9418594 Battista Members of the family Deinococcaceae are distinguished by their extraordinary resistance to the lethal and mutagenic effects of many DNA damaging agents including mitomycin C, ultraviolet (UV) light, and ionizing radiation. For example, an exponential phase culture of Deinococcus radiodurans R1, will withstand 5000 Gray (500 krad) of ( radiation without loss of viability. The capacity to survive such massive insults to their genetic integrity suggests that the Deinococcaceae have evolved distinctive mechanisms of DNA damage tolerance and available evidence argues that efficient DNA repair is an integral part of this tolerance. The enzymology of the deinococci's DNA repair processes is poorly understood, however. It is the long term objective of this project to develop a comprehensive explanation for the deinococci's remarkable resistance to ionizing radiation. Satisfying this objective will entail identifying the types of proteins involved in the repair of ionizing radiation-induced DNA damage, establishing how they interact with each other and determining how they are regulated. This research initiates the characterization of two recently isolated ionizing radiation sensitive mutants of Deinococcus radiodurans R1 with the intention of determining the nature of the defect in each mutant strain. This objective will be pursued through completion of the following stages: (1) Sequence the irrB and irrI loci and use this information to determine how their expression is regulated and how the Irrb and IrrI gene products influence the activity of the D. radiodurans repair protein, endonuclease (. (2) Evaluate each mutant strain for the ability to recognize and repair ionizing and ultraviolet radiation induced DNA damage by determining a) if cell extracts from these strains can bind DNA damage and b) if these strains can repair damaged transforming DNA. (3) Evaluate each mutant for its effect on the extensive DNA degradation observed post-irradiation. For each mutant, it wil l be determined: a) whether DNA damage is exported from the cell into the growth medium, b) what the size of the exported fragments are c) if the kinetics of damage export differs from wild type export during the first and second phases of DNA degradation, and d) if degradation terminates when repair should be complete. %%% Active oxygen species, in particular hydroxyl radicals, are the agents that mediate the majority of ionizing radiation-induced cellular damage in aerobic aqueous environments. It is, therefore, expected that the study of the deinococci will yield general insights into how the lethal effects of oxidative cellular damage is best avoided. ***
