Biomedical subjects
Publications and source records attributed to K C Smith.
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A much higher yield of DNA single-strand breaks was obtained in the DNA polymerase-deficient mutant Escherichia coli K-12 pol A1 after a given dose of x-rays than had been found before in Escherichia coli. The increased yield of single-strand breaks was due to the absence of a rapid repair system, which had not been described in Escherichia coli K-12. This absence probably accounts for the x-ray sensitivity of the pol A1 mutant. The rapid repair system can be reversibly inhibited in pol+ cells.
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Late log-phase Escherichia coli B/r cells are 1.6 times more sensitive to killing by X rays than are stationary-phase cells when grown in Brain Heart Infusion (BHI) + glucose. The number of single-chain breaks formed per krad is the same for log- and stationary-phase cells. Stationary-phase cells show a somewhat greater ability to repair single-chain breaks (especially after high doses of X rays) than do log-phase cells. The rapidity and extent of postirradiation deoxyribonucleic acid (DNA) degradation are greater in log-phase cells than in stationary-phase cells. The enhanced viability exhibited by stationary-phase cells thus appears to correlate both with enhanced single-chain break repair and the reduced degradation of DNA. Cells grown to stationary phase in peptone medium (PO cells) are 3.4 times more sensitive to killing by X rays than cells grown to stationary phase in peptone medium supplemented with glucose and phosphate buffer (PG cells). The yield of single-strand breaks is the same for both types of cells (but the absolute yield is about two times higher than in the cells grown in BHI + glucose). The kinetics for the repair of single-chain breaks are the same for both types of cells for about 30 min. After this time period, further repair ceases in the PO cells but continues in the PG cells, provided that glucose is present in the medium. Postirradiation DNA degradation is both more rapid and more extensive in PO cells than in PG cells whether or not glucose is present in the postirradiation incubation medium. The survival of stationary-phase E. coli B/r grown in PO or PG medium is likewise unaffected by the presence of glucose in the plating medium, and thus correlates better with the lower DNA degradation seen in the PG cells than with the increased strand rejoining, since this latter process requires the presence of glucose.
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Mutants of Escherichia coli K-12 unable to excise pyrimidine dimers from their deoxyribonucleic acid (DNA) because of a uvr mutation show a higher survival when plated on a minimal salts medium after exposure to ultraviolet radiation than when plated on a complex medium such as nutrient agar containing yeast extract. This response has been called minimal medium recovery (MMR). Recovery of uvr mutants can take place in liquid as well as on solid medium, but not in buffer or under conditions of amino acid starvation that do not permit cell growth and normal DNA replication. MMR can thus be distinguished from the recovery of recombination-deficient (rec(-)uvr(+)) derivatives of K-12 which can occur under conditions where growth is not possible. Because MMR is characteristic of excision-defective mutants, it evidently reflects a type of repair independent of excision. We have obtained genetic evidence that MMR is determined by the rec genes, which also control recombination in K-12. Cells carrying a uvr mutation together with recA13, recA56, recB21, or recC22 failed to show MMR and were more sensitive to ultraviolet radiation than either their rec(+)uvr(-) or rec(-)uvr(+) parents. The rec(+)uvr(-) derivatives obtained from recA uvr(-) strains by transduction or by reversion regained the capacity for MMR. Our results indicate that inactivation of any one of the three genes, recA, recB, or recC, prevents cells from showing MMR.
Strains of Escherichia coli K-12 mutant in the genes controlling excision repair (uvr) and genetic recombination (rec) have been studied with reference to their radiosensitivity and their ability to repair X-ray-induced single-strand breaks in deoxyribonucleic acid (DNA). Mutations in the rec genes appreciably increase the radiosensitivity of E. coli K-12, whereas uvr mutations produce little if any increase in radiosensitivity. For a given dose of X-rays, the yield of single-strand breaks has been shown by alkaline sucrose gradient studies to be largely independent of the presence of rec or uvr mutations. The rec(+) cells (including those carrying the uvrB5 mutation) could efficiently rejoin X-ray-induced single-strand breaks in DNA, whereas recA56 mutants could not repair these breaks to any great extent. The recB21 and recC22 mutants showed some indication of repair capacity. From these studies, it is concluded that a correlation exists between the inability to repair single-strand breaks and the radiosensitivity of the rec mutants of E. coli K-12. This suggests that unrepaired single-strand breaks may be lethal lesions in E. coli.
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The uvr mutations of Escherichia coli K-12 decrease the ability of cells to survive ultraviolet light (UV), to excise pyrimidine dimers from their deoxyribonucleic acid and to reactivate bacteriophage exposed to UV. The rec mutations decrease the ability of the cells to survive UV and to undergo genetic recombination. Certain rec mutations, including recA1, rec-12, recA13, and rec-56, are necessary for the expression of liquid-holding recovery (LHR), observed as an increase in colony-forming ability when irradiated cells are held in buffer in the dark. These rec mutations appear to act indirectly to permit the detection of LHR rather than to affect its occurrence directly. We have tested the effect of uvr markers on LHR in cells containing one of these rec mutations. Recombinants containing rec-56 together with a uvr marker were constructed and tested for LHR. None of the 39 recombinants examined, carrying uvrA6, uvrB5, or uvrC34, showed LHR. Three rec(-)uvr(-) strains were also tested for photoreactivation. In all three, photoreactivation was observed, indicating that they contained detectable amounts of pyrimidine dimers. Our results are consistent with the idea that uvr mutations inactivate LHR, and suggest that LHR reflects excision-dependent repair of pyrimidine dimers.
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