Milena Sedliaková

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.

Changes in the radioresistance of bacteria after the inhibition of proteosynthesis in the preirradiation phase

The strain ofEscherichia coli WP2 (tryv) was irradiated with UV light, at a dosage of 240 erg/mm. Proteosynthesis was inhibited by the elimination of the essential amino acid from the cultivation medium. Changes in radioresistance were followed during 45 minutes of starvation and during the subsequent 45 minutes of restitution after the addition of the essential amino acid. The radioresistance of the cells showed a linear increase immediately after the removal of the essential amino acids, proportional to the duration of the inhibition of proteosynthesis. The increase in radioresistance was shown to be reversible. After the addition of the essential amino acid there was an immediate decrease in radioresistance which was most marked in the first 15 minutes.

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.

RELATIONSHIP BETWEEN SURVIVAL AND THYMINE‐DIMER EXCISION IN ULTRAVIOLET‐IRRADIATED ESCHERICHIA COLI

Abstract— The influence of amino acid prestarvation on both the resistance to u.v. light and excision of thymine dimers of bacterial strains E. coli B/r hcr+ thy‐ trp‐, E. coli B/r hcr‐thy‐trp‐, and E, coli 15 T‐ 555–7 thy‐meth‐trp‐arg‐ has been studied.

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.

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].

A NON-EXCISION UVR-DEPENDENT DNA REPAIR PATHWAY OF ESCHERICHIA COLI (INVOLVEMENT OF STRESS PROTEINS)

In UV-irradiated excision-proficient (uvr+) Escherichia coli, pre-induced by simultaneous pre-starvation for thymine (T) and amino acids (AAs), and/or a low UV pre-dose applied after prestarvation for AAs, pyrimidine dimer excision (PDE) is reduced without an adequate increase of UV sensitivity and UV mutagenesis. The unexcised lesions are tolerated by a putative repair pathway that is uvr dependent but does not involve excision. The process consists of PDE inhibition, which requires outer membrane protease OmpT, and subsequent pyrimidine dimer (PD) toleration, which may be mediated by interaction with a sister duplex using a number of SOS and stress-inducible proteins.