Maintenance of the genetic information through DNA replication and repair has evolutionary consequences as well as significant impact on health-related issues including carcinogenesis and genetic diseases. Induced mutagenesis results when cellular processes forfeit their fidelity upon interaction with damaged DNA and allow the inclusion of altered genetic information. Toward a better understanding of DNA alterations induced by alkylating chemicals, this AREA application seeks to investigate the effect of neighboring bases on the production of specific base substitutions generated by ENU and correlate these alterations with the production of specific O-alkylthymine residues. The tyrA14 allele of E. coli B/r strain FX-11 will be used as the target DNA sequence. This allele is defective because of a UAA nonsense codon at position 161 in the TyrA polypeptide renders FX11 cells Tyr-. To determine how neighboring bases influence ENU mutagenesis, site-directed sequencesubstitutions on both sides of the TAA sequence in tyrA14 have been created to produce a set of strains that have all possible bases around the TAA. At least seven of these strains will be studied. Excision repair defective E.coli cells harboring these altered sites will be grown and exposed to ENU. Following isolation of numerous Tyr mutants, genomic DNA will be directly sequenced using PCR to determine the base changes, and computer assisted quantitative analyses will be used to determine the effect of the base context. The production of specific O-alkylthymine residues will be analyzed by HPLC of ENU-treated oligonucleotides. These analyses will associate specific adducts with precise base changes. To corroborate these findings, strains that either lack or overexpress alkylation-specific DNA repair enzymes and harbor altered tyrA14 alleles will be examined. The principal investigator indicates that these studies will provide valuable research experience for undergraduate students in the biological sciences and have the potential to reveal important new insights regarding the etiology of human cancer and genetic disease.
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