Structural and kinetic studies of the effect of guanine-N7 alkylation and metal cofactors on DNA replication.

A wide variety of endogenous and exogenous alkylating agents attack DNA to preferentially generate N7-alkylguanine (N7-alkylG) adducts. Studies of the effect of N7-alkylG lesions on biological processes have been difficult in part because of complications arising from the chemical lability of the positively charged N7-alkylG, which can readily produce secondary lesions. To assess the effect of bulky N7-alkylG on DNA replication, we prepared chemically stable N7-benzylguanine (N7bnG)-containing DNA and evaluated nucleotide incorporation opposite the lesion by human DNA polymerase β (polβ), a model enzyme for high-fidelity DNA polymerases. Kinetic studies showed that the N7-benzyl-G lesion greatly inhibited dCTP incorporation by polβ. The crystal structure of polβ incorporating dCTP opposite N7bnG showed a Watson–Crick N7bnG:dCTP structure. The polβ-N7bnG:dCTP structure showed an open protein conformation, a relatively disordered dCTP, and a lack of catalytic metal, which explained the inefficient nucleotid...