The Sequence Dependence of Human Nucleotide Excision Repair Efficiencies of Benzo[ a]pyrene-derived DNA Lesions: Insights into the Structural Factors that Favor Dual Incisions

Abstract

Nucleotide excision repair (NER) is a vital cellular defense system against carcinogen–DNA adducts, which, if not repaired, can initiate cancer development. The structural features of bulky DNA lesions that account for differences in NER efficiencies in mammalian cells are not well understood. In vivo, the predominant DNA adduct derived from metabolically activated benzo[ a]pyrene (BP), a prominent environmental carcinogen, is the 10 S (+)- trans- anti-[BP]- N 2-dG adduct ( G*), which resides in the B-DNA minor groove 5′-oriented along the modified strand. We have compared the structural distortions in double-stranded DNA, imposed by this adduct, in the different sequence contexts 5′-…CG G*C…, 5′-…C G*GC…, 5′-…CI G*C… (I is 2′-deoxyinosine), and 5′-…C G*C…. On the basis of electrophoretic mobilities, all duplexes manifest moderate bends, except the 5′-…CG G*C…duplex, which exhibits an anomalous, slow mobility attributed to a pronounced flexible kink at the site of the lesion. This kink, resulting from steric hindrance between the 5′-flanking guanine amino group and the BP aromatic rings, both positioned in the minor groove, is abolished in the 5′-…CI G*C…duplex (the 2′-deoxyinosine group, I, lacks this amino group). In contrast, the sequence-isomeric 5′-…C G*GC…duplex exhibits only a moderate bend, but displays a remarkably increased opening rate at the 5′-flanking base pair of G*, indicating a significant destabilization of Watson–Crick hydrogen bonding. The NER dual incision product yields were compared for these different sequences embedded in otherwise identical 135-mer duplexes in cell-free human HeLa extracts. The yields of excision products varied by a factor of as much as ∼ 4 in the order 5′-...C G*GC…> 5′...CG G*C…≥ 5′...CI G*C…≥ 5′-…C G*C…. Overall, destabilized Watson–Crick hydrogen bonding, manifested in the 5′-...C G*GC...duplex, elicits the most significant NER response, while the flexible kink displayed in the sequence-isomeric 5′-...CG G*C...duplex represents a less significant signal in this series of substrates. These results demonstrate that the identical lesion can be repaired with markedly variable efficiency in different local sequence contexts that differentially alter the structural features of the DNA duplex around the lesion site.