependent, error-free, pathway of post-replication repair.     

Requirement of the Escherichia coli dnaK gene for thermotolerance and protection against H2O2

Thermotolerance in Escherichia coli is induced by exposing cells to a brief heat shock (42 degrees C for 15 min). This results in resistance to the lethal effect of exposure to a higher temperature (50 degrees C). Mutants defective in the recA, uvrA and xthA genes are more sensitive to heat than the wild-type. However, after development of thermotolerance these mutants are like the wild-type in their heat sensitivity. This suggests that thermotolerance is an inducible response capable of protecting cells from the lethal effects of heat, independently of recA, uvrA and xthA. Thermotolerance does not develop in a dnaK mutant. In addition, the dnaK mutant is sensitive to heat and H2O2, but is resistant to UV irradiation. This implies that the E. coli heat-shock response includes a mechanism that protects cells from heat and H2O2, but not from UV.     

Difference in superoxide toxicity between 4,7-dicyanobenzofurazan and paraquat.

The O2-. production by aerobically cultured Escherichia coli in the presence of benzofurazan (1), 4,7-dimethylbenzofurazan (2), 4,7-dibromobenzofurazan (3), 4-bromo-6-cyanobenzofurazan (4), and 4,7-dicyanobenzofurazan (5) was examined by using the cytochrome c reduction method in order to elucidate the mechanism of cytotoxicity of benzofurazans. Adding compound 5 to E. coli cell suspension caused cytochrome c reduction, which was completely inhibited by superoxide dismutase. The rate of cytochrome c reduction was in the order of 1 = 2 = 3 less than 4 less than 5, which correlates well with that of the reduction potentials of these benzofurazans. Adding glucose to the E. coli cell suspension-compound 5-cytochrome c system accelerated the rate of cytochrome c reduction. The formation of 4,7-dicyanobenzofurazan anion radical in the cell suspension-compound 5-glucose system in the absence of O2 was followed by ESR spectroscopy. The ESR signal of the anion radical disappeared when O2 was added. Compound 5 was shown to have an approximately 10-fold greater increasing effect on the flux of O2-. by E. coli than paraquat (PQ) by the cytochrome c reduction method. The results were confirmed by the electrochemical method with an oxygen electrode. However, compound 5 had a bacteriostatic, but not lethal, effect, while PQ had both effects. The effect of compound 5 and PQ on lethality of E. coli showed a dramatic difference when E. coli was exposed to these two compounds and washed prior to testing the effects of that exposure. This difference probably arose because compound 5 readily leaked from the cells during dilution and plating. Also, the reduced form of compound 5 exits from the cells more readily than the reduced form of PQ and then generates O2-. in the medium by autoxidation. This suggests the importance of the intracellular production of O2-., rather than the extracellular production of O2-., for lethal effect.     

Intergeneric conjugational hybridization of Escherichia coli and Salmonella typhimurium. 1. Obtaining a salmonella hybrid possessing greater recipient activity in crosses with Escherichia coli]

Intergeneric hybrids were selected from mating HfrH Escherichia coli with F- Salmonella typhimurium. The hybrid obtained from E. coli leu+ and pro+ genes possessed the increased recipient ability in the mating with E. coli HfrR1 (O--ilv--metE--ara). This hybrid lacked the ability to restrict the phage P1 DNA propagated on E. coli K-12. The replacement of mutated uvrA gene of Salmonella for uvrA+ gene of E. coli restore uvr+ phenotype of Salmonella mutant.     

The oxygen effect in E. coli cells. Role of the photoreactivated component in modifying the oxygen effect in E. coli WP2 uvrA6 cells

A study was made of a contribution of photoreactivated lesions to the oxygen effect (OE) manifestation in cells of UV-sensitive E. coli WP2 uvrA6. It was shown that the contribution of the photoreactivated lesions depends upon radiation energy and gas conditions in which radiation is delivered. The OE value in the uvrA6-mutant may be modified in the irradiation conditions preventing induction of the photoreactivated lesions (gamma-irradiation in the presence of nigrosine, and X-irradiation).     

Escherichia coli and Bacillus subtilis PriA proteins essential for recombination-dependent DNA replication: involvement of ATPase/helicase activity of PriA for inducible stable DNA replication

The E. coli PriA protein, a DEXH-type DNA helicase with unique zinc finger-like motifs interrupting the helicase domains, is an essential component of the phiX174-type primosome and plays critical roles in RecA-dependent inducible and constitutive stable DNA replication (iSDR and cSDR, respectively) as well as in recombination-dependent repair of double-stranded DNA breaks. B. subtilis PriA (BsPriA) protein contains the conserved helicase domains as well as zinc finger-like motifs with 34% overall identity with the E. coli counterpart. We overexpressed and purified BsPriA and examined its biochemical properties. BsPriA binds specifically to both n'-pas (primosome assembly site) and D-loop and hydrolyzes ATP in the presence of n'-pas albeit with a specific activity about 30% of that of E. coli PriA. However, it is not capable of supporting n'-pas-dependent replication in vitro, nor is it able to support ColE1-type plasmid replication in vivo which requires the function of the phiX174-type primosome. We also show that a zinc finger mutant is not able to support recombination-dependent DNA replication, as measured by the level of iSDR after a period of thymine starvation, nor wild-type level of growth, cell morphology and UV resistance. Unexpectedly, we discovered that an ATPase-deficient mutant (K230D) is not able to support iSDR to a full extent, although it can restore normal growth rate and UV resistance as well as non-filamentous morphology in priA1::kan mutant. K230D was previously reported to be fully functional in assembly of the phiX174-type primosome at a single-stranded n'-pas. Our results indicate that ATP hydrolysis/ helicase activity of PriA may be specifically required for DNA replication from recombination intermediates in vivo.     

Expression of Erwinia chrysanthemi ENA49 uvr gene in Escherichia coli K12 cells

The uvrA gene of Erwinia chrysanthemi ENA49 similar to uvrA gene of Escherichia coli K12 has been cloned in vivo in Escherichia coli AB1886 uvrA6 cells using the plasmid pULB113 (RP4mini Mu). The presence of pULB113 carrying uvrA gene of Erwinia in Escherichia coli K12 uvrA- cells resulted in suppression of this mutation while uvrB and uvrC are not suppressed by this locus. The genetic control of excision repair of UV-damage in Erwinia chrysanthemi ENA49 is concluded to be similar to the one in Escherichia coli K12.     

Contrasting fates of the paraquat monocation radical in Escherichia coli B and in Dunaliella salina

Anaerobic cultures of Escherichia coli exposed to paraquat (PQ2+) accumulated the corresponding monocation radical PQ+., both within the cells and in the suspending medium. The green alga, Dunaliella salina, which is susceptible to a light- and O2-dependent toxicity of PQ2+, was nevertheless unable to cause accumulation of PQ+. when illuminated anaerobically and could, moreover, discharge the ESR signal and the blue color of PQ+. accumulated by E. coli. Spin trapping allowed demonstration of the photoproduction of O2- within D. salina and of the augmentation of that O-2 production by PQ2+. D. salina appears to contain an electron sink and a heat-labile mechanism for transferring electrons from PQ+. to that sink. This mechanism was demonstrable anaerobically but did not prevent PQ+.-mediated O2- production under aerobic conditions.