Postirradiation recovery dependent on the uvr-1 locus in Bacillus subtilis

Abstract

A mutant (uvr-1) of Bacillus subtilis that is deficient in excision of ultraviolet (UV)-induced pyrimidine dimers from deoxyribonucleic acid (DNA) shows a marked increase in ability to survive UV irradiation when plated on amino acid-supplemented agar medium compared with its survival ability when plated on nutrient plating medium, the effect is considered to be one of growth-dependent lethality. Irradiated stationary phase uvr-1 cells, incubated in liquid medium lacking amino acids required for growth, recover from this sensitivity to rich medium within 3 to 4 h after irradiation. Recovery is greatly reduced in the absence of glucose oiminated. Exponentially growing cells have a limited ability to recover from sensitivity to rich medium. Growth-dependent lethality can also occur in liquid medium. In nutrient broth the ability of irradiated stationary-phase uvr-1 cells to form colonies on defined agar medium decreases during postirradiation incubation, but treatmeth with chloramphenicol inhibits the loss of colony-forming ability. Recovery from sensitivity to rich media is inhibited by caffeine but not by 6-(p-hydroxyphenylazo)-uracil, and inhibitor of DNA replication. Alkaline sucrose gradient profiles show that conditions allowing recovery also favor maintaining intact DNA strands, whereas DNA strand breakage or degradation is associated with loss of viability. Recovery from sensitivity to rich medium has not been observed in the Ur+ parent or in strains carrying the mutations uvs-42 (another deficiency in dimer excision), recA1, or polA59. A uvr-1 recA1 mutants shows a higher level of recovery than does the recA1 single mutant, but a much lower level than the uvr-1 single mutant. Apparently, both the uvr-1 defect and Rec+ and PoII+ functions are essential for recovery from sensitivity to rich medium. For optimal recovery, growth immediately after irradiation must be delayed. The process requires energy, apparently involves recombination, and probably results in rejoining of DNA strands in which incision but not excision has occurred.

Heat and UV light resistance of vegetative cells and spores of Bacillus subtilis Rec-mutants

“…With respect to Bacillus subtilis, a number of genetic loci, including polA, rec, and uvr (5, 9,11,20), have been shown to be important in determining DNA repair efficiency. Vegetative cells of B. subtilis strains with mutations in these genes have been shown to be less resistant to the DNA-damaging effects of physical mutagens, particularly UV light irradiation, and chemical mutagens (4, 9,11,19,23). In all cases, sensitivity to the mutagens was related to the genetic inability of these strains to repair DNA damage induced by these agents.…”

Postreplication repair of ultraviolet-irradiated transforming deoxyribonucleic acid in Bacillus subtilis

“…There is abundant evidence that the uvr-1 and uvr-42 mutations cause distinctly different phenotypes. Strains bearing these mutations are different in recovery from UV damage in minimal medium (16), in rate and extent of dimer excision (19), and in repair resynthesis (17), but not in postreplication repair (9). However, evidence has been presented by Munakata (28) that the two mutations map within clusters of mutations in close proximity.…”

“…Cells were grown in the casein hydrolysate-supplemented medium described previously (2). Cells in mid-log phase were harvested and irradiated as described previously (16), except that the concentration of cells being irradiated was around 2 x 107/ml. Samples were plated on nutrient agar for enumeration of colony-forming units.…”

“…Because UV-irradiated uvr-1 cells are able to excise dimers slowly (17,19) and survive better on defined medium than uvr-42 cells (16), it seemed likely that very small amounts of integrated damage might be repaired in uvr-1 cells by excision rather than by postreplication repair. To study the effects of saturating the excision repair system, I compared the kinetics of accumulation of transformants in Uvr+ and Uvrcells.…”

Restriction-like phenomena in transformation of Bacillus subtilis recA

“…Exposure to DNA was terminated by treatment with deoxyribonuclease (Calbiochem, San Diego, Calif.) at 20 gg/ml for 5 min. Cells were plated on the synthetic amino acid medium as described previously (16).…”