Contribution of DNA photoproducts to UV mutagenesis

Fix, Douglas

Abstract

The maintenance of genetic information through the accuracies of DNA replication and DNA repair has evolutionary consequence as well as significant impact on health-related concerns including carcinogenesis and genetic disease. 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 carcinogenic agents, this AREA (R15) application seeks to investigate the contribution of DNA photoproducts produced by ultraviolet (UV) irradiation of bacterial cells to the specificity of mutations that result. This general area of basic research has provided the foundation for understanding repair and mutagenesis in eukaryotes and the role of DNA damage to UV-induced skin cancers in human cells. The proposed studies will employ the tyrA14 allele of Escherichia coli B/r strain FX-11 as a target DNA sequence. This allele is defective because of a UAA nonsense codon in the mRNA encoding position 161 of the TyrA polypeptide, rendering FX-11 Tyr-. Using strains that have different bases neighboring the nonsense defect will produce different potential damage profiles. E. coli cells harboring these alleles will be grown and exposed to UV. Following isolation of numerous TyrA+ mutants, genomic DNA will be directly sequenced using PCR-amplification to determine the base changes. Preliminary studies indicated several important findings. First, a potentially significant role for the TA* photoproduct was discovered. Second, a very different mutational pattern for a 5'-CT photoproduct was revealed. Third, a possible difference in the mutagenic processing of photoproducts by the leading and lagging DNA replication complexes was suggested. To further investigate these findings, this application proposes three specific aims. First, strains that only allow the formation of a TA* targeting photoproduct will be used in the presence or absence of two different DNA photolyases, which recognize only cyclobutane pyrimidine dimers (CPDs) or [6-4] photoproducts. Excision repair proficient strains will also be used. Second, strains that permit the formation of a 5'-CT targeting photoproduct in both DNA strands will be used. Again, the two different DNA photolyases and excision repair will be used to confirm the photoproduct identity. Third, the tyrA gene will be recombined onto the E. coli genome in opposite orientations relative to the origin of replication, and the effect on mutagenesis will be determined. These studies will also discover if the DNA damage distribution differs within identical sequences located in opposite DNA strands. Furthermore, these AREA-funded studies will provide valuable research experience for undergraduate students in the biological sciences.