gamma-Ray mutagenesis in bacteriophage T4 is not enhanced by oxygen.

As in the induction of r mutants in bacteriophage T4 by gamma-rays, the radiation-induced reversion of T4 amber mutants to wild-type was found to depend on the product of the DNA-repair gene x of the phage. Neither the efficiency of induction of r mutants nor the efficiency of reversion of ambers was enhanced by the presence of oxygen during irradiation. T4 differed in this respect from phage T7, for which no indication has been found that gamma-ray mutagenesis results from error-prone repair of DNA damage. Notwithstanding the substantial contribution of misrepair to mutation induction in T4, the efficiency of induction per base-pair observed for irradiation under oxygen was lower than that found previously for T7.     

Ultraviolet mutagenesis in bacteriophage T4. II. Photoreversal of mutational lesions

Drake, John W. (University of Illinois, Urbana). Ultraviolet mutagenesis in bacteriophage T4. II. Photoreversal of mutational lesions. J. Bacteriol. 92:144-147. 1966.-T4r mutations were induced by ultraviolet irradiation of extracellular phage particles, using a phage mutant, v, which is particularly susceptible to photoreactivation. Most of the induced r mutations could be subsequently photoreversed intracellularly with white light. Ultraviolet irradiation induces both transitions and sign mutations, and both were susceptible to photoreversal. The results suggest that two very different types of mutational lesions may arise from a common type of photochemical lesion.     

Clone size distributions of mutations induced by ethyl methanesulfonate in bacteriophage T4

Size distributions of mutant clones can reveal important aspects of the mutation process. Previously published data on mutant clones induced by ethyl methanesulfonate (EMS) in bacteriophage T4 generated a distribution that was essentially flat, implying a mutagenic mechanism involving only rare mispairing by reacted bases. Here, methods for estimating the spontaneous component of such a distribution are used to generate a corrected distribution. The corrected distribution is strongly peaked, implying frequent (but not obligatory) mispairing. Frequent mispairing is in accord with current views of the fates of DNA lesions believed to mediate EMS-induced mutagenesis.     

Cytotoxicity of alkylating agents towards sensitive and resistant strains of Escherichia coli in relation to extent and mode of alkylation of cellular macromolecules and repair of alkylation lesions in deoxyribonucleic acids

1. A quantitative study was made of the relationship between survival of colony-forming ability in Escherichia coli strains B/r and B(s-1) and the extents of alkylation of cellular DNA, RNA and protein after treatment with mono- or di-functional sulphur mustards, methyl methanesulphonate or iodoacetamide. 2. The mustards and methyl methanesulphonate react with nucleic acids in the cells, in the same way as found previously from chemical studies in vitro, and with proteins. Iodoacetamide reacts only with protein, principally with the thiol groups of cysteine residues. 3. The extents of alkylation of cellular constituents required to prevent cell division vary widely according to the strain of bacteria and the nature of the alkylating agent. 4. The extents of alkylation of the sensitive and resistant strains at a given dose of alkylating agent do not differ significantly. 5. Removal of alkyl groups from DNA of cells of the resistant strains B/r and 15T(-) after alkylation with difunctional sulphur mustard was demonstrated; the product di(guanin-7-ylethyl) sulphide, characteristic of di- as opposed to mono-functional alkylation, was selectively removed; the time-scale of this effect suggests an enzymic rather than a chemical mechanism. 6. The sensitive strain B(s-1) removed alkyl groups from DNA in this way only at very low extents of alkylation. When sensitized to mustard action by treatment with iodoacetamide, acriflavine or caffeine, the extent of alkylation of cellular DNA corresponding to a mean lethal dose was decreased to approximately 3 molecules of di(guanin-7-ylethyl) sulphide in the genome of this strain. 7. Relatively large numbers of monofunctional alkylations per genome can be withstood by this sensitive strain. Iodoacetamide had the weakest cytotoxic action of the agents investigated; methyl methanesulphonate was significantly weaker in effect than the monofunctional sulphur mustard, which was in turn weaker than the difunctional sulphur mustard. 8. Effects of the sulphur mustards on nucleic acid synthesis in sensitive and resistant strains were studied. DNA synthesis was inhibited in both strains at low doses in a dose-dependent manner, but RNA and protein synthesis were not affected in this way. 9. DNA synthesis in E. coli B(s-1) was permanently inhibited by low doses of mustards. In the resistant strains 15T(-) and B/r a characteristic recovery in DNA synthesis was observed after a dose-dependent time-lag. This effect could be shown at low doses in the region of the mean lethal dose. 10. Cellular DNA was isotopically prelabelled and the effect of mustards on stability of DNA was investigated. With resistant strains a dose-dependent release of DNA nucleotide material into acid-soluble form was found; this was much more extensive with the difunctional mustard (about 400 nucleotides released per DNA alkylation) than with the monofunctional mustard (about 10 nucleotides per alkylation). With the sensitive strain no dose-dependent release was found, though the DNA was less stable independent of cellular alkylation. 11. The results are discussed in terms of the concepts that alkylation of cellular DNA induces lesions which interfere with DNA replication, but which can be enzymically ;repaired'. The possible nature of these lesions is discussed in terms of the known reactions of the alkylating agents with DNA.     

Altered properties of deoxyribonucleic acid polymerase I isolated from the membrane of bacteriophage T4-infected Escherichia coli.

Previous studies have shown that a large fraction of the host cell deoxyribonucleic acid (DNA) polymerase I (EC 2.7.7.7) becomes associated with the cell membrane shortly after infection with bacteriophages T4 and T7. The present investigation of the bound enzyme revealed that the polymerase activity can be eluted from the membrane with chelating agents, and that the material thus obtained shows many properties that distinguish it from purified DNA polymerase I. These include its chromatographic behavior, sedimentation rate, sensitivity to anti-DNA polymerase I antiserum, and activity with synthetic and natural DNA primers. Several of these physical and biological parameters were shown to revert slowly during storage to those exhibited by the purified enzyme. Efforts to determine whether the unusual properties of the membrane enzyme resulted from its association with DNA failed to support that possibility. These observations suggest that either the cause or the result of membrane binding of DNA polymerase I is a transient change in conformation or structure of the enzyme, with a resultant change in its enzymatic activity.     

Characterization of the ribonucleic acid primers and the deoxyribonucleic acid product synthesized by the DNA polymerase and gene 4 protein of bacteriophage T7.

The DNA polymerase and gene 4 protein of phage T7, in the presence of helix-destabilizing protein (DNA binding protein), catalyze DNA synthesis on duplex templates. As has been previously shown (Kolodner, R. D., and Richardson, C. C. (1978) 4. Biol. Chem. 253, 574-584), in the absence of ribonucleoside 5'-triphosphates DNA synthesis is initiated at nicks, and all of the newly synthesized DNA is covalently attached to the template. In this paper we characterize the DNA synthesized in the presence of ribonucleoside 5'-triphophates and show that, in contrast, the major portion of the newly synthesized DNA is not attached to the template, having an average chain length of 5000 to 6000 nucleotides. In addition, each chain of newly synthesized DNA is terminated at its 5'-end by a covalently attached tetranucleotide RNA primer whose sequence is predominantly pppApCpCpC and pppApCpCpA. The results of isotope transfer experiments are in agreement with the number of initiation events determined by the incorporation of [gamma-32P]ATP and indicate that each of the four deoxyribonucleotides is present at the RNA-DNA junction.