Date of Award

2013

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Pharmaceutical Sciences

First Advisor

Bongsup P. Cho

Abstract

2-Acetylaminofluorene (AAF) is a prototype aryl amine carcinogen that forms C8-substituted dG-AAF and dG-AF as the major DNA lesions. The bulky N-acetylated dG-AAF lesion can induce various frameshift mutations depending on the base sequence around the lesion. We hypothesized that the thermodynamic stability of bulged-out slipped mutagenic intermediates (SMIs) is directly related to deletion mutations. The objective of the present study was to probe the structural/conformational basis of various dG-AAF–induced SMIs formed during a translesion synthesis. We performed spectroscopic and thermodynamic studies of several AAF-modified 16-mer model DNA duplexes, including fully paired and -1, -2, and -3 deletion duplexes of the 5'-CTCTCGATG[FAAF]CCATCAC-3' sequence and an additional -1 deletion duplex of the 5'-CTCTCGGCG[FAAF]CCATCAC-3' NarI sequence. Modified deletion duplexes existed in a mixture of external B and stacked S conformers, with the population of the S conformer being ‘GC’-1 (73%) > ‘AT’ -1 (72%) > full (60%) > -2 (55%) > -3 (37%). Thermodynamic stability was in the order of -1 deletion > -2 deletion > fully paired > -3 deletion duplexes. These results indicate that the stacked S-type conformer of SMIs was thermodynamically more stable than the conformationally flexible external B conformer. This order of lesion stability was in good agreement with the efficiencies of the frameshift mutations obtained previously by primer extension assays with the human DNA polymerase η [Schorr and Carell, ChemBioChem, 11, 2534-2537 (2010)]. Taken together, these results support a hypothesis that the conformational and thermodynamic stabilities of the SMIs are critical determinants for the induction of frameshift mutations.

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