František Mašek

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.

Thymine-dimer excision after the preirradiation inhibition of DNA synthesis.

Ultraviolet radiation impairs cells mainly by the production of pyrimidine dimers in DNA molecules. These lesions can either be removed by excision or tolerated by postreplication repair (for review, see [1] ). Excision has been assumed to be a highly efficient tool enabling the cells to cope with a great number of UV lesions [2]. Thus it was surprising to find that the excision of dimers was rather depressed after amino acidless pretreatment which considerably enhanced the fraction of surviving cells [3]. In this paper the influence of the pre-irradiation inhibition of DNA synthesis on thymine-dimer excision is reported. As demonstrated, the excision process can be considerably inhibited by the inhibition of DNA synthesis before irradiation.

Dependence of DNA dark repair on protein synthesis inEscherichia coli

SummaryWe investigated the influence of aminoacidless treatments applied prior and after UV irradiation on survival, dimer excision, postirradiation DNA degradation, DNA synthesis and sedimentation profiles of parental DNA ofE. coli B/r Hcr+ cells. In dependence on the treatment applied, the fluence 50 J/m2 yielded distinctly different fractions of survivors within 0,03–85%. In all cases dimers were completely excised. The rate of DNA degradation was similar during a 30–40 min period after UV during which the bulk of dimers was excised. Degradation ceased, however, earlier in the prestarved cells than in exponentially growing ones; it was prolonged by aminoacidless postincubation. Sedimentation profiles of parental DNA did not differ during the whole period of dimer excision. In cells DNA synthesis was not restored for several hours after addition of amino acids. In cells addition of amino acids resulted in a fast resumption of DNA synthesis. We conclude that removal of dimers and repair of gaps were similar in all cases. We believe that aminoacidless treatments influence production and repair of damage to the sites of DNA replication. The treatment appears to prevent this damage when applied before UV irradiation, but interferes with its restoration when applied after UV irradiation. Consequently, the former treatment increases survival of cells while the latter produces an opposite effects.

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.

Effect of amino acid starvation on the degradation of DNA in Escherichia coli B/r after UV irradiation.

Resistance of bacterial cells to UV irradiation can be considerably influenced by the inhibition of protein synthesis during their preirradiation growth [l] . The inhibition of protein synthesis, for example by omitting essential amino acids, results in a delay of DNA synthesis and a delay of cell division after W irradiation. Thus the cells are given more time for repair of damaged genetic material prior to its replication, so that before its replication the UV damaged DNA molecule is more or less repaired [2] . As was shown later, a condition for the increase of bacterial UV resistance is that the cells are capable of repair; the UV sensitive E. coli B/s, mutant, containing bothexr-and hermutations does not display any increase of resistance after amino acid prestarvation [3] . From this it is possible to conclude that the pretreatment in some way affects the repair processes. It was found that the increase of W resistance after amino acid prestarvation does not result in increase of the repair synthesis [4]. Similarly no direct relationship was found between increase 6f UV resistance following amino acid prestarvation and excision of pyrimidine dimers [S] . These findings led to consideration of the size of the gaps after the excision of dimers. It was postulated that the activity of exonucleases after prestarvation could be partially inhibited, so that the gaps, after dimer excision, would be smaller than in logarithmic cells. Therefore, the same amount of repair synthesis would be sufficient to repair a greater number of gaps [4].

Inhibition of pyrimidine dimer excision in ultraviolet-irradiated Escherichia coli overproducing RecA protein.

Escherichia coli Br Hcr+ cells transformed with the recombinant multicopy plasmid pBR322 carrying recA gene contain increased amounts of RecA protein. When these cells were UV-irradiated, excision of pyrimidine dimers was reduced by about 50%. It is suggested that the damaged DNA strands may be coated with RecA protein which makes them insensitive to the action of the uvrABC excision nuclease.

Error-free uvr+-dependent Inducible DNA Repair in Escherichia Coli B/r Hcr+ Cells

The frequency of suppressor (tryptophan reversions) and of true (streptomycin-resistant and dependent) mutations has been followed in E. coli cells irradiated with a single dose or two separate doses of ultraviolet (U.V.) radiation. Under these conditions dimers were efficiently excised after a single dose, while about 40 per cent of the dimers remained unexcised after two doses. Although the level of unexcised dimers in the latter case increased proportionally with the second U.V. dose, the mutation frequency increased by 1.5-2-fold, but did not continue to increase with the level of unexcised dimers. A comparison of excision-proficient and excision-deficient cells containing similar amounts of persisting dimers has shown that proficient cells can tolerate a high level of dimers without an adequate increase in mutation frequency. Our results suggest the existence of an error-free uvr+-dependent inducible repair in E. coli B/r Hcr+ cells.

Inhibition of dimer excision in repeatedly UV-irradiated Escherichia coli: Its requirement for RecA protein and de novo protein synthesis

In UV-irradiated Escherichia coli dimer excision was found to be inhibited by predamage (M. Sedliaková, F. Masek and J. Brozmanová, FEBS Lett., 23 (1972) 325-326) or overproduction of RecA protein, which suggests that the coating of the dimers by this protein may make them inaccessible to the excision nuclease (M. Sedliaková, K. Kleibl and F. Masek, Mutat. Res., 191 (1987) 13-16). We measured the levels of RecA protein and dimer excision in cells irradiated with (i) a single dose of 50 J m-2, (ii) two separate doses of 30 and 50 J m-2, post-incubated with chloramphenicol; (iii) two separate doses of 30 and 50 J m-2, post-incubated without chloramphenicol. Dimer excision was complete in the first two cases, but in the latter it was inhibited by 40%. At the time of active dimer excision, there were marked differences in RecA protein content between the cells irradiated with a single dose and cells irradiated with two separate doses (both post-incubated without chloramphenicol), which might account for the differences in dimer excision. However, relatively small differences in RecA protein content were found in cells irradiated with two doses and post-incubated with or without chloramphenicol, which could therefore not account for the differences in dimer excision. The data suggest that the inhibition of dimer excision involves some short-lived component(s) other than RecA protein.

Inducible stable DNA replication in Escherichia coli uvr− and uvr− cells treated with genotoxic chemicals

Inducible stable DNA replication (iSDR) provoked by a damaging treatment with MMS, MNU, MNNG, NFAA, NFN, 4NQO, NAL or MMC, was followed in both repair-competent E. coli PQ35 and its uvrA derivative E. coli PQ37. In contrast to SOS-inducible mutagenesis, which is more pronounced in excision-deficient cells, iSDR was more obvious in repair-competent cells. This may be due to special features of iSDR and need not indicate involvement of the uvrA gene product in it.