Cells at all phylogenetic levels are capable of repairing damage to DNA. The DNA repair systems that carry out this repair function to maintain the integrity of the cells hereditary material by protecting the DNA from the consequences of damage by external agents or errors that occur during replication. One such DNA repair system in Escherichia coli acts when it is treated with sublethal, submutagenic concentrations of alkylating agents and renders the cells resistant to the lethal and mutagenic effects of subsequent treatments with higher concentrations of alkylating agents. This induced resistance has been called the adaptive response to alkylating agents and results from the induced expression of DNA repair activities that function to remove or repair alkylated bases present in the DNA, or remove alkyl groups from phosphotriesters in the sugar phosphate backbone. I will focus on the genetic aspects of the regulation, expression and function of the genes that carry out this induced repair of alkylation damage to DNA in E. coli. To date we have isolated lac operon fusion to promoters of at least five different genes or operons that are induced by methylation treatments. I propose to continue the phenotypic characterization of these fusion mutants, begin the isolation and characterization of regulatory mutants that control these alkylation inducible fusions. In addition we will clone the fusions along with their promoters in order to compare promoter sequences and learn what the important features of the promoters of the alkylation inducible genes are, and to learn how the regulatory proteins interact with the promoters. The cloned fusions will also be used as DNA probes to identify clones of the corresponding wild type genes which will then be used to identify their products. This project is designed to learn more about the types of DNA damage that cause the induction of the alkylation inducible genes and the types of DNA repair processes carried out by the induced gene products. In addition, this study will also provide a more complete picture of how cells repair their DNA and mitigate the effects of genetic damage.
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