Karol Kleibl

Molecular mechanisms of adaptive response to alkylating agents in Escherichia coli and some remarks on O6-methylguanine DNA-methyltransferase in other organisms

Alkylating agents are environmental genotoxic agents with mutagenic and carcinogenic potential, however, their properties are also exploited in the treatment of malignant diseases. O(6)-Methylguanine is an important adduct formed by methylating agents that, if not repaired, can lead to mutations and death. Its repair is carried out by O(6)-methylguanine DNA-methyltransferase (MTase) in an unique reaction in which methyl groups are transferred to the cysteine acceptor site of the protein itself. Exposure of Escherichia coli cells to sublethal concentrations of methylating agents triggers the expression of a set of genes, which allows the cells to tolerate DNA lesions, and this kind of inducible repair is called the adaptive response. The MTase of E. coli, encoded by the ada gene was the first MTase to be discovered and one of best characterised. Its repair and regulatory mechanisms are understood in considerable detail and this bacterial protein played a key role in identification of its counterparts in other living organisms. This review summarises the nature of alkylation damage in DNA and our current knowledge about the adaptive response in E. coli. I also include a brief mention of MTases from other organisms with the emphasis on the human MTase, which could play a crucial role in both cancer prevention and cancer treatment.

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.

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.

Effect of expression of the Escherichia coli nth gene in Saccharomyces cerevisiae on the toxicity of ionizing radiation and hydrogen peroxide.

Purpose: To examine the contribution of endonuclease III (Nth)‐repairable lesions to the cytotoxicity of ionizing radiation (IR) and hydrogen peroxide (H2O2) in the yeast Saccharomyces cerevisiae. Materials and methods: A selectable expression vector containing the E. coli nth gene was transformed into two different wild‐type strains (7799‐4B and YNN‐27) as well as one rad52 mutant strain (C5–6). Nth expression was verified by Western analysis. Colony‐forming assay was used to determine the sensitivity to IR and H2O2 in both stationary and exponentially growing cells. Results: The pADHnth‐transformed wild‐type (7799‐4B) strain was considerably more resistant than vector‐only transformants to the toxic effects of IR, in both stationary and exponential growth phases, although this was not the case in another wild‐type strain (YNN‐27). In contrast, there were no significant effects of nth expression on the sensitivity of the wild‐type cells to H2O2. Moreover, nth expression caused no effects on the H2O2 sensitivity in the rad52 mutant cells, but it led to a slight increase in sensitivity in these cells following IR, particularly at the highest dose levels used. Conclusions: Whilst other damage‐processing systems may play a role, DNA lesions that are substrates for Nth can also make a contribution to the toxic effects of IR in certain wild‐type yeast. Hence, DNA double‐strand breaks should not be considered the sole lethal lesions following IR exposure.

In UV-irradiated Escherichia coli PQ35 overproducing the RecA protein, expression of the sfiA gene and dimer excision are alleviated

SummaryEscherichia coli PQ 35 cells carrying thesfiA-:lacZ operon fusion were transformed either with a multicopy plasmid containing therecA gene (pHSG262recA) or with a multicopy plasmid alone (pHSG262). Both transformants were UV irradiated. Then induction of thesfiA gene and dimer excision were followed. Amplification of therecA gene partly inhibited bothsfiA gene induction and dimer excision. The following interpretation of this phenomenon is proposed. When the RecA protein is in bundance, pyrimidine dimers are quickly masked by it. The masked dimers are less efficiently distinguished by excision nuclease and do not provide the induction signal. Due to this, induction of thesfiA gene as well as dimer excision are inhibited early.

COOPERATION OF UV INDUCIBLE AND EXCISION REPAIR SYSTEMS IN DNA DARK REPAIR OF ESCHERICHIA COLI

ABSTRACT Our previous data have indicated that in wild type E. coli dimers can be either excised or tolerated by a uvr, recA, lex-dependent mechanism both beeing similarly efficient. In this report it is demonstrated that dimers which occur in DNA replicated after UV are no more sensitive to the extract from M. luteus although they can be detected by paper radiochromatography. It appears that in uvr+ cells dimers (or DNA distortions) are modified on replication thus becoming insensitive to the endonucleases. We suppose that in wild type cells lesions are tolerated through an incision-dependent copy choice bypass.

A recA-ada hybrid gene inducible by DNA damage

A damage-inducible expression vector was constructed in which the original recA structural gene was replaced by the protein-coding region of the ada gene. The O6-alkylguanine-DNA alkyltransferase encoded by the ada gene can be measured by a rapid and highly sensitive assay. The introduction of this construct into an appropriate host cell provides an effective bacterial assay for genotoxins.

O6-Alkylguanine-DNA-Alkyltransferase Gene Expression and the Cytotoxicity of Triazenes

DNA repair gene transfection has been used to examine the contribution of specific alkylation products in DNA to the toxic effects of triazenes. Expression of the E.coli O 6-alkylguanine (O 6-AlkG)-DNA-alkyltransferases (ATase) encoded by ada and ogt has been achieved in Chinese hamster lung fibroblasts by means of mammalian cell expression vectors. In comparison with parent vector transfected control cells such cells are more resistant to killing by the chloroethylating triazeno compound mitozolomide indicating that O 6-AlkG and/or O 4-alkylthymine (O 4-AlkT) are critical lesions. These results support the idea that in normal and in tumour cells, endogenous ATase gene expression may be a major factor in resistance to such agents.