No abstract

SUMMARYTwo temperature-sensitive mutants of Micrococcus radiodurans defective in DNA synthesis, which were very resistant to the lethal effect of ultraviolet and ionizing radiation at permissive temperatures, became sensitive to radiation and also to the action of N-methyl-N'-nitro-N-nitrosoguanidine when held at the restrictive temperature of 39 "C. With M. radiodurans tsr the sensitization began soon after transfer to 39 "C and reached a maximum 4 h later. During this period there was no loss of viability. After 4 h the shoulders of the ultraviolet and ionizing radiation survival curves had almost completely disappeared and the exponential part of the curves had doubled in slope. The size of the shoulder fell exponentially with the time the bacteria were held at 39 "C. Sensitization occurred in the presence of chloramphenicol. During the period the bacteria were held at 39 "C their ability to effect recombination as measured by transformation fell exponentially and was correlated with the rate of loss of the shoulder. This suggests that the repair which gives rise to the large shoulders of the radiation survival curves is of the post replication recombination type.The recovery of radiation resistance at 30 "C in bacteria which had been exposed to 39 "C for 75 min did not begin immediately. For 55 min there was no measurable increase in resistance but after 75 min substantial recovery had occurred and by 105 min was complete. Recovery of resistance did not occur in the presence of chloramphenicol even when the chloramphenicol was added 30 min after the bacteria had been at 30 "C.The sensitization to radiation was not a general property of temperaturesensitive (ts) mutants.

SUMMARYThe measurement of the time at which normal colony-forming ability returns in irradiated cultures of Micrococcus radiodurans tsI held at 30 "C can be used to estimate the time of completion of recombination repair. By comparing the times to complete such repair in populations given increasing radiation doses it is possible to calculate the rate of recombination repair. The rate was independent of the radiation dose; recombination could repair in one minute the damage caused either by 1.2 krad gamma radiation or 4 x I O -~ J mm-2 U.V. radiation.The time taken for the normal rate of DNA synthesis to return in irradiated M. radiodurans tsr was measured under conditions identical to those used to measure recombination repair. The delay in DNA synthesis was 1.0 min per 1 -2 krad gamma radiation and 1-0 min per 5-6 x I O -~ J mm-2 U.V. radiation. The data suggest that the normal rate of DNA synthesis resumes immediately after the completion of recombination repair of gamma-induced damage, but before the completion of recombination repair of u.v.-induced damage. It is postulated that cell death at the lethal dose of U.V. radiation is caused by a second round of replication of DNA which is still being repaired by recombination.

A mutant of Micrococcus radiodurans which is deficient in recombination has been isolated after treatment of the wild type with N-methyl-N'-nitro-Nnitrosoguanidine. We have called this mutant Micrococcus radiodurans rec3O. The efficiency of recombination in this mutant, as measured by transformation, is less than 0.01% that of the wild type. It is 15 times more sensitive to the lethal action of ultraviolet radiation, 120 times more sensitive to ionizing radiation, and 300 times more sensitive to mitomycin C (MMC) than the wild type. It is probably inactivated by a single MMC-induced deoxyribonucleic acid cross-link per genome. The excision of ultraviolet-induced pyrimidine dimers is normal. There is no radiation-induced degradation of deoxyribonucleic acid. All spontaneous revertants selected for resistance to low levels of MMC had wild-type resistance to radiation and MMC, and the same efficiency of recombination as the wild type, suggesting that the recombination deficiency of the strain is due to a single mutation. Deoxyribonucleic acid from this mutant can transform M. radiodurans UV17 presumed deficient in an exr type gene to wild type. The isolation of recombination-deficient mutants of Escherichia coli and the discovery that they were more sensitive to radiation damage than the wild type (7) led to the finding that recombination repair mechanisms are involved in the repair of radiation-and chemically damaged deoxyribonucleic acid (DNA) in recombination-proficient strains (20). It has been suggested that a repair mechanism involving recombination operates in the very radiationresistant bacterium Micrococcus radiodurans, and that the large shoulders on the dose response curves, i.e., 11,000 ergs/mm2 for ultraviolet (UV) radiation and 640 krad for ionizing radiation, are largely a reflection of this type of repair (19). We have now isolated a mutant of M. radiodurans, by N-methyl-N'-nitro-Nnitrosoguanidine (NTG) mutagenesis, which is deficient in recombination ability as measured by transformation, and report here some of its properties. MATERIALS AND METHODS Bacteria. M. radiodurans wild type, originally isolated by Anderson et al. (2), and M. radiodurans UV17, a radiation-sensitive mutant (16) thought to be equivalent to an exr mutant of E. coli (18), were used. Both of these strains are sensitive to 2.5 gg of acriflavin per ml, 20 ug of erythromycin per ml, and 100 gg of streptomycin per ml in TGY agar. A multiple mutant of M. radiodurans resistant to 50 Ag of acriflavin per ml, 40 ,g of erythromycin per ml, and 250 Ag of streptomycin per ml was used for the preparation of transforming DNA. Media. TGY medium for growth contained: tryptone (Difco), 5 g; glucose, 1 g; yeast extract (Difco), 3 g; and distilled water to 1 liter. TGY agar was made by solidifying this medium with 15 g of agar per liter.