The rate of recombination repair and its relationship to the radiation-induced delay in DNA synthesis in Micrococcus radiodurans.

The measurement of the time at which normal colony-forming ability returns in irradiated cultures of Micrococcus radiodurans tsI held at 30 degrees 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 10(-6) J mm-2 u.v. radiation. The time taken for the normal rate of DNA synthesis to return in irradiated M. radiodurans tsI 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 10(-6) 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.

Isolation and properties of a recombination-deficient mutant of Micrococcus radiodurans.

A mutant of micrococcus radiodurans which is deficient in recombination has been isolated after treatment of the wild type with N-methyl-N'-nitro-N-nitrosoguanidine. We have called this mutant Micrococcus radiodurans rec30. 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.

Involvement of a recombination repair function in disciplined cell division of Micrococcus radiodurans.

When a culture of the temperature-sensitive DNA mutant Micrococcus radiodurans tsI is irradiated with a sublethal dose of ultraviolet or ionizing radiation and is plated immediately, all the bacteria give rise, after 36 h incubation, to colonies identical to those derived from unirradiated bacteria. However, when the irradiated population is held at its restrictive temperature (39 degrees C) (restrictive temperature holding) for 3 h before being plated, less than 0-1% of the surviving bacteria give rise to normal colonies, the rest producing, after incubation for 96 h, small malformed colonies. Qualitatively, the same effect is observed when u.v.-irradiated wild-type M. radiodurans is incubated at 39 degrees C in the presence of nalidixic acid before plating. Compared with the loss of viability, the loss of normal colony development as a function of the radiation dose is sensitive, having I/e values of 210 ergs/mm2 for u.v. radiation and of 4 to 5 krad for 60Co gamma-radiation. These are identical to the radiation dose-response values of a recombination-deficient mutant of M. radiodurans. At first the abnormal colonies consist entirely of giant bacteria but eventually a few bacteria with normal morphology appear and because of their much faster generation time a highly sectored colony results. These colonies can be "rescued" by plating the irradiated bacteria held at 39 degrees C on agar containing pantoyl lactone, their growth being identical to that of unirradiated bacteria. Abnormal colony development is not a general phenomenon in temperature-sensitive mutants of M. radiodurans but occurs in those mutants which are sensitized to radiation when held at 39 degrees C. It is concluded that these abnormal colonies are produced as a result of a defect in a recombination function and that this function is also involved in the regulation of normal cell division.