Date of Completion

Spring 5-2-2014

Thesis Advisor(s)

Ashis K. Basu

Honors Major

Molecular and Cell Biology

Disciplines

Biochemistry | Molecular Biology | Structural Biology

Abstract

It is well established that clustered DNA damages or multiply damaged sites (MDS) are the result of ionizing radiation and that they are characterized by an enhanced mutagenic potential. As a model MDS, we have evaluated the mutagenic and cytotoxic properties of the ubiquitous oxidative DNA damage 8-oxoguanine (G8-oxo) adjacent to the abasic site lesion (Z) using a single stranded M13mp7L2 vector. The recombinant DNA was used to transform wild type E. coli strains and strains deficient in the translesion DNA polymerase of the Y-family, DNA polymerase IV, in the presence or absence of SOS induction. The percent survival of the 5’ZG8-oxo lesion-containing M13, measured in comparison to an undamaged vector, was slightly higher in the DNA polymerase IV knockout strain compared to the wild type indicating that translesion polymerases other than DNA polymerase IV may be more efficient in bypassing the damage. The mutagenesis of 8-oxoguanine was enhanced in the tandem lesion compared to the single 8-oxoguanine lesion suggesting that the abasic site alters the replication fidelity of its neighboring lesions. The major mutations observed for the abasic site were deletions across all strains with a smaller proportion of Z-to-T mutations. Interestingly, the polymerase IV knockout strain under SOS induction showed an approximately equal percentage of Z-to-T mutations and Z-to-Δ deletions. Overall, these results support the hypothesis that DNA polymerase V, another translesion DNA polymerase of the Y-family, may be involved in Z-to-T mutations and more efficient bypass of the tandem lesion.

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