The NarI sequence (5'-G1G2CG3CC-3') is a mutational hot spot for frameshift mutagenesis, where arylamine adduction at G3 exerts a high frequency of -2 deletion mutations. We have shown that the thermodynamic stability of bulged-out slipped mutagenic intermediates (SMIs) is directly related to deletion mutations. Acetylaminofluorene (AAF) is a prototype arylamine carcinogen that forms C8-substituted dG-AAF and dG-AF as the major DNA lesions. There have been conflicting NMR results as to which flanking cytosine (5? or 3?) loops out with modified-dG to form a SMI during translesion synthesis: (i) 3'-flanking dC loops out with AAF modified- dG3 (ii) 5'- flanking dC loops out with AF modified-dG3. Here we investigated the AF and AAF induced -2 bulge structures using fluorescence spectroscopy by substituting the flanking dC with a fluorescent analog pyrrolo-deoxycytidine (PC). We prepared two NarI templates; 5'--G1G2PCG3CC--3' and 5'--G1G2CG3PCC--3' with PC located at 5' or 3' flanking position to G3,respectively. Our results suggest that AAF prefers 3'-PC whereas AF favors 5'-PC in forming -2 deletion bulge structures, consistent with the above-referenced NMR studies. We further confirmed similar lesion effect on non-NarI sequences (G1G2 was replaced by CT). Overall, these results indicate that the 'N-acetyl' factor (e.g., N-acetylated AAF vs. N-deacetylated AF) plays a key role in inducing different types of bulge structures.

DNA lesions in different conformations produce alternate bulge structures that can result in different frameshift mutations

246th National Meeting (2013)

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