Temperature-sensitive recA mutant of Escherichia coli K-12: deoxyribonucleic acid metabolism after ultraviolet irradiation.

A mutant of Escherichia coli K-12 temperature sensitive for genetic recombination was investigated and found to carry a mutation that could be cotransduced with cysC and hence could be in the recA gene. To determine whether recA+ can complement this mutation, matings were carried out at 35 and 40 C between Hfr donors that transfer recA+ or recA1 early and recipients carrying wild-type or mutant alleles. It was found that recA+ but not recA1 complements this mutation in zygotic temporary partial diploids. The mutant allele was accordingly designated recA44. A transductant carrying recA44 behaved normally at low temperatures but more like recA- strains at high temperatures with respect to recombinant colony formation in Hfr matings, cell survival, and deoxyribonucleic acid (DNA) synthesis after ultraviolet irradiation, cellular DNA breakdown, and prophage induction when lysogenic for lambda. Alkaline sucrose sedimentation studies of DNA from recA44 cells showed that short DNA molecules synthesized immediately after ultraviolet irradiation increased in molecular weight during subsequent incubation at 32 C but not at 45 C. Hence, recA+ is required for this molecular weight increase. Cells exposed to ultraviolet light synthesized DNA that remained of low molecular weight during a 40-min incubation at 32 C. This material increased in molecular weight in recArut not in recA44 cells during subsequent incubation at 45 C. Thus, the availability of recA+ during the first 40 min at 32 C after irradiation did not obviate the need for recA+ in the subsequent phases of this post-replication repair process.     

Repair of ultraviolet light-damaged deoxyribonucleic acid in sbc-A strains of Escherichia coli K-12.

An Escherichia coli strain carrying both rec+ and sbcA has been constructed. Repair of ultraviolet light-induced deoxyribonucleic acid damage was examined by measuring survival and thymine-dimer excision in the rec+ sbcA strain as well as rec+ sbcA+ and recB recC sbcA strains. The sbcA mutation restores normal survival in both recB recC uvrB and recB recC uvr+ strains. Excision of thymine-containing dimers does not occur in uvrB mutants, regardless of the rec or sbcA genotype. Survival, after ultraviolet-light damage, of a rec+ sbcA strain is quantitatively similar to rec+ sbcA+ and recB recC sbcA strains.     

tif-Stimulated deoxyribonucleic acid repair in Escherichia coli K-12

Bacterial survival is significantly increased after ultraviolet irradiation in tif sfi cells, provided that the thermosensitive tif mutation has been expressed at 41 degrees C before irradiation. This tif-mediated "reactivation of ultraviolet irradiated bacteria" needs de novo protein synthesis, as is the case for the tif-mediated reactivation of ultraviolet-irradiated phage lambda. However, in striking contrast to the phage reactivation process, this tif-mediated reactivation is no longer associated with mutagenesis. It also requires the presence of the uvrA+ excision function. These results strongly suggest the existence in Escherichia coli K-12 of a repair pathway acting on bacterial deoxyribonucleic acid which is inducible, error free, and uvr dependent.     

Role of deoxyribonucleic acid polymerases and deoxyribonucleic acid ligase in x-ray-induced repair synthesis in toluene-treated Escherichia coli K-12.

The biosynthesis of ribosomal ribonucleic acid (rRNA) In wild-type Neurospora crassa growing at 25 degrees C was investigated by continuous-labeling and pulsechase experiments using [5-3H]uridine. The results of these experiments suggest the following precursor-product relationships: the first RNA molecule to be synthesized in significant quantities is the 2.4 X 10(6)-dalton (2.4-Mdal) ribosomal precursor RNA. This RNA is cleaved to produce two species of RNA with weights of 0.7 and 1.4-Mdal. The former is the mature 17S rRNA of the 37S ribosomal subunit. The 1.4-Mdal RNA is subsequently cleaved to produce the mature 1.27-Mdal (25S) and 61,000-dalton (5.8S) rRNA's of the 60S ribosomal subunit. In the maturation process, approximately 15 to 20% of the 2.4-Mdal ribosomal precursor rRNA molecule is lost. As in other eukaryotes that have been examined, 5S rRNA is not derived from this precursor molecule.     

Location of the Escherichia coli K-12 ruv gene affecting septum formation after inhibition of deoxyribonucleic acid synthesis.

Escherichia coli ruv gene was located at 36.1 min on the chromosome by P1 transduction experiments and the gene order his - supD - uvrC, dar4 - ruv - eda - fadD - pps was proposed. Complementation analysis by an F' factor carrying genes in the his region indicated that ultraviolet light sensitivity genes, ruv and uvrC, consist of different cistrons and wild-type alleles of these genes are dominant over the mutant alleles.     

Repair of hydrogen peroxide-induced single-strand breaks in Escherichia coli deoxyribonucleic acid.

Near-ultraviolet (300 to 400 nm) irradiation of L-tryptophan yielded H2O2 (a toxic photoproduct) that was selectively lethal for rec and polA1 Escherichia coli mutants. H2O2 treatment of cells resulted in the induction of single-strand deoxyribonucleic acid breaks. These breaks were repaired to only a small extent in polA1, recA recB, and recA mutants, but were efficiently repaired in wild-type strains. We conclude that H2O2 deoxyribonucleic acid lesions require both the polA+ and recA+ pathways for repair.     

The duration of recovery and DNA repair in excision deficient derivatives of Escherichia coli K-12 after ultraviolet irradiation

Summary Our results indicate that cells of excision deficient (uvr) mutants of Escherichia coli K-12 which survive exposure to ultraviolet radiation may require several hours to complete their recovery. For example, the duration of the recovery period for cells exposed to 63 ergs mm^-2 at 254 nm was about 5 hours, the equivalent of slightly more than 4 generations of the unirradiated controls. During the recovery period the rate of cell division was reduced (Figs. 3 and 4), the cells gradually regained resistance to complex medium (Figs. 1 and 3), and they became refractory to photoreactivation (Fig. 1). Over the same period of time their pattern of DNA synthesis changed. More intact molecules, similar to those found in unirradiated controls, and relatively fewer discontinuous molecules were synthesized (Figs. 6 and 7).     

Cell division in Escherichia coli BS-12 is hypersensitive to deoxyribonucleic acid damage by ultraviolet light.

Escherichia coli BS-12 uvrA lon is hypersensitive to ultraviolet light. On minimal agar plates at densities in excess of about 10(7) bacteria per plate, as few as one or two photoreversible pyrimidine dimers in the entire genome are sufficient to cause inhibition of cell division. Most of the resulting filaments are unable to divide or form a viable colony. Inhibition of cell division appears to be a rapid consequence of replication of deoxyribonucleic acid containing a pyrimidine dimer. Photoreversibility of the inhibition of cell division persists indefinitely, indicating that the continued presence of the pyrimidine dimers (or the continued generation of daughter strand gaps) is necessary to maintain the division-inhibited state. In view of the kinetics for the production of filamentation by ultraviolet light and the extremely low average inducing fluence (0.03 J/m2), it is concluded that the initiating signal is not the same as that causing other inducible phenomena such as prophage induction or Weigle reactivation.     

Repair of x-ray-induced deoxyribonucleic acid single-strand breaks in xth mutants of Escherichia coli.

An exonuclease III-deficient strain of Escherichia coli K-12, BW2001 (xthA11), was unable to perform rapid repair of X-ray-induced deoxyribonucleic acid single-strand breaks and appeared to have a defect in the priming of the 3'-termini necessary for initiation of repair synthesis at the breaks. This defect cannot be explained solely by the lack of exonuclease III activity, because other xth mutants tested, including a deletion mutant, repaired radiation-induced strand breaks at close to the normal rate.     

Characterization of arithmetic deoxyribonucleic acid synthesis at restrictive temperature in a dnaE mutant of Escherichia coli K-12.

The dna-293 mutation is shown to be a dnaE allele. The linear deoxyribonucleic acid synthesis previously observed in this mutant has been further characterized. The production of small deoxyribonucleic acid intermediates and their subsequent joining were identical in the mutant and its dnaE+ parent at 42.5 degrees C. Though the mutant cells continued to divide at the nonpermissive temperature, the rate of division was reduced. The data are consistent with a lack of production of replicationally active deoxyribonucleic acid polymerase III at the restrictive temperature.     

Metabolism of arginine-specific messenger ribonucleic acid in Escherichia coli K-12.

Ribonucleic acid-deoxyribonucleic acid (RNA-DNA) hybridization was employed for the determination of the level of messenger RNA (mRNA) transcribed from seven of the nine genes of the arginine regulon of Escherichia coli K-12. The quantity of RNA complexing with each of the separated DNA strands of the argA, argF, argE, and argCBH operons carried on specialized transducing phages was measured. The derepressed:repressed ratio of mRNA formed in vivo was found to vary between about 3 and 4 when measured by hybridization to DNA isolated from specialized transducing phages carrying the argA, argE, argCBH, argF, and argI operons.