Repair of radiation-induced damage in Escherichia coli: I. Effect of rec mutations on post-replication repair of damage due to ultraviolet radiation

Abstract

The rec mutants of Escherichia coli K12 are more sensitive to ultraviolet radiation than corresponding rec + derivatives. We have tested several of these rec mutants for post-replication repair after irradiation with ultraviolet light. The mutants carried recA13, recA56, recB21 and recC22. Cultures growing exponentially were exposed to ultraviolet light (63 ergs/mm 2) and then labeled for ten minutes with [ 3H]thymidine. The radioactive medium was removed and the culture divided into two portions, one of which was chilled while the other was reincubated in non-radioactive medium for various times. The DNA from the cells was then analysed on alkaline sucrose gradients. Labeled DNA from irradiated cells which had not been reincubated sedimented as short molecules. After reincubation, DNA from the recA mutants still behaved as short molecules, while DNA from rec +, recB and recC cells assumed a position similar to that of DNA from unirradiated control cells. These results indicate that the two recA mutants tested were defective in their capacity for post-replication of damage induced by ultraviolet radiation. However, no deficiency in the recB and recC mutants was detected under the conditions used. Even in uvr + cells, which are capable of excision-dependent repair, the formation of short molecules could be demonstrated immediately after irradiation. However, after a 40-minute post-irradiation incubation before labeling with [ 3H]thymidine, only “normal-sized” DNA was formed. In contrast, in a uvr − strain, short molecules were still formed after a 90 minute post-irradiation incubation. These results indicate that excision-dependent repair can remove the lesions that lead to the synthesis of short pieces of DNA after ultraviolet-irradiation. Similarly, the exposure of ultraviolet cells to conditions of photoreactivation (which repairs pyrimidine dimers in situ) before labeling greatly diminished but did not completely eliminate the synthesis of short pieces of DNA.