Jan Retèl

The Ambivalent Role of Glutathione in the Protection of Dna Against Singlet Oxygen

Glutathione (GSH) was examined with respect to its ability to protect DNA against 1O2 damage. We have found that GSH protected, at least partly, the DNA against inactivation by 1O2. Up to 10 mM the protection increased as a function of GSH concentration. Above 10 mM the protection remained constant and less than expected on the basis of scavenging/quenching of 1O2, in contrast to the protection offered by sodium-azide. Especially at the higher concentrations of GSH the protection against the biological inactivation is accompanied by an increase in single-strand breaks and also probably lethal base damage. However, all together the data suggest that at least in the physiologically important range (0.1-10 mM) GSH is able to protect efficiently against 1O2-induced inactivating DNA damage.

Effects of the ortho-quinone and catechol of the antitumor drug VP-16-213 on the biological activity of single-stranded and double-stranded ΦX174 DNA

We have studied the effects of the recently reported two new metabolites of the antitumor agent VP-16-213, the ortho-dihydroxy derivative or catechol and the ortho-quinone, on the biological activity of single-stranded and double-stranded phi X174 DNA, the binding of the metabolites to calf thymus DNA and the conversion of the catechol into the ortho-quinone. Evidence was obtained for the oxidation of the catechol into the ortho-quinone and for the fact that the ortho-quinone is the metabolite of VP-16-213 responsible for its binding to rat liver microsomal proteins. The catechol and ortho-quinone of VP-16-213 were found to bind 7-9 times more strongly to calf thymus DNA than VP-16-213 itself. In contrast to the parent compound VP-16-213, the catechol as well as the ortho-quinone inactivated both single-stranded (ss) and double-stranded (RF) biologically active phi X174 DNA. The mean T37-values for inactivation of ss and RF phi X174 DNA by 2.2 x 10(-4)M catechol at 37 degrees and pH 7.4 were 96 and 640 min, respectively. Reduction of the ortho-quinone by NADPH cytochrome P-450 reductase resulted in formation of the catechol. The system ortho-quinone/NADPH cytochrome P-450 reductase inactivated ss phi X174 DNA with a mean T37-value of 454 min, and this inactivation was inhibited by DMSO. The mean T37-value for inactivation of ss phi X174 DNA by 1.8 x 10(-4) M ortho-quinone at 37 degrees and pH 4.0 was 24 min. The chemical stability of the ortho-quinone and the extent of inactivation of ss phi X174 DNA by the ortho-quinone were both pH-dependent: at higher pH the ortho-quinone was less stable and gave less inactivation of DNA. The aqueous decomposition product(s) of the ortho-quinone formed at pH 7.4 inactivated ss phi X174 DNA with a mean T37-value of 175 min. The rate of inactivation of RF phi X174 DNA by the ortho-quinone at pH 4.0 was twice as low as the rate of inactivation of ss phi X174 DNA: T37 = 49 min. When using excision repair deficient E. coli mutants (uvrA- or uvrC-), a higher inactivation of RF phi X174 DNA was found: T37 = 29 min for uvrA- E. coli, indicating that a part of the DNA damage introduced by the incubation with ortho-quinone is removed by excision repair.(ABSTRACT TRUNCATED AT 400 WORDS)

Structure and transcription specificity of yeast RNA polymerase A.

DNA-dependent RNA polymerase A (Nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) was isolated from whole yeast cells and purified to a nearly homogeneous state. The subunit structure as well as the transcription specificity of the purified enzyme were investigated. Polyacrylamide gel electrophoresis under denaturating conditions revealed that yeast polymerase A is made up of two large subunits having mol. wts of 190 000 and 135 000, and five smaller subunits with mol. wts of 54 000, 44 000, 35 000, 25 000 and 16 000, respectively. The molar ratios of all these polypeptides were found to be about unity. The transcription specificity of yeast polymerase A was tested using homologous nuclear DNA as a template. The in vitro synthesized RNA was characterized by determining its degree of self-complementarity and its ability to compete with purified ribosomal RNA in hybridization experiments. It was found that yeast polymerase A is capable of a highly selective transcription in vitro of the rRNA cistrons, provided DNA of high integrity is used as a template.

Inactivation of φX174 DNA by the ortho-quinone derivative or its reduction product of the antitumor agent VP 16-213

Biologically active phi X174 DNA is inactivated by the ortho-quinone derivative of the antitumor agent VP 16-213, but not by VP 16-213 itself, VP 16-213 phenoxy radical or aqueous decomposition product(s) of the ortho-quinone. Reduction of the ortho-quinone by cytochrome P-450 reductase and NADPH results in deactivation of the ortho-quinone towards anti-phi X174 DNA activity. However, compared with the parent compound VP 16-213, reduction of the ortho-quinone results in substantial damage towards DNA.

C/G to A/T transversions represent the main type of mutation induced by γ-irradiation in double-stranded M13mp10 DNA in a nitrogen-saturated solution

To get more insight into the possible mutagenic consequences of DNA damage induced by radiation-generated H radicals (.H), a nitrogen-saturated solution of double-stranded (ds) M13mp10 DNA in phosphate buffer was irradiated with gamma-rays. Under these conditions 55% of the DNA-damaging species consists of H radicals and 45% of OH radicals (.OH). The mutations were investigated in a 144-bp mutational target sequence inserted into the lacZ alpha gene. A very specific mutation spectrum was obtained with respect to the type of mutations. Twenty out of the 28 radiation-induced mutations were C/G to A/T transversions; the remaining 8 mutations were 4 C/G to G/C transversions, 2 C/G to T/A transitions, one T/A to A/T transversion and only one -1 bp deletion. The mutations were rather randomly distributed along the 144-bp mutation target sequence with no clear mutational hot spots. When these results are compared with those we have obtained previously after irradiation of ds M13mp10 DNA under O2 (100% .OH) or N2O (90% .OH; 10% .H) (Hoebee et al., 1988, 1989), the data strongly suggest that H radicals may be responsible for the observed C/G to A/T transversions but not for -1 bp deletions.

Influence of nucleotide excision repair of Escherichia coli on radiation-induced mutagenesis of double-stranded M13 DNA

To investigate a possible role of nucleotide excision repair (NER) of E. coli in the removal of gamma-radiation-induced DNA lesions, double-stranded M13mp10 DNA, which contains a part of the lac operon, including the promoter/operator region, the lacZ alpha gene and a 144 basepair (bp) inframe insert in the lacZ alpha gene, as mutational target was gamma-irradiated in a phosphate buffer under N2. Subsequently, the radiation-exposed DNA was transfected to wild-type or NER-deficient (uvrA-) E. coli, mutants in the mutational target selected, followed by characterization of the mutants by sequence analysis. Both the mutations obtained from wild-type and uvrA- E. coli appeared to consist mainly of bp substitutions. However, in contrast to wild-type cells, a relatively large proportion of the mutations obtained from the NER-deficient cells (about 25%) is represented by -1 bp deletions, indicating that NER may be responsible for the removal of lesions which cause this particular type of frameshift. Comparison of the bp substitutions between both E. coli strains showed considerable differences. Thirty per cent of all bp substitutions in the NER-deficient host are T/A-->C/G transitions which are virtually absent in wild-type E. coli. This indicates that NER is involved in the elimination of lesions responsible for these transitions. This may also be true for a part of the lesions which cause C/G-->T/A transitions, which make up 52% of the bp substitutions in uvrA- cells versus 17% in wild-type cells. Strikingly, C/G-->G/C transversions appeared to be only formed in wild-type, where they make up 22% of all bp substitutions, and not in the NER-deficient E. coli. This result suggests, that due to the action of NER, a particular type of mutation may be introduced. A similar indication holds for C/G-->A/T transversions, which are predominant in wild-type (58%) and in the minority in uvrA- cells (15%).

Effect of the sulfhydryl compound cysteamine on gamma-radiation-induced mutations in double-stranded M13 DNA

Sulfhydryl compounds can protect DNA against free-radical-induced DNA damages not only by scavenging of radicals, but also by chemical non-enzymatic repair or modification of such damages by hydrogen-donation. To investigate the influence of chemical repair and modification on mutations, induced by gamma-radiation-generated free radicals (.OH, .H), phosphate-buffered aqueous solutions of double-stranded (ds) M13 DNA were exposed to gamma-rays under N2 in the presence of 5 mM cysteamine. The exposed DNA was subsequently transfected to wild-type E. coli and mutations in the mutational target were characterized. This target in fact contains three different target sequences, i.e., the lac promoter/operator, the lacZ alpha gene and a 144 bp inframe insert. The mutation spectrum obtained was compared with those in the absence of cysteamine under N2 and N2O. In the latter case, the ratio of .OH and .H available for reacting with DNA is about the same as under N2 + cysteamine. The results show that chemical repair and/or modification by cysteamine of potentially lethal lesions takes place, leading to a much higher survival of ds M13 DNA in the presence of cysteamine than could be expected on basis of scavenging of .OH and .H alone. This higher survival appeared to be accompanied with a higher mutation induction. However, the N2 + cysteamine mutation spectrum shows a remarkable resemblance with the N2O-spectrum. This holds for the total mutation target, as well as each of the three targets, although the mutations obtained in each of the three targets under the same irradiation conditions are quite different. Thus, it can be concluded that cysteamine is mainly effective on radiation-induced potentially lethal DNA lesions, and not so much on (pre)mutagenic damages. Moreover, the type of mutation appeared to be strongly dependent on the mutational target sequence.

Topographical analysis of yeast ribosomal DNA by cleavage with restriction endonucleases

Yeast ribosomal DNA (rDNA) was digested with the restriction enzymes Hind III, Hind II and a mixture of Hind II and Hind III. The cleavage products were analyzed by electrophoresis on 1.5% agarose gels. Several distinct bands could be observed, which are derived from the redundant ribosomal transcription units. They are superimposed on a rather broad smear of background DNA, representing the heterogenous ‘spacer’ sequences. From the restriction maps, together with data obtained by partial digestion, a physical map for the ribosomal transcription unit in yeast could be constructed.

Interaction of RSU 1069 and 1137 with DNA in vitro: Biological implications and mechanistic aspects

We have examined the capacity of the nitroimidazole aziridine antitumour drug RSU 1069 to react with DNA in vitro in order to get a better understanding of its mechanism of action. Moreover, we have utilized biologically active phi X174 DNA to investigate the biological relevance of the chemical DNA modification induced by the drug. Incubation of RSU 1069 in the presence of single-stranded phi X174 DNA resulted in extensive inactivation of the DNA, which is dependent on the concentration of drug and temperature. Only about 2% of the inactivating damage can be attributed to strand breakage. The main damage most probably consists of base damage, of which a part is non-lethal and alkali-labile which in turn can be converted into lethal lesion and subsequently into a break applying a post-incubation alkali treatment. Furthermore, from the dependence of the inactivation and also the formation of breaks on pH and ionic strength, it is concluded that the reaction most probably takes place between a protonated RSU 1069 and a negative DNA coil and that the damage pattern reflects the difference in reactivity of RSU 1069 with the phosphate groups and the bases in DNA. Comparison between RSU 1069 and its ring-open hydrolysis product RSU 1137 revealed that (lethal) damage induced in the DNA must be ascribed to the alkylating properties of the aziridine moiety.

Inactivation and repair of double-stranded DNA damaged by the aziridinyl nitroimidazole RSU 1069

Incubation of RSU 1069 in the presence of biologically active double-stranded phi X174 DNA resulted in, depending on pH, ionic strength and concentration of drug, inactivation of the DNA. A variety of lesions are induced including a high number of single-strand breaks and alkali-labile lesions, which are at most partly lethal. The main inactivating damage consists probably of base damage, induced by alkylation. A considerable part of the damage induced by RSU 1069 can be repaired by the various repair enzymes of the bacterial host of the phi X174 DNA. Finally the damage (pattern) depends considerably on the ionic composition of the reaction solution, which can be explained by an equilibrium model presented in this paper.