Kazunori Shimada

Mechanism of repair of DNA in bacteriophage: I. Excision of pyrimidine dimers from ultraviolet-irradiated DNA by an extract of T4-infected cells☆

Abstract When [14C]thymine-labeled T4 DNA, after exposure to ultraviolet light, was incubated with an extract of T4-infected Escherichia coli, pyrimidine dimers were selectively released into the acid-soluble fraction; the rate of release of dimers was about five times greater than that for thymine. The reaction requires Mg2+ and proceeds at pH 7 to 8 in the dark. The amount of thymine and thymine-containing dimers released from the ultraviolet-irradiated DNA into the acid-soluble fraction was determined by Dowex 1 column chromatography. Dimers lost from the DNA were almost quantitatively recovered, in the form of nuoleotides but not of free bases or nucleosides, in the acid-soluble fraction. As a result of the specific release of dimers, the content of dimers in the DNA decreases progressively during the incubation. Thus the excision in vitro is similar to the reaction observed in vivo in ultraviolet-resistant cells. An extract of normal cells does not exhibit the preferential release of dimers even when the homologous E. coli DNA is used as substrate. Only a small amount of dimers is released into the acid-soluble fraction during the incubation of irradiated E. coli or T4 DNA with the normal cell extract. Thus the level of dimer-excising activity of normal cells, if any, must be very low compared with that of T4-infected cells. It is likely that enzyme(s) responsible for excision of dimers is induced by infection with T4.

The effect of caffeine on the repair of ultraviolet-damaged DNA in bacteria

Abstract 1. 1. The effect of caffeine on host cell reactivation of ultraviolet-irradiated λ phage and on ultraviolet-induced degradation of bacterial DNA was investigated using an ultraviolet-sensitive and host cell reactivation negative mutant of Escherichia coli in which ultraviolet-induced DNA breakdown takes place. 2. 2. The photo-reactivation was rapidly impaired after infection of the mutant with ultraviolet-irradiated λ phage. In the presence of 1.0 % caffeine the photo-reactivatability of the ultraviolet-irradiated phage was almost completely preserved in this strain. 3. 3. Caffeine inhibited ultraviolet-induced DNA breakdown in bacteria. 4. 4. The intracellular state of the ultraviolet-irradiated λ phage DNA was studied using the sucrose density-gradient centrifugation technique, and it was observed that caffeine inhibited the introduction of a first clip in the λ phage DNA pre-exposed to ultraviolet light. 5. 5. It was suggested that caffeine depresses the excision of pyrimidine dimers from ultraviolet-irradiated DNA.

Effects of bleomycin on Escherichia coli strains with various sensitivities to radiations.

Abstract 1. The mode of action of bleomycin was investigated using Escherichia coli K12 strain W3623 and its ultraviolet light-sensitive mutants, N17-9 uvr A-54, N3-1 uvr B-51, N17-7 uvr C-56 and N14-4 uvr D-3. The last one is also sensitive to γ-rays. In the experiments on the damage of intracellular DNA by bleomycin, thyminerequiring mutants of these strains were also employed. 2. The lethal effects of bleomycin on these strains were similar. 3. The bacteriocidal effect of bleomycin was counteracted by the concomitant addition of a high concentration of 2-mercaptoethanol. Moreover, cultures survived when they were incubated with the sulfhydryl compound after incubation with bleomycin. With N17-9 and W3623 the antibacterial activity could be counteracted by the subsequent addition of 2-mercaptoethanol for up to 5 and 3 h of incubation with bleomycin, respectively. With N14-4, the surviving fraction was not restored by subsequent treatment with the sulfhydryl compound and was decreased as the time of incubation with the antibiotic increased. 4. Incubation of the cells with bleomycin caused little breakdown of cellular DNA in W3623 thy − or N17-9 thy − , but caused marked degradation of that in N14-4 thy − . 5. Studies by alkaline sucrose gradient centrifugation showed that bleomycin caused extensive single-strand scissions of the DNA of N14-4 thy − , fewer in that of W3623 thy − , and very few in N17-9 thy − . 6. Studies by neutral sucrose gradient centrifugation indicated that the number of double-strand scissions in the DNA of N14-4 thy − increased with increased times of incubation of the cells with bleomycin, but this could not be detected in either W3623 thy − or N17-9 thy − even after incubation with bleomycin for 1 h. 7. Strand scissions caused by the action of bleomycin on the DNA of W3623 thy − and N17-9 thy − were repaired when the cells were treated with 2-mercaptoethanol and subsequently incubated in the absence of both compounds. However, no repair of the DNA of N14-4 thy − was observed under the same incubation conditions.

Packaging of ColE1 DNA having a lambda phage cohesive end site

SummaryThe mechanism of λ phage-mediated transduction of hybrid colicin E1 DNAs of various lengths was studied, and factors influencing the formation of these transducing particles were investigated. The results were as follows: 1)The presence of a cohesive end site of \gl phage (cos\gl) on colicin E1 DNA was essential for packaging of the DNA2)Packaging of colicin E1 DNAs, which carry cos\gl with molecular sizes corresponding to 68% of that of \gl phage DNA, was observed in the absence of all known recombination functions of E. coli K-12 and of \gl phage.3)Hybrid colicin E1 DNAs having cos\gl with molecular sizes corresponding to 28% of that of \gl phage DNA were packaged within \gl phage particles as trimers; hybrid DNAs with cos\gl of 40 and 47% of the length of \gl phage DNA were packaged as dimers; and those with molecular sizes of 68% of that of \gl phage DNA were packaged mostly as monomers. These results demonstrated that two factors are essential for the packaging of DNAs within λ phage particles; the presence of cosλ on the DNA molecule and an appropriate size of DNA.

Fine structure of the recB and recC gene region of Escherichiacoli

Abstract For genetic and enzymatic study of recBC DNase, the Escherichia coli thyA , recC , recB and argA gene region cloned in a cosmid was subcloned into the pBR322 plasmid vector and its fine restriction map was made. Complementation analysis showed that these genes were located in a 19kb Bam HI fragment in the order thyA recC recB argA , so far as estimated. The recA recB recC cells harboring plasmid subcloned with this Bam HI fragment exhibited 12.3-fold increase in recBC DNase activity. Clones carrying the recB and the recC gene expressed recB and recC function, respectively, and the initiation sites of transcription of these genes were detected by S1 nuclease mapping, proving that the recB and recC genes consist of independent cistrons.

Expression of the guanine operon of Escherichia coli as analyzed by bacteriophage lambda-induced mutations

SummaryStudies were made on two guaninerequiring strains of Escherichia coli isolated independently as a result of insertion of prophage λ into one of the structural genes of the guanine operon. These mutants do not exhibit any detectable guaB function but express the guaA function constitutively at a low level, presumably due to transcription from the pI promoter on the prophage. Various types of plaque-forming gua-transducing phages were generated from these lysogens. The approximate location and the mode of substitution of the gua genes in the phage genome were determined. These results clearly indicate that the gene guaB is located closer to the operatorpromoter region of the gua operon than is guaA, and the gene order is “operator”-guaB-guaA.

Colicinogenic mutant of ColE1 plasmid that fails to confer immunity to colicin E1

Insertion of the ampicillin transposon (Tn3) into ColE1 DNAs causes various mutations in the plasmids. Escherichia coli K-12 cells carrying one of these mutants showed novel properties; they were sensitive to colicin E1 and were able to produce active colicin E1. The site and the orientation of Tn3 insertion in this mutant ColE1 DNA were determined by heteroduplex analysis and by enzymatic digestion with restriction endonucleases. The potential usefulness of this mutant ColE1 DNA as a cloning vehicle is discussed.

Molecular nature of an invitro recombinant molecule: Colicin el factor carrying genes for synthesis of guanine

Summary The molecular nature of an in vitro recombinant DNA, which consists of colicin El DNA, a bacterial gene for xanthosine 5′-monophosphate aminase and a part of the γ phage genome, was studied by electron microscopy, restriction DNAse digestion, and electrophoresis in agarose gel. This molecule existed as a monomer plasmid with an average molecular weight of 21.6 × 106 daltons within E . coli . The present results confirmed the unique structure of the molecule and its potential use as a cloning vehicle.

Replication process of single-stranded DNA of bacteriophage φX174: V. Production of replicative form in Escherichia coli cells irradiated by ultraviolet light☆☆☆

Abstract The process of replicative form production was studied in ultraviolet-irradiated bacteria infected with φX174, and the results were compared with the process in non-irradiated bacteria. It was observed that in irradiated bacteria, double-stranded parental replicative form is produced as a result of conversion of the injected phage single-stranded DNA, but subsequent production of infectious progeny replicative form does not take place. On the other hand, in non-irradiated cells the injected phage single-stranded DNA is converted to double-stranded parental replicative form, which then replicates semiconservatively to accumulate progeny replicative form molecules (Denhardt & Sinsheimer, 1965; Matsubara, Shimada & Takagi, 1967). Several lines of evidence suggest that the double-stranded parental replicative form produced in irradiated cells is normal, but its subsequent replication is blocked in ultraviolet-irradiated cells.

Repair of ultraviolet-light damaged ColE1 factor carrying Escherichia coli genes for guanine synthesis

SummaryHybrid ColE1 plasmids called ColE1-cosλ-guaA or ColE1-cosλ-gal can be efficiently transduced into various E. coli K-12 cells through packaging into λ phage particles. Using these plasmids, repair of ultraviolet-light (UV) damaged ColE1 DNAs was studied in various UV sensitive E. coli K-12 mutants. (1) The host mutations uvrA and uvrB markedly reduced host-cell reactivation of UV-irradiated ColE1-cosλ-guaA. (2) Pre-existing hybrid ColE1 plasmids had no effect on the frequency of λ phage-mediated transduction of another differentially marked hybrid ColE1 DNAs. (3) ColE1-cosλ-guaA and ColE1-cosλ-gal DNAs could temporarily but not stably co-exist in E. coli K-12 recA cells. (4) The presence of ColE1-cosλ-gal in uvrB cells promoted the repair of super-infected UV-irradiated ColE1-cosλ-guaA about 7-fold. (5) The same ColE1-cosλ-gal plasmid in a uvrB recA double mutant did not have this promoting effect. These results indicate that the effect of resident hybrid ColE1 plasmids is manifested by the host recA+ gen function(s) and suggest that ColE1 plasmid itself provides no recA+-like functions.