Recognition and incision of site-specifically modified C8 guanine adducts formed by 2-aminofluorene, N-acetyl-2-aminofluorene and 1-nitropyrene by UvrABC nuclease

Nucleotide excision repair plays a crucial role in removing many types of D NA adducts formed by UV light and chemical carcinogens. We have examined th e interactions of Escherichia coli UvrABC nuclease proteins with three site -specific C8 guanine adducts formed by the carcinogens 2-aminofluorene (AF) , N-acetyl-2-acetylaminofluorene (AAF) and l-nitropyrene (1-NP) in a 50mer oligonucleotide, Similar to the AF and AAF adducts, the l-NP-induced DNA ad duct contains an aminopyrene (AP) moiety covalently linked to the C8 positi on of guanine, The dissociation constants for UvrA binding to AF-, AAF- and AP-DNA adducts, determined by gel mobility shift assay, are 33 +/- 9, 8 +/ - 2 and 23 +/- 9 nM, respectively, indicating that the AAF adduct is recogn ized much more efficiently than the other two. Incision by UvrABC nuclease showed that AAF-DNA was cleaved similar to 2-fold more efficiently than AF- or AP-DNA (AAF > AF approximate to AP), even though AP has the largest mol ecular size in this group. However, an opened DNA structure of six bases ar ound the adduct increased the incision efficiency for AF-DNA (but not for A P-DNA), making it equivalent to that for AAF-DNA, These results are consist ent with a model in which DNA damage recognition by the E,coli nucleotide e xcision repair system consists of two sequential steps, It includes recogni tion of helical distortion in duplex DNA followed by recognition of the typ e of nucleotide chemical modification in a single-stranded region. The diff erence in incision efficiency between AF- and AAF-DNA adducts in normal DNA sequence, therefore, is a consequence of their difference in inducing stru ctural distortions in DNA. The results of this study are discussed in the l ight of NMR solution structures of these DNA adducts.