Structural and Conformational Probing of 2-Acetylaminofluorene-Modified -1, -2 and -3 Slipped Mutagenic Intermediates

Author

Anusha Sandineni, University of Rhode IslandFollow

Date of Award

2013

Degree Type

Thesis

Degree Name

Master of Science in Pharmaceutical Sciences

Department

Biomedical and Pharmaceutical Sciences

First Advisor

Bongsup P. 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.

Recommended Citation

Sandineni, Anusha, "Structural and Conformational Probing of 2-Acetylaminofluorene-Modified -1, -2 and -3 Slipped Mutagenic Intermediates" (2013). Open Access Master's Theses. Paper 18.
https://digitalcommons.uri.edu/theses/18