Date of Award

1996

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

John R. Battista

Abstract

Deinococcus radiodurans is extremely resistant to ionizing radiation and can withstand 5 kGy $\gamma$ radiation without loss of viability or evidence of mutation. Available evidence argues that an efficient DNA repair mechanism is responsible for the resistance these organisms possess to ionizing radiation. The enzymes which coordinate DNA repair in D. radiodurans are poorly characterized. Two new loci have been detected in D. radiodurans, namely, irrB and irrI which provide resistance. Mutational inactivation of either locus resulted in a partial loss of resistance to ionizing radiation. The extent of sensitivity was locus specific and differentially affected by inactivation of the uvrA gene product. An irrB uvrA double mutant was more sensitive to ionizing radiation than was an irrB mutant. In contrast, an irrI uvrA double mutant and irrI single mutant were equally sensitive to ionizing radiation. irrB and irrI mutations also reduced resistance to UV radiation in D. radiodurans which was more pronounced in the presence of an $uvrA\sp+$ background. Subclones derived from each gene were generated and the loci of each gene were mapped relative to each other. The irrB and irrI genes were separated by approximately 20 kb of intervening sequence in which the uvrA and pol genes are located. A mutation which inactivates the irrB gene does not impair repair of transforming DNA which has been damaged and contains thymine glycol or 8-hydroxyguanosine lesions. Examination of irrB cell extracts for novel DNA binding proteins revealed little information. irrB strains do produce a 23 kD novel protein (IrrB*) in large amounts but IrrB* did not bind to either ssDNA or dsDNA in the presence of 50 mM NaCl. A mutation which impairs the function of the irrI gene product did not affect its potential to reactivate 8-hydroxyguanosine containing transforming DNA, but it failed to reactivate thymine glycol containing transforming DNA. In the absence of the irrI gene product D. radiodurans was extremely sensitive to UV radiation and its DNA underwent extensive degradation. The irrI gene product appears to regulate endonuclease $\alpha$ mediated DNA degradation which occurs following exposure to UV radiation.

Pages

95

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