Crystal structures of 2-acetylaminofluorene and 2-aminofluorene in complex with T7 DNA polymerase reveal mechanisms of mutagenesis

Abstract

The carcinogen 2-acetylaminofluorene forms two major DNA adducts: N-(2-deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) and its deacetylated derivative, N-(2-deoxyguanosin-8-yl)-2-aminofluorene (dG-AF). Although the dG-AAF and dG-AF adducts are distinguished only by the presence or absence of an acetyl group, they have profoundly different effects on DNA replication. dG-AAF poses a strong block to DNA synthesis and primarily induces frameshift mutations in bacteria, resulting in the loss of one or two nucleotides during replication past the lesion. dG-AF is less toxic and more easily bypassed by DNA polymerases, albeit with an increased frequency of misincorporation opposite the lesion, primarily resulting in G 3 T transversions. We present three crystal structures of bacteriophage T7 DNA polymerase replication complexes, one with dG-AAF in the templating position and two others with dG-AF in the templating position. Our crystallographic data suggest why a dG-AAF adduct blocks replication more strongly than does a dG-AF adduct and provide a possible explanation for frameshift mutagenesis during replication bypass of a dG-AAF adduct. The dG-AAF nucleoside adopts a syn conformation that facilitates the intercalation of its fluorene ring into a hydrophobic pocket on the surface of the fingers subdomain and locks the fingers in an open, inactive conformation. In contrast, the dG-AF base at the templating position is not well defined by the electron density, consistent with weak binding to the polymerase and a possible interchange of this adduct between the syn and anti conformations. N umerous carcinogenic aromatic amines, including a variety of environmental and dietary carcinogens and heterocyclic aromatic amines present in tobacco smoke condensate, are known to react with DNA to form adducts at the C8 position of guanine (1). 2-Acetylaminofluorene (AAF) is the best-studied example of this class of carcinogen (2). Originally developed as a pesticide, toxicity tests showed that this compound and related derivatives are potent liver carcinogens (3). Thus, the compound was never introduced as a pesticide. Instead, AAF has become a model compound for the study of the mutagenic and carcinogenic effects of aromatic amines (4).Metabolic activation of AAF in vivo generates intermediates that form two related adducts bound to the C8 position of guanine DNA: the N-(2Ј-deoxyguanosin-8-yl)-AAF (dG-AAF) adduct and the corresponding deacetylated N-(2Ј-deoxyguanosin-8-yl)-2-aminofluorene (dG-AF) derivative (Fig. 1) (3). The mutagenic consequences of these adducts are quite distinct in Escherichia coli. The dG-AF adduct predominately produces randomly distributed base-substitution mutations (5, 6), whereas the dG-AAF adduct results in frameshift mutations that frequently target specific repetitive sequences (4, 7-9). In vitro studies using templates modified with either a dG-AF or dG-AAF adduct have shown that the 2-aminofluorene (AF) adduct is bypassed much more readily than the corresponding AAF adduct by a variety of DNA pol...

Examination of the Long-range Effects of Aminofluorene-induced Conformational Heterogeneity and Its Relevance to the Mechanism of Translesional DNA Synthesis

“…The newly formed dG[AF]:dC base pair, however, blocks the next template base from occupying the template preinsertion site, thereby stalling DNA synthesis. Dutta et al (53) have studied a crystal from the T7 DNA polymerase treated with ddCTP or ddATP and found sketchy electron density around the template AF-adduct. The observed disorder reflects the dynamic mobility of the AF-adduct within the active site of a crystalline polymerase and is consistent with our 19 F NMR finding, indicating that the n dC-match model adopts an S*/B*-equilibrium (Fig.…”

“…The dramatic differences in the rates of nucleotide insertion (~10 4 -fold) observed for dG [FAF]:dC match and dG [FAF]:dA mismatch emphasize the importance of adduct-induced steric constraints in the DNA binding area, especially the minor groove interaction between the template-primer DNA and the polymerase, for determining replication fidelity (52)(53)(54)(55). Altering the adduct-induced conformational equilibria is important, but the complex features of enzyme structure and interaction with the lesion must be taken into consideration.…”

Shotgun metagenomics indicates novel family A DNA polymerases predominate within marine virioplankton

“…The amino acid sequence of ctg_CBS_polA_1019 was modeled onto structure 1 Â 9WA of T7 DNA polymerase (Dutta et al, 2004).…”

Biochemical Basis of Genotoxicity of Heterocyclic Arylamine Food Mutagens

“…Gill and Romano (20), in contrast, reported that Klenow fragment (exonuclease Ϫ ) produced a GC deletion when copying the NarI sequence with an AAF adduct at G 3 but did not provide specific evidence. Crystal structures of two replicative DNA polymerases bound to AAF-modified DNA have been solved, with Bacillus stearothermophilus pol I (45) and bacteriophage pol T7 (46). AF-adducted G appears to pair with C; in both reports the C 8 -G AAF adduct induces an anti to syn shift in the glycosidic bond, which in the case of pol T7 leads to intercalation of the AAF fluorene ring into the hydrophobic pocket of the polymerase and locks it in the "open" conformation (46).…”