Date of Award

1989

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Veterinary Medical Sciences - Pathobiological Sciences

First Advisor

H. Douglas Braymer

Abstract

In Escherichia coli, the SOS response and two 3-methyladenine DNA glycosylases (TagI and TagII) are required for repair of DNA damaged by alkylating agents such as methyl methanesulfonate (MMS). Mutations of the recA gene eliminate the SOS response. TagI and TagII are encoded by the tag and alkA genes, respectively. A gene (rpr) encoding 3-methyladenine DNA glycosylase activity was isolated from the Gram-negative bacterium Serratia marcescens. The gene, localized to a 1.5-kilobase pair SmaI-HindIII restriction fragment, was cloned into plasmid pUC18. The clone complemented E. coli tag alkA and recA mutations for MMS resistance. The rpr gene did not, however, complement recA mutations for resistance to ultraviolet light or the ability to perform homologous recombination reactions, nor did it complement E. coli ada or alkB mutations. Two proteins of molecular weights 42,000 and 16,000 were produced from the rpr locus. Analysis of deletion and insertion mutants of rpr suggested that the 42kD molecule is the active protein. The 16kD protein may either be a breakdown product of the 42kD species or may be encoded by another gene overlapping the reading frame of the rpr gene. Biochemical assays showed that the rpr gene product (Rpr) possesses 3-methyladenine DNA glycosylase activity. Moreover, Rpr acts exclusively on 3-methyladenine, thus exhibiting the same substrate specificity as E. coli TagI. Furthermore, Rpr was as effective as TagI in host cell reactivation of bacteriophage lambda which had been exposed to MMS. Finally, rpr did not complement a triple mutation in E. coli (tag alkA recA) which abolished 3-methyladenine DNA glycosylase activity and the SOS response. Southern blot hybridization experiments evidenced no detectable homology between rpr and various known E. coli DNA repair genes. Furthermore, the E. coli tag and alkA genes did not hybridize to S. marcescens chromosomal DNA. Gene dosage experiments demonstrated no differences in recA complementation profiles whether rpr was on a low or high copy plasmid vector. However, rpr on the high copy vector pUC18 sensitized wild type E. coli and E. coli mutants deficient in exonuclease III (E. coli xth) and/or endonuclease IV (E. coli xth nfo) to MMS.

Pages

66

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