Substrate of a UV-induced repair system providing for W-reactivation of lambda phage]

Escherichia coli uvrA, polA and uvrD cells carrying non-UV-inducible prophage lambdac1857ind- were infected with 3H-thymidine labelled homoimmune phage lambdac1857, and the effect of UV-irradiation of super-infecting phage and lysogenic bacterial cells on the content of intracellular covalently-closed lambda DNA circles (cccDNA) and pyrimidine dimer content in lambda DNA are studied. UV-irradiation of host cells results in two-fold increase of relative content of cccDNA of UV-irradiated phage lambda in uvrD mutant, while there is no such an effect in uvrA and polA mutants. In UV-irradiated or intact uvrA lysogens cccDNA molecules, forming after the infection with UV-irradiated phage lambda, contain pyrimidine dimers, but in uvrD mutant cccDNA in free of dimers. The data indicate that the repair system induced by UV-irradiation of uvrA and polA cells acts exclusively on the DNA defects appearing after (or in the course) of phage genomes replication. UV-inducible repair system in uvrD mutant can operate also on some intermediates of abortive excision repair, possibly on long single straided excision gaps.     

pBR322 plasmid DNA modified with 2-acetylaminofluorene derivatives: transforming activity and in vitro strand cleavage by the Escherichia coli uvrABC endonuclease

Covalently closed circular plasmid DNA was treated with three reactive derivatives of 2-acetylaminofluorene: N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF), its 7-iodo derivative (N-Aco- AAIF ) and N-hydroxy-N-2-aminofluorene (N-OH-AF), and tested as substrates for the Escherichia coli uvrABC endonuclease and for transformation frequencies on wild-type, uvrA, recA, uvrArecA and polA mutant strains. The uvrABC endonuclease reacted with all three substrates with high efficiency, implicating this enzyme in the repair of DNA containing all three types of adducts. However, only AAF- and AAIF -DNA showed greatly reduced survival on uvrA mutants (five adducts/lethal hit) relative to wild-type (20 adducts/lethal hit). AF-DNA survived equally well on uvrA mutant and wild-type cells, and at a much higher level of modification (60 adducts/lethal hit). A mutation in recA had only a minor effect on the survival of either DNA. The polA mutation reduced the survival of the AAF-treated DNA to the same extent as the uvrA mutation (five adducts/lethal hit). Also AF-DNA showed reduced survival on polA mutant cells versus wild-type. However, many more adducts (20/lethal hit) were tolerated than for AAF-DNA, indicating that AF lesions in the template do not efficiently block replication of DNA.     

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.     

Inactivation of Escherichia coli, F′ Episomes at Transfer, and Bacteriophage Lambda by Psoralen Plus 360-nm Light: Significance of Deoxyribonucleic Acid Cross-Links

We have investigated some biological consequences of light-induced psoralen-deoxyribonucleic acid (DNA) adducts and find that for several Escherichia coli functions (killing of strain AB2480 recA13 uvrA6, inactivation of phage lambda plaque-forming ability in wild type and uvrA6 hosts, loss of ability to transmit intact Flac(+) episomes), a light exposure sufficient for production of a single cross-link per DNA molecule correlates well with the biological consequence. Although one cross-link per genome is apparently lethal to recA13 uvr(-) strains, mutants carrying the recA13 or uvrA6 markers survive light exposures producing 6.7 and 16 cross-links per genome, respectively, and wild-type cells recover from 65 psoralen cross-links. Evidently, the excision and recombinational repair systems complement one another in reconstructing an intact genome from cellular DNA containing psoralen photoproducts. The above bacterial and phage strains, in which DNA repair processes are minimized, are also extremely sensitive to pyrimidine dimer-forming 254-nm UV light (without psoralen), and were expected to respond similarly to formation of psoralen-pyrimidine base monoadducts in their DNA. Since the biological inactivation by psoralen correlates well with cross-link formation, we suggest that the sensitizing action of this drug primarily derives from its ability to form DNA cross-links.     

Regulation and SOS induction of division inhibition in Escherichia coli K12

Summary When Escherichia coli is subjected to treatments that damage DNA or perturb DNA replication considerable cell filamentation occurs. It has been postulated that this phenomenon is associated with the presence of a division inhibitor induced coordinately with the SOS functions. The role of this induction would be to delay septation during DNA repair to prevent the formation of DNAless cells. In this communication, we present evidence for such a division inhibitor based on the properties of a division mutant which is hyperactive in the septation delay. Cells of this mutant filament extensively after a nutritional shift-up, have drastically reduced colony-forming abilities on a rich medium but not on a minimal medium following treatment with ultraviolet radiation and, are deficient in the lysogenization of phage lambda; phenotypes which are characteristic of but expressed to a much lower extent in another type of division mutant called lon. Cells harboring the division mutation plus either one of the lexA mutant alleles, spr-51 or tsl-1, are filamentous suggesting that they are permanently derepressed for division inhibition. These results are in agreement with models that assign the regulation of cell division to a division inhibitor which is regulated by the lexA repressor protein.     

Genotoxicity of stannous chloride in yeast and bacteria

Stannous chloride was found genotoxic in microbial test systems of the yeast Saccharomyces cerevisiae, in one strain of Salmonella typhimurium and in the Mutoxitest of Escherichia coli. Five isogenic haploid yeast strains differing only in a particular repair-deficiency had the following ranking in Sn2+ -sensitivity: rad52delta>rad6delta>rad2delta>rad4delta>RAD, indicating a higher relevance of recombinogenic repair mechanisms than nucleotide excision in repair of Sn2+ -induced DNA damage. Sn2+ -treated cells formed aggregates that lead to gross overestimation of toxicity when not undone before diluting and plating. Reliable inactivation assays at exposure doses of 25-75 mM SnCl2 were achieved by de-clumping with either EDTA- or phosphate buffer. Sn2+ -induced reversion of the yeast his1-798, his1-208 and lys1-1 mutant alleles, in diploid and haploid cells, respectively, and putative frameshift mutagenesis (reversion of the hom3-10 allele) was observed. In diploid yeast, SnCl2 induced intra-genic mitotic recombination while inter-genic (reciprocal) recombination was very weak and not significant. Yeast cells of exponentially growing cultures were killed to about the same extend at 0.1% of SnCl2 than respective cells in stationary phase, suggesting a major involvement of physiological parameters of post-diauxic shift oxidative stress resistance in enhanced Sn2+ -tolerance. Superoxide dismutases, but not catalase, protected against SnCl2-induced reactive oxygen species as sod1delta had a three-fold higher sensitivity than the WT while the sod2delta mutant was only slightly more sensitive but conferred significant sensitivity increase in a sod1delta sod2delta double mutant. In the Salmonella reversion assay, SnCl2 did not induce mutations in strains TA97, TA98 or TA100, while a positive response was seen in strain TA102. SnCl2 induced a two-fold increase in mutation in the Mutoxitest strain IC203 (uvrA oxyR), but was less mutagenic in strain IC188 (uvrA). We propose that the mutagenicity of SnCl2 in yeast and bacteria occurs via error-prone repair of DNA damage that is produced by reactive oxygen species.     

Copper ions mediate the lethality induced by hydrogen peroxide in low iron conditions in Escherichia coli

Iron ions mediate the formation of lethal DNA damage by hydrogen peroxide. However, when cells are depleted of iron ions by the treatment with iron chelators, DNA damage can still be detected. Here we show that the formation of such damage in low iron conditions is due to the participation of copper ions. Copper chelators can inhibit cell inactivation, DNA strand breakage and mutagenesis induced by hydrogen peroxide in cells pre-treated with iron chelators. The Fpg and UvrA proteins play an important role in the repair of DNA lesions formed in these conditions, as suggested by the great sensitivity of the uvrA and fpg mutant strains to the treatment when compared to the wild type strain.     

RecN and RecG are required for Escherichia coli survival of Bleomycin-induced damage

The sensitivity of a panel of DNA repair-defective bacterial strains to BLM was investigated. Escherichia coli recA cells were far more sensitive than were uvrA, dam-3, and mutM mutY strains, underscoring the importance of RecA to survival. Strains recBCD and recN, which lack proteins required for double strand break (DSB) repair, were highly sensitive to BLM, while recF cells were not. The requirement for DSB-specific enzymes supports the hypothesis that DSBs are the primary cause of bleomycin cytotoxicity. The acute sensitivity of recN cells was comparable to that of recA, implying a central role for the RecN protein in BLM lesion repair. The Holliday junction processing enzymes RecG and RuvC were both required for BLM survival. The recG ruvC double mutant was no more sensitive than either mutation alone, suggesting that both enzymes participate in the same pathway. Surprisingly, ruvAB cells were no more sensitive than wildtype, implying that RuvC is able to perform its role without RuvAB. This observation contrasts with current models of recombination in which RuvA, B, and C function as a single complex. The most straightforward explanation of these results is that DSB repair involves a structure that serves as a good substrate for RecG, and not RuvAB.     

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.     

Unbalanced growth as a normal feature of development of Bdellovibrio bacteriovorus

In this study we have investigated the rates and spatial patterns of chromosome replication and cell elongation during the growth phase of wild-type and facultatively prey-independent mutant strains of Bdellovibrio bacteriovorus. For the facultatively prey-independent mutants, the total DNA content of synchronously growing cultures was found to increase exponentially, as the multiple chromosomes within each filamentous cell replicated simultaneously. Cell mass, measured as total cellular protein, also increased exponentially during this period, apparently by means of multiple elongation sites along the filament wall. The relative rates of DNA and protein synthesis were unbalanced during growth, however, with the cellular concentration of DNA increasing slightly faster than that of protein. The original cellular DNA: protein ratio was restored in the progeny cells by continued protein synthesis during the septation period that follows the termination of DNA replication. Because of technical problems, these experiments could not be conducted on the wild-type cells, but similar results are assumed. This unusual pattern of unbalanced growth may represent an adaptation by bdellovibrios to maximize their progeny yield from the determinate amount of substrate available within a given prey cell.     

Repair of damage induced by ultraviolet light in DNA polymerase-defective Escherichia coli cells

Cells of the Escherichia coli mutant polA1, which lack DNA polymerase activity in vitro, are four times as sensitive as wild-type to ultraviolet irradiation. Cells of the mutant uvrA6, which are unable to excise dimers, are 12 times as sensitive as wild-type. We have shown that the double mutant polA1 uvrA6 is only slightly more sensitive to u.v. than the uvrA6 single mutant and conclude, therefore, that the u.v. sensitivity associated with the defect in DNA polymerase is primarily the result of a reduction in the efficiency of the excision-repair pathway. Observations on the effect of u.v. irradiation on the ability of polA1 cells to support the growth of phage λ suggest that the post-u.v. repair function of polymerase is subsequent to the action of the uvr⁺ gene products. Evidence is presented that the recA repair system is involved in excision-repair in polA1 cells, and we propose that it can substitute for DNA polymerase in repairing the gaps produced by dimer excision. This would account for the relatively slight effect of the polA1 mutation on u.v. sensitivity.     

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 Lethal Sectoring, a Further Feature of Escherichia coli Strains Deficient in the Function of rec and uvr Genes

Eight recombination-deficient (Rec(-)) mutants of Escherichia coli were studied. Progeny lines were obtained on solid media, by means of micromanipulation, and the colony-forming ability of individual cells was analyzed. Cells of all eight strains gave rise to colony-forming as well as non-colony-forming descendants ("lethal sectoring"). Lethal sectors, i.e., groups of non-colony-forming cells which originate from a common ancestor, appeared with frequencies per generation ranging between 4 and 20% in Rec(-) strains, whereas lethal sectors were rare in Rec(+) strains (less than 1%). A strain carrying a mutation (uvrA6) in one of the genes involved in pyrimidine dimer excision from deoxyribonucleic acid (DNA) showed twice as many lethal sectors per generation as a strain with the genotype uvrA(+). Similarly, a double mutant (AB2480, uvrA6, recA13) showed twice as much spontaneous lethal sectoring as the corresponding Rec(-) strain (uvrA(+), recA13). The kinetics of growth curves obtained in nutrient broth and the frequency of non-colony-forming units in stationary-phase broth cultures indicate clearly that lethal sectors occur in liquid cultures too. The causes for spontaneous lethal sectoring are unknown at present. It seems reasonable to assume that gene uvrA and the rec genes are somehow involved in the repair of spontaneously occurring DNA lesions, since a deficiency in this type of repair may cause lethal sectors. The extent to which spontaneous lethal sectoring (observed in all Rec(-) strains of E. coli studied) may contribute indirectly to the failure to form recombinants is discussed.     

Formation of additional contacts of chromosome with membrane in the process of DNA repair synthesis in bacterial cells

An increase in the amount of membrane-bound DNA was found in B. subtilis cells with UV-induced DNA repair synthesis as compared to untreated cells. It was shown that DNA repair synthesis occurred in DNA membrane complexes (DMC) formed during UV-irradiation. UV-induced formation of DMC was observed in cells of wild type strains which were capable of repairing damaged DNA but not in a mutant defective in DNA-polymerase I. It was demonstrated that DNA-polymerase I is located on the membrane of B. subtilis cells. This suggested a participation of DNA-polymerase I in binding of the chromosome to the membrane in UV-irradiated cells. UV-induced DMC did not dissociate when the cells were treated with inhibitors of DNA-gyrase. It, therefore, was qualitatively different from the DMC found during replication. The mechanisms of binding of the damaged DNA to the membrane in UV-irradiated cells of B. subtilis are discussed.     

Inhibition of Escherichia coli K12 by short-chain organic acids: lack of evidence for induction of the SOS response

Sublethal concentrations of formic acid (10 mmol/l) and propionic acid (5 mmol/l) at pH 5.0 preferentially inhibit DNA synthesis and stop cell multiplication in the absence of a corresponding cessation in the increase of culture turbidity. The possibility that the acids induce the SOS response by starving cells of thymine or by causing physical damage to the DNA molecule has now been investigated. Accumulation of thymine into the cytoplasm of whole cells was not inhibited by either acid. Mutants defective in excision repair ( uvrA6 ), recombination repair ( recA56 ) and polymerase activity ( polA1 ) were not more sensitive to the acids than their isogenic parent. No significant increase in cell length was observed from measurements of transmission electron microscope images of acid-treated cells. It is concluded, therefore, that sublethal concentrations of formic and propionic acid inhibit DNA synthesis without physically damaging DNA molecule, or starving the cell of essential thymine or otherwise inducing an SOS response.     

Epistatic analysis of the roles of the RAD27 and POL4 gene products in DNA base excision repair in S. cerevisiae

The cellular role of the DNA polymerase encoded by the Saccharomyces cerevisiae POL4 gene is unclear. We have used an epistasis analysis to investigate whether the proteins encoded by the POL4 and RAD27 genes participate in alternative, non-redundant subpathways of DNA base excision repair (BER). We constructed strains in which the genes were deleted singly or in combination and have examined their sensitivity to DNA damaging agents as well as spontaneous mutation frequency. The double deletion strain is no more sensitive to damaging agents and has no higher spontaneous mutation frequency than the most sensitive single mutant. These data indicate that the protein encoded by the POL4 gene does not participate in a non-redundant subpathway of base excision repair under these conditions. We discuss the implications of these results in light of the recent classification of the POL4 gene product as a member of the DNA polymerase lambda family.     

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

Postreplication DNA repair (PRR) in UV-irradiated Escherichia coli WP2 uvrA (tryptophan-dependent strain) and K12 AB1886 uvrA6 pre-irradiated by gamma-rays in low doses (radioadaptation, the first stress effect) has been investigated. PRR was found to be more effective after incubation in the growth medium (for 45-60 min) than in non-radioadapted cells: the repair of postreplication gaps increased by 6-15%. If cells of WP2 uvrA strain were incubated after UV-irradiation in media lacking tryptophan or casamin acids (the second stress effect), PRR was seen to increase as early as within 15 min of incubation and it is more effective than at the first stress. After a 30-60 min incubation the double stress effect leads to an increase in postreplication gap repair by 23-45%. In this case almost all the gaps prove to be repaired. The second stress alone exerts no influence on PPR efficiency. It is supposed that a preliminary radioadaptation may stimulate synthesis of a protein (proteins) of the SOS-response (presumably DNA polymerase V). The second stress effect apparently induces synthesis of an unknown factor (or depreesses synthesis of a MmrA-like protein), and this in cooperation with a protein newly synthesized during radioadaptation significantly increases the efficiency of PPR.     

Induction of SOS functions by nitrogen dioxide in Escherichia coli with different DNA-repair capacities

The effect of gaseous nitrogen dioxide (NO2) on cytotoxicity, induction of synthesis of UmuC and RecA proteins, and mutagenesis was studied in Escherichia coli strains with different capacities of DNA repair. Gaseous NO2 (90, 180 microliter/l) killed Escherichia coli. The recA mutant was most sensitive, the lexA mutant moderately sensitive, and the uvrA mutant and the wild-type the least sensitive. When 90 microliter/l NO2 gas was bubbled into bacterial suspensions for 30 min at a flow rate of 100 ml/min, the induction of umuC gene expression increased in the wild-type strain. NO2 also induced the recA gene expression in the wild-type strain. The synthesis of neither RecA nor UmuC proteins was induced in the recA and lexA mutants. We further investigated the NO2 mutagenesis in the cells treated with bubbling of NO2 gas. NO2 caused mutation to Trp+ of WP2.     

Gene responsible for protecting Escherichia coli from sodium dodecyl sulfate and toluidine blue plus light.

Escherichia coli cells were killed by visible light irradiation in the presence of the photosensitizing dye, toluidine blue. Two uvrB mutant strains of E. coli K-12 (AB1885 and N3-1) were much more sensitive than the isogenic uvrA and uvrC strains to treatment with toluidine blue plus light, suggesting that the uvrB+ gene product was involved in repair of DNA damage induced by the treatment. The uvrB+ gene cloned in a high- or low-copy-number plasmid was transformed into the uvrB strain (AB1885). Although all the transformants showed the same resistance as its wild-type strain (AB1157) to UV irradiation, they were as sensitive as AB1885 was to treatment with toluidine blue plus light. The two uvrB strains were more sensitive to sodium dodecyl sulfate than the other strains, suggesting that these strains had a defect in the cell surface. A sodium dodecyl sulfate-resistant revertant obtained from AB1885 was more resistant than AB1885 was to treatment with toluidine blue plus light. The two uvrB strains (AB1885 and N3-1) appear to have a defective gene (tentatively called dvl) different from uvrB. Its map position was around 7 min on the E. coli map.