Miroslav Piršel

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.

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.

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.

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.

The pre-UV nutritional stresses increase UV resistance, decrease UV mutagenesis and inhibit excision repair.

Nutritional stresses applied to E. coli prior to UV irradiation increase UV resistance and decrease UV mutagenesis. This effect is uvrA-dependent and might reflect a more efficient excision of pyrimidine dimers [1]. The data presented here, however, indicate that after prestarvation for glucose or amino acids pyrimidine dimer excision (PDE) was partly inhibited. It appears that the stress conditions stimulate a mode of uvr-dependent tolerance of lesions, efficient and precise. Possible modes of PDE inhibition and lesion tolerance are discussed.

DNA of Chinese hamster V79 cells newly synthesized after ultraviolet irradiation contains alkali-labile sites

SummaryPulse-labeled daughter DNA of UV-irradiated Chinese hamster V79 cells was denatured in alkaline or neutral conditions and analysed by sucrose gradient centrifugation. A comparison of the sedimentation profiles of DNA treated in alkaline or neutral conditions has shown that in UV-irradiated cells some alkali-labile sites are produced during replication of damaged templates.

Inducible stable DNA replication (iSDR) and the uvr-dependent tolerance of pyrimidine dimers in UV-irradiated Escherichia coli are two uncoupled processes

Inducible stable DNA replication (iSDR) is dependent on recombination and is supposed to play a role in DNA repair of Escherichia coli. Our previous data suggested that iSDR may be involved in the tolerance of UV lesions, which remain unexcised in excision-proficient E. coli exposed to some UV pretreatments. Now, the tolerance of unexcised lesions has been followed in E. coli recB21 and in E. coli priA1 sup mutants, incapable of iSDR. The obtained data do not confirm the previous hypothesis about the involvement of iSDR in the putative uvr-dependent lesion tolerance. They rather suggest that iSDR and the uvr-dependent lesion tolerance are two uncoupled processes.