The goal of this program is to characterize the DNA repair pathways that are critical to the radioresistance of Deinococcus radiodurans. This bacterium is the most radioresistant organism known. Furthermore, its repair of extensive DNA damage of almost all types is error-proof, producing no mutations. Our emphasis is on repair of DNA double-strand breaks (DSBs), the most severe form of radiation-induced DNA damage. During the current Project Period we placed reporter sequences into D. radiodurans chromosomes that allowed us to asses DSB-repair postirradiation in vivo. Surprisingly, we detected two DSB- recombinational repair pathways, one that is recA-dependent and another that is recA-independent. After an exposure of 15,000 Gy60Co (100 percent survival under our conditions) producing 130 DSBs per chromosome we detected 150 recombinational events per chromosome, evidently sufficient to repair 130 DSBs. The recA gene was cloned, and found to be novel in its regulation. We also found that the RecA protein is exceptional in its properties. In the next Project Period we will: 1) Physically characterize recombination events in a totally in vitro cell-free system that we have developed to monitor radiation-induced recombination. Radiosensitive mutants will be examined using this system, allowing us to identify, by complementation, proteins required for recombination; 2) The extraordinary RecA of D. radiodurans has now been purified allowing us to assess its recombinase activities in vitro; and 3) The organization of D. radiodurans' chromosomes will be probed to determine whether chromosome structure is important in this organism's supremely high capacity for DSB-repair recombination. These studies will delineate the genetics and biochemistry of radioresistance in D. radiodurans. Insight into its strategies may have broad implications for understanding radioresistance and DSB-metabolism throughout nature.
