Abstract
Aromatic amines have been studied for more than a half-century as model carcinogens representing a class of chemicals that form bulky adducts to the C8 position of guanine in DNA. Among these guanine adducts, the N-(2'-deoxyguanosin-8-yl)-aminofluorene (G-AF) and N-2-(2'-deoxyguanosin-8-yl)-acetylaminofluorene (G-AAF) derivatives are the best studied. Although G-AF and G-AAF differ by only an acetyl group, they exert different effects on DNA replication by replicative and high-fidelity DNA polymerases. Translesion synthesis of G-AF is achieved with high-fidelity polymerases, whereas replication of G-AAF requires specialized bypass polymerases. Here we have presented structures of G-AF as it undergoes one round of accurate replication by a high-fidelity DNA polymerase. Nucleotide incorporation opposite G-AF is achieved in solution and in the crystal, revealing how the polymerase accommodates and replicates past G-AF, but not G-AAF. Like an unmodified guanine, G-AF adopts a conformation that allows it to form Watson-Crick hydrogen bonds with an opposing cytosine that results in protrusion of the bulky fluorene moiety into the major groove. Although incorporation opposite G-AF is observed, the C:G-AF base pair induces distortions to the polymerase active site that slow translesion synthesis.
Highlights
Aromatic amines have been studied for more than a half-century as model carcinogens representing a class of chemicals that form bulky adducts to the C8 position of guanine in DNA
Acetylaminofluorene (G-AAF)1 adduct belongs to the class of lesions that blocks replication [3,4,5,6,7] and requires that replicative polymerases be transiently replaced by specialized polymerases, most of which belong to the Y family of DNA polymerases, to achieve translesion synthesis [8]
The conformations of aminofluorene adducts in DNA duplexes in the absence of protein have been studied by NMR and molecular dynamics simulations (16, 19, 30 –34), there is no a priori expectation that these conformations are adopted in the context of a polymerase active site, because it is not known what effect the polymerase structure has on the allowed conformations of G-AF and G-AAF
Summary
Replication of G-AF and G-AAF in Solution and in BF Crystals—AF- and AAF-modified DNA duplexes were used to determine the ability of BF to incorporate nucleotide opposite G-AF and G-AAF in solution and in the crystal. The ability to observe DNA synthesis in BF polymerase crystals allowed us to determine structures of G-AF before (Fig. 3) and after (Fig. 4) dCTP incorporation These structures, solved to 2.1 and 2.0 Å resolution, respectively (Table I), show G-AF positioned at two sites: the template preinsertion site (n) and the postinsertion site (n-1). Hydrogen bonds between protein residues that read the DNA minor groove (Gln-797 and Arg-615) and the n-1 base pair are retained Both the primer and template bases adopt conformations identical to those of cognate bases at this site. This conformation resembles the “external” conformation described in structures of AF in duplex
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