Date of Award
Master of Science
Molecular Biology Microbiology and Biochemistry
LEADING STRAND DNA SYNTHESIS INCREASES THE FREQUENCY OF UV-INDUCED MUTATIONS IN Escherichia coli Ultraviolet (UV) radiation can cause a variety of alterations in the chemical structure of DNA strands. The most common lesions formed are the thymine-thymine cyclopyrimidine dimers (CPDs), which result in the covalent linking of adjacent thymine bases in the same strand. These lesions, if left uncorrected, may eventually lead to cell death or mutations. In previous research, the Escherichia coli strain FX-11-TC, an excision repair deficient derivative of FX-11, was shown to produce an unequal distribution of UV-induced mutations within the palindromic target sequence 5'-GTTAAC-3'. The results suggested a potential difference in the fidelity of DNA replication between the leading and the lagging strands. In this research, the possibility of unequal rates of UV mutagenesis in the leading strand of the replicon as opposed to the lagging strand was investigated. Two strains of E.coli were constructed, each having the target gene in opposite orientations with respect to the origin of chromosomal replication. First, a deletion strain (FX-33 δtyrA) was constructed by in vivo replacement of the chromosomal tyrA gene with the δtyrA deletion. Then, specialized vectors, harboring the target gene tyrA-TC were constructed to integrate the gene into the chromosome of the cells at the λ attachment site, attB. The target gene contained a specific sequence of bases (5'-GTTAAC-3') that encoded a stop codon (UAA) that resulted in the termination of the polypeptide chain. This rendered the strains auxotrophic for tyrosine. After treatment with 1 J/m2 of UV radiation, survival and reversion frequencies for both the strains were measured. The resulting revertant tyrosine prototrophs were isolated on minimal media and differentiated between backmutants and tRNA suppressor mutants. The backmutants were sequenced using the dideoxynucleotide method to determine the exact base changes that gave rise to the mutation. Finally, the strains were transformed using a plasmid harboring a gene encoding the Drosophila melanogaster (6-4) phr photolyase, to test the effect of photoreactivation on the mutation frequencies. The results illustrated that more mutations occurred in the leading strand as compared to the lagging strand. However, photoreactivation had little effect on the overall frequency of mutagenesis in the two strains but did have mutation-specific effects that were dependent upon gene orientation.
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