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K Kleibl

Publications and source records attributed to K Kleibl.

At least 19 recordsLinked to original sources
PubMed2003

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 (77994B) 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.

Cell Survival↗
PubMed1998

Increasing DNA repair capacity in bone marrow by gene transfer as a prospective tool in cancer therapy.

Resistance of tumor cells to alkylating anticancer agents that produce adducts at the O6 position of guanine in DNA, the O6-alkylating agents, correlates with the expression of O6-alkylguanine-DNA alkyltransferase (ATase). O6-benzylguanine and related pseudosubstrates are able to inactivate human ATase in vitro and in vivo and they are being tested as chemotherapeutic adjuvants for enhancing the effectiveness of O6-alkylating drugs. On the other hand, the clinical consequences of ATase depletion may be fatal for some sensitive systems e.g. hematopoiesis. To overcome this problem, strategies for the protection of primary bone marrow cells by targeted transfer of pseudosubstrate-resistant ATase genes have been considered and recently achieved at the laboratory level. This approach could therefore be now extended to a clinical cancer gene therapy program.

Alkylation↗
PubMed1995

Escherichia coli, Saccharomyces cerevisiae, rat and human 3-methyladenine DNA glycosylases repair 1,N6-ethenoadenine when present in DNA.

The human carcinogen vinyl chloride is metabolized in the liver to reactive intermediates which generate various ethenobases in DNA. It has been reported that 1,N6-ethenoadenine (epsilon A) is excised by a DNA glycosylase present in human cell extracts, whereas protein extracts from Escherichia coli and yeast were devoid of such an activity. We confirm that the human 3-methyladenine-DNA glycosylase (ANPG protein) excises epsilon A residues. This finding was extended to the rat (ADPG protein). We show, at variance with the previous report, that pure E.coli 3-methyladenine-DNA glycosylase II (AlkA protein) as well as its yeast counterpart, the MAG protein, excise epsilon A from double stranded oligodeoxynucleotides that contain a single epsilon A. Both enzymes act as DNA glycosylases. The full length and the truncated human (ANPG 70 and 40 proteins, respectively) and the rat (ADPG protein) 3-methyladenine-DNA glycosylases activities towards epsilon A are 2-3 orders of magnitude more efficient than the E.coli or yeast enzyme for the removal of epsilon A. The Km of the various proteins were measured. They are 24, 200 and 800 nM for the ANPG, MAG and AlkA proteins respectively. These three proteins efficiently cleave duplex oligonucleotides containing epsilon A positioned opposite T, G, C or epsilon A. However the MAG protein excises A opposite cytosine much faster than opposite thymine, guanine or adenine.

Adenine↗
PubMed1994

Repair of O6-methylguanine and O4-methylthymine by the human and rat O6-methylguanine-DNA methyltransferases.

In order to compare the ability of the human and rat O6-methylguanine-DNA methyltransferases (transferases) to repair in vitro O6-methylguanine (O6-MeGua) and O4-methylthymine (O4-MeThy) residues, which are two mutagenic DNA adducts formed by alkylating agents, we have purified both proteins to homogeneity. Gel electrophoresis of the proteins shows that the O4-MeThy repair is due to the transfer of the methyl group from the alkylated base to the transferase molecules. However, both proteins repair with different efficiencies the O6-MeGua and O4-MeThy residues present in alkylated DNA, poly[d(G.C)], poly(dG.dC), or in alkylated poly[d(A.T)] and poly(dA.dT), respectively. Reaction of both proteins with either methylated residues follows a second-order kinetics. The rate constants are 1 x 10(9) M-1 min-1 for both proteins acting on O6-MeGua and 4.8 x 10(6) or 1.8 x 10(5) M-1 min-1 for the rat or human protein acting on O4-MeThy, respectively. The activity of the mammalian transferases on O4-MeThy present in a poly(dA.dT) substrate is inhibited by double-stranded DNA.

Animals↗
PubMed1993

Enhancement of the uvrA gene dosage reduces pyrimidine dimer excision in UV-irradiated Escherichia coli.

E. coli possesses an efficient repair mechanism able to remove pyrimidine dimers from UV-irradiated DNA, which is catalyzed by UvrABC endonuclease. In E. coli B/r Hcr+ cells transformed with a multicopy plasmid harboring a gene coding for UvrA, the excision capacity was greatly reduced. The course of thymine dimer excision was investigated using the enzymatic as well as the radiochromatographic method and the results are discussed in term of nonspecific interaction between the excess of UvrA protein and undamaged DNA duplex.

DNA Repair↗
PubMed1993

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.

Bacterial Proteins↗
PubMed1992

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.

Cloning, Molecular↗
PubMed1992

Increased resistance to the toxic effects of alkylating agents in tobacco expressing the E. coli DNA repair gene ada.

The protein coding region of the E. coli gene ada has been transferred to tobacco plants by a leaf disc transformation procedure involving an Agrobacterium tumefaciens Ti plasmid. Transformed plants were shown to be transgenic for the ada message and had increased levels of O6-alkylguanine DNA alkyltransferase activity. The N-methyl-N-nitrosourea- or taurinechlorethylnitrosourea-induced inhibition of growth of calluses or of cells in suspension was considerably lower in ada-transformed than in non-transformed plants. This indicates that O6-alkylguanine, O4-alkylthymine or phosphotriesters are growth-inhibitory lesions in tobacco.

Agrobacterium tumefaciens↗
PubMed1990

Expression of the E.coli ada gene in yeast protects against the toxic and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine.

The E.coli ada gene protein coding region has been ligated into an extrachromosomally replicating yeast expression vector downstream of the yeast alcohol dehydrogenase gene promoter region to produce pADH06C. The yeast strains SX46A, 7799-4B and VV-6 are deficient in endogenous O6-alkylguanine-DNA-alkyltransferase and transformation of these strains with this shuttle vector resulted in the expression of 1730, 1260 and 374 fmoles ada-encoded ATase/mg protein in stationary phase yeast: transformation with the parent vector had no effect on endogenous ATase activity which remained less than 2 fm/mg. In comparison with parent vector transformed yeast, all of the pADH06C-transformed strains showed an increase in the resistance to the toxic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). In addition, 7799-4B and VV-6 were more resistant to the mutagenic effects of this agent. These results indicate that the toxic and mutagenic effects of MNNG in yeast are mediated, at least in part, by DNA lesions than can be repaired by the E.coli ada gene product.

Alcohol Dehydrogenase↗
PubMed1989

Expression of the ogt gene in wild-type and ada mutants of E. coli.

O6-alkylguanine (O6-AlkG) DNA alkyltransferase (ATase) and alkylphosphotriester (AlkP) ATase activity have been quantitated individually in extracts of various E. coli strains by means of ATase specific DNA substrates. O6-AlkG ATase activity was higher than AlkP ATase activity in the wild-type strains F26, AB1157 and SB229 and in the ada- mutants PJ1, PJ3, PJ5 and PJ6 indicating a 5-70 times higher level of expression of the ogt gene than the ada gene. The ada- mutant strains BS23, BS73 and GW5352 expressed O6-AlkG ATase but not AlkP ATase activity indicating expression only of the ogt gene. Southern analysis of DNA from F26, BS23, BS73, PJ1 and GW5352 showed a consistent pattern of hybridisation to an ogt probe but not to an ada probe. Exposure of E. coli to adaptive doses of N-methyl-N-nitro-N-nitroso-guanidine (MeNNG) caused an increase in AlkP ATase activity in F26, AB1156, SB229, PJ1, PJ3, PJ5 and PJ6. O6-AlkG ATase activity also increased in F26, AB1157 and SB229 but decreased to almost undetectable levels in all other strains examined except PJ3 where it remained constant. MeNNG increased ada mRNA abundance in F26 but no ada mRNA was detected in BS23, BS73 or GW5352: there was no evidence for increased ogt mRNA in any of the strains examined. In a limited survey, other bacterial strains have been shown to possess an ogt-like ATase activity.

Blotting, Southern↗
PubMed1989

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

Escherichia coli PQ35 cells carrying the sfiA-::lacZ operon fusion were transformed either with a multicopy plasmid containing the recA gene (pHSG262 recA) or with a multicopy plasmid alone (pHSG262). Both transformants were UV irradiated. Then induction of the sfiA gene and dimer excision were followed. Amplification of the recA gene partly inhibited both sfiA gene induction and dimer excision. The following interpretation of this phenomenon is proposed. When the RecA protein is in abundance, 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 the sfiA gene as well as dimer excision are inhibited early.

Bacterial Proteins↗
PubMed1988

Transfection of murine multi-potent haemopoietic stem cells with an E. coli DNA alkyltransferase gene confers resistance to the toxic effects of alkylating agents.

O6-alkylguanine-DNA-alkyltransferase (ATase)-deficient murine haemopoietic stem cells were transfected, following electroporation, with a G418-selectable expression vector containing the protein coding region of the Escherichia coli ATase gene ada. Clones of cells that were resistant to G418 or the chloroethylating agent mitozolomide (Mz) were selected and most were shown to express very high levels of bacterial gene-encoded ATase. In comparison with control cells that were transfected with the parent vector, the ATase-expressing clones were considerably more resistant to the toxic effects of the methylating agents N-methyl-N-nitrosourea and methylmethanesulphonate or the chloroethylating agents Mz or taurine chloroethylnitrosourea, but unchanged in their susceptibility to the bis-chloroethylating agent nitrogen mustard. Thus alkylation damage in DNA that can be repaired by the E. coli ATase constitutes the principal lethal lesion produced by alkylating agents in murine haemopoietic stem cells and the ATase deficiency in these cells can be complemented by electroporation-mediated gene transfection.

Alkyl and Aryl Transferases↗
PubMed1987

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.

DNA Repair↗
PubMed1981

Evidence that dimers remaining in preinduced Escherichia coli B/r Hcr+ become insensitive after DNA replication to the extract from Micrococcus luteus.

In Escherichia coli B/r Her+ irradiated with two separate fluences, dimer excision is prematurely interrupted. The present study was designed to follow tha fate of dimers remaining unexcised. The results imply that these dimers (or distortions containing dimers) are transformed on replication from the state of sensitivity to the state of insensitivity to endonuclease from Micrococcus luteus. This conclusion is based on the following findings: (a) dimers were radiochromatographically detectable in DNA replicated after UV, which indicated that they were tolerated on replication. (b) Similar amounts of dimers were detected radiochromatographically both in DNA remaining unreplicated and DNA twice replicated after UV, This along with the low transfer of parental label into daughter DNA, indicated that dimers remained in situ in parental chains. (c) Immediately after UV, all parental DNA contained numerous sites sensitive to the extract from M. luteus. 2 h after UV, a portion of parental DNA still contained a number of endonuclease-sensitive (Es) sites, while another portion of parental DNA and all daughter DNA were free of Es sites. (d) The occurrence of parental DNA free of Es sites was not temporally correlated with dimer excision, but with the first round of DNA replication. (e) The amount of DNA free of Es sites corresponded to the amount of replicated DNA. (f) Separation of replicated and unreplicated DNA, and detection of Es sites in both portions separately showed that the replicated DNA was almost free of Es sites, whereas unreplicated DNA contained a number of such sites.

DNA Replication↗