Date of Award

2013

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Pharmaceutical Sciences

First Advisor

Bongsup Cho

Abstract

2-Acetylaminofluorene (AAF) is a prototype arylamine carcinogen that forms C8-substituted dG-adduct (dG-C8-AAF) as a major DNA damage. The bulky dG-C8-AAF lesion is known to induce -1, -2, or -3 frameshift mutations depending on the base sequences around the lesion. We hypothesize that the stability of bulged-out structures slipped mutagenic intermediate (SMI) facilitates primer elongation, hence manifestation of frameshift mutations. The objective of the present study was to probe the structural/conformational basis of various dG-C8-AAF induced frameshift mutations. Here, we describe spectroscopic (19F NMR and CD) and thermodynamic (UV-melting and DSC) studies of several dG-C8-FAAF-modified 16-mer DNA duplexes containing fully-paired, -2, and -3 deletion duplexes on the 5'-CTCTCGATG[FAAF]CCATCAC-3' sequence and -1 deletion duplexes on either the 5'-CTCTCGATG[FAAF]CCATCAC-3' or 5'-CTCTCGGCG [FAAF]CCATCAC-3' sequences. The results were analyzed to determine the conformational and thermodynamic basis of AAF-induced frameshift mutagenesis. We found that the dG-AAF lesion exists in a mixture of external binding B and inserted/bulge conformers and the population of the latter was in order of ‘GC’-1(73%) > ‘AT’ -1 (72%) > full (60%) > -2 (55%) > -3 (37%). Thermodynamic stability was found to be in order of -1 deletion > -2 deletion > fully paired > -3 deletion duplexes. These results indicate that the stacked conformer especially in the deletion duplexes is thermodynamically more stable than the conformationally flexible external B-conformer. Previous primer extension results involving the human DNA polymerase η have shown that the frequency of deletion was in order of -1 > -2 > -3. Taken together, our results support the hypothesis that the conformational and thermodynamic stability of the SMI is a critical determinant for the induction of various frameshift mutations.

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