The effect of spermine binding on the reactivity of DNA towards carcinogenic alkylating agents

The effect of spermine binding on the electrostatic potential of DNA is evaluated. The calculations are performed for the essential reactive sites, atoms N7 and O6 of guanine, N3 and N7 of adenine, of the nucleic acid and for its surface envelope. An important weakening of the potential is found affecting all the important reactive sites in both grooves and spreading moreover along the polynucleotide chain far away from the site of binding of the ligand. These results are discussed in connection with the experimentally observed inhibitory effect of spermine binding on DNA methylation by carcinogenic agents.     

Synthesis of paf-acether by E. coli K12

Paf-acether (platelet-activating factor) is one of the most potent mediator of inflammation released from and acting on most cells that participate in inflammatory diseases. Its molecular structure is 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine. Two metabolic steps are involved in its biosynthesis: the action of a phospholipase A2 on choline-containing membrane alkyl-ether lipids results in the production of lyso paf-acether and acetylation of the lyso compound by an acetyltransferase yields the biologically active molecule. Membrane alkyl-ether lipids can therefore be considered as potential precursors of paf-acether and their composition has been studied in various cell types. In this work, we investigated the presence of paf-acether in E. coli. Our results showed that paf-acether can be obtained from E. coli K12 under a variety of bacterial growth conditions. Paf-acether from E. coli exhibited the same physicochemical and biological characteristics as synthetic paf-acether and that from eucaryotic cells. Therefore, it appears that E. coli itself has the ability of producing paf-acether, a result that could be of some importance with respect to the pathogenesis of Enterobacteria and the use of E. coli in the recombinant DNA technology.     

A theoretical evaluation of the effect of netropsin binding on the reactivity of DNA towards alkylating agents.

The effect of netropsin binding on the electrostatic potential of DNA reactive sites is presented. Calculations are performed for atoms N7 and O6 of guanine, N3 and N7 of adenine of model, 25 base pair long, DNA-netropsin complexes. An important weakening of the potential is found spreading along all the oligonucleotide chain studied. The results are discussed in connection with the inhibitory effect of a related ligand, distamycin A, on DNA methylation.     

Pathways of mutagenesis and repair in Escherichia coli exposed to low levels of simple alkylating agents.

Mutagenesis by simple alkylating agents is thought to occur by either a lexA+-dependent process called error-prone repair or a lex-independent process often attributed to mispairing during replication. We show here that error-prone repair is responsible for the majority of mutants formed after a large dose of alkylating agent, but it is unlikely that it contributes significantly to mutagenesis during exposure to low concentrations of these chemicals. The mutagenicity of these low doses of alkylating agent is reduced by a repair system constitutively present in lexA+ cells but absent in lexA mutants. This system reduces mutagenesis until a second error-free system, called the adaptive responses, can be induced [P. Jeggo, M. Defais, L. Samson, and P. Schendel, Mol. Gen. Genet, 157:1-9, 1977; L. Samson and J. Cairns, Nature (London) 267:281-283, 1977]. The adaptive response is capable of dealing with a much larger amount of alkylation damage than the constitutive system and, when induced, appears to be able to reduce mutagenesis by both decreasing the number of sites available for mutagenesis and delaying the induction of error-prone repair enzymes. Finally, we discuss a model of chemically induced mutagenesis based on these findings which maintains that the observed mutation frequency is dependent on a "race" between these two error-free systems and the two mutagenic pathways.     

Optimal conditions for mutagenesis by ozone in Escherichia coli K12

A procedure for the quantitative determination of ozone-induced maltose and methionine mutants in Escherichia coli K12 was defined. High yields of induced mutants with over 90% survival have been obtained when washed cells, taken from logarithmic phase of growth, were aerated by a stream of ozone (0.05–1.0 ppm) in buffer at pH 5.0 for 30 to 60 min. It is postulated that ozone itself is probably exerting directly both lethal and mutagenic effects on the cells via a primary effect on the permeability of the cellular membrane.     

Synergistic effect of an Escherichia coli mutator gene on mutagenesis by ultraviolet radiation and by alkylating agents

E. coli strains differing in a gene responsible for high spontaneous mutability (mut HI) were compared for their mutability by UV radiation and by the alkylating agents ethyl methanesulfonate and methyl methanesulfonate. All three exogenous mutagenic agents induced significantly higher frequencies of mutants with impaired carbohydrate-fermenting ability when the mutator allele rather than the wild-type allele was present. Thus the mut HI gene product possibly increases the probability of replication error due to alterations in the structure of the template strand of DNA. An attempt to detect an synergistic effect for UV-induced suppressor mutations was unsuccessful. The failure may have been due to the particular method used for scoring this type of mutation.     

The role of the gene umuC and plasmid muc genes in mutagenesis induced by dioxidine in E. coli K12

The mutability induced by dioxidine in E. coli cells has been shown to be stringently dependent on a function of chromosomal umuC+ gene. Suppression of an umuC mutation by plasmids pKM101 or ColIb, restoring the dioxidine induced mutability, proves the possibility of umuC gene functional complementation by the plasmid muc+ genes.     

Weigle reactivation and mutagenesis of bacteriophage lambda in lexA(Def) mutants of E. coli K12

Summary The SOS response in UV-irradiated bacteria enhances the survival and mutagenesis of infecting damaged bacteriophage . In a lexA(Def) strain, SOS bacterial genes are fully derepressed by an inactivating mutation in the LexA repressor gene. We tested several lexA(Def) derivative strains for their capacity to constitutively promote high survival and mutagenesis of irradiated . We showed that UV irradiation of the lexA(Def) host bacteria is still necessary for optimal efficiency of both these SOS functions, which are dependent on the umuC gene product and an activated form of RecA protein.     

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.     

Cell division and DNA synthesis in uvrA recA double mutants of E. coli K12

Summary Cell division and incorporation of ³H-thymidine into acid-insoluble fraction were investigated for three uvrA recA double mutants of E. coli K12 irradiated with UV at 1.5 ergs/mm², producing about ten pyrimidine dimers per genome (about 0.01% survival). Cell division was measured both in M9 medium and in the same medium which was made very viscous by the addition of Metlose (the same product as Methocel used by Lin et al., 1971). It was found that a major fraction of irradiated bacteria continues to divide once or twice and stops thereafter. Incorporation of ³H-thymidine proceeded at a considerable rate for a short period following irradiation and then stopped. During subsequent incubation, the incorporation gradually decreased and after 4 h incubation most of the early incorporated radioactivity disappeared from the acid-insoluble fraction. These results indicate that cell division occurs after irradiation without parallel DNA synthesis as in a recA thy mutant of E. coli K12 deprived of thymine (Inouye, 1971). These results suggest that UV irradiation increases lethal sectoring due to the reckless cell division without parallel DNA synthesis. Since DNA synthesis took place only for a short period after irradiation, it may be assumed that the recA gene normally has at least a dual function; 1. elimination of damage induced by UV to support elongation or initiation of DNA, and 2. maintenance of coordination between DNA synthesis and cell division.