Viera Slezáriková

UV-inducible repair: Influence on survival, dimer excision, DNA replication and breakdown in Escherichia coli B/r Hcr+ cells

SummaryUsing a model of double-UV-irradiation with inducing1 (non-lethal) and lethal fluences2 we have studied involvement of UV-inducible functions in post-UV-irradiation restoration processes and survival of Escherichia coli B/r thy-thy- Hcr+. Cells irradiated with both inducing and lethal fluences differed from cells irradiated with lethal fluence in the following respects: They were more UV resistant; they did not die during postincubation with chloramphenicol3; they exhibited a significant reduction in dimer excision; they were able to resume DNA replication and produce normal-sized DNA molecules in the presence of chloramphenicol. Since induction was provoked in cell prestarved for amino acids it was not associated with damage to points active in replication. However, the inducible product was more important for repair of replicating than non-replicating cells. The data indicate that protein necessary for resumption of DNA synthesis after UV is not constitutive but inducible.

Uv-inducible repair II: its role in various defective mutants of Escherichia coli K-12.

SummaryInvolvement of UV-inducible protein(s) in repair of various E. coli K-12 cell strains has been investigated using a procedure of double UV irradiation and postincubation with chloramphenicol.From the course of dose survival curves the following conclusions concerning significance of a UV-inducible protein have been drawn: 1. It is very improtant for wild type cells; in these cells its early occurrence is necessary to prevent killing. 2. It is involved in repair of excision-deficient cells; however, its action early after UV is less urgent. 3. It is not involved at all in reapir of lex mutant cells; 4. It exhibits some effect on survival of recA as well as recB mutant cells.We conclude that the protein is involved in excision repair as well as in resumption of DNA replication.

Depression of thymine dimer excision in various excision-proficient strains of Escherichia coli

Abstract A simultaneous starvation of thymine and amino acid applied prior to ultraviolet irradiation may cause a depression of thymine dimer excision. The starvation need not cause either death of the cells during the treatment or a decrease of the surviving ability after irradiation. The above effects may be obtained in various excision-proficient cells of Escherichia coli when proper starvation conditions are employed.

THE EFFECT OF THE OMPT PROTEASE ON EXCISION REPAIR IN UV-IRRADIATED ESCHERICHIA COLI

The extent of pyrimidine dimer excision (PDE) was inhibited in UV-irradiated E. coli KS272 (ompT+) cells when they were preinduced by a low UV predose preceded by a nutrition stress but not in the preinduced E.coli SF100 (ompT-) mutants. The preinduction, however, markedly inhibited PDE in the ompT- cells transformed with a multicopy plasmid carrying ompT gene. The data are consistent with the hypothesis that the inducible OmpT protease (controlled by rpoH) might terminate the SOS period of excision repair so that when cells are preinduced PDE might be inhibited prematurely.

DNA synthesis in pretreated and ultraviolet-irradiated cells ofEscherichia coli B/rHcr + andHcr −

DNA synthesis after the ultraviolet irradiation was followed in the excision proficient strainEscherichia coli B/rHcr+, in which the ability to excise thymin dimers was suppressed by a preirradiation inhibition of DNA and protein syntheses and in the excision deficient strainEscherichia coli B/rHcr−. Synthesis of pulse-labeled DNA, its stability and semiconservative DNA synthesis were compared in both strains. It was found that cells of theHcr+ strain restore semiconservative DNA synthesis and the pulselabeled DNA appears stable, in spite of the fact that dimers are not excised under these conditions. On the other hand, cells of theHcr− strain are unable to restore semiconservative DNA synthesis and the pulselabeled DNA is degraded. As the repair by the excision of dimers under the used experimental conditions may be excluded in both strains, it is possible to assume that activity of enzymes included in theHcr+ marker is prerequisite for restoring the DNA synthesizing system in theHcr+ strain.

Effect of plasmid pKM101 in ultraviolet irradiated uvr+ and uvr- Escherichia coli

The effect of plasmid pKM101 on UV irradiated excision proficient and excision deficient cells was investigated. The plasmid increased the survival of excision proficient cells while partially inhibiting thymine dimer excision. The frequency of mutations was almost unchanged. In excision deficient cells the effect of the plasmid on survival was less pronounced while cell mutability was increased. Our data indicate that the mucAB genes (carried by the plasmid) influence the two types of cells in a different way.

The presence of alkali-labile sites in DNA daughter chains of UV-irradiated Escherichia coli

DNA newly synthesized in UV irradiated Escherichia coli B/r Hcr+ was 2 min pulse‐labeled at various periods, then denatured and analysed by sucrose gradient centrifugation either in neutral or in alkaline conditions. Data indicate that in DNA of damaged cells alkali‐labile sites are produced. In cells saturated with inducible proteins production of alkali‐labile sites disappears in ∼1 h. In the absence of inducible proteins production of alkali‐labile sites continues.

Constitutive increase of RecA protein: its influence on pyrimidine dimer excision and survival of UV-irradiated Escherichia coli

Transformation of E. coli with the plasmid pRA containing recA gene increased the constitutive level of RecA protein 50-67 fold. This slightly inhibited pyrimidine dimer excision and reduced cell survival in three investigated, UV-irradiated E. coli strains. Our data support the view that RecA protein prematurely present at a high level may mask the dimers. The masking subsequently reduces the dimer excision and switches off the inducing signal.

Role of UV-inducible proteins in repair of various wild-type Escherichia coli cells

Abstract 3 wild-type strains of E. coli , namely K12 AB2497, B/r WP2 and 15 555-7v proficient in excision and post-replication repair, differ markedly in their UV resistance. To elucidate this difference, the influence was investigated of induction by application of inducing fluence (IF) before lethal fluence (LF) on repair processes after LF. In cells distinguished by low UV resistance ( E. coli 15 555-7; E. coli B/r WP2), dimer excision was less complete in cultures irradiated with IF + LF than in cultures irradiated with LF only. The highly resistant E. coli K12 AB2497 performed complete excision both after IF + LF or after LF alone. All 3 types of cell survived better after IF + LF than after LF only. Because, in most strains so far investigated, the application of IF reduced dimer excision and increased survival, dimer excision per se does not appear important for survival. We conclude that the rate and completeness of dimer excision can serve as a measure of efficiency of the excision system whose action is necessary for repair of another lesion. Cells of all investigated strains could not resume DNA replication and died progressively when irradiated with LF and post-incubated with chloramphenicol (LF CAP + ). Thus, it appears that inducible proteins are necessary for repair in all wild-type E. coli cells give with potentially lethal doses of UV irradiation.

Repair, replication and survival in UV-irradiatedEscherichia coli

The influence of dimer removal through excision or photoreactivation on the kinetics of DNA synthesis, sedimentation profiles of DNA molecules and survival of cells was investigated in excision-deficient and excision-proficientEscherichia coli K-12 after a flux of 20 J m−2. In excision-deficient cells photoreactivation did not influence the kinetics of DNA synthesis for a long period and the sedimentation properties of DNA synthesized immediately after photoreactivation were influenced only slightly. However, survival was increased remarkably. In excision-proficient cells where dimers were removed through excision, the kinetics of DNA synthesis increased rapidly, normal-sized DNA molecules were synthesized 60 min after irradiation and survival was substantially higher than in the above-mentioned case. This can hardly be interpreted as a more complete repair of dimers by excision because the persistence of dimers in these cells did not significantly influence either the kinetics of DNA synthesis or normalization of DNA molecules and/or survival of cells. It is concluded that persisting dimers play an important role in excision-deficient but not in excision-proficient cells, that a non-dimer damage to DNA causes inhibition of DNA synthesis after UV and that this damage ia of primary importance for excision-proficient cells which can easily cope with persisting dimers.