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. 2004 Nov 16;101(46):16186-91.
doi: 10.1073/pnas.0406516101. Epub 2004 Nov 4.

Crystal Structures of 2-acetylaminofluorene and 2-aminofluorene in Complex With T7 DNA Polymerase Reveal Mechanisms of Mutagenesis

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Crystal Structures of 2-acetylaminofluorene and 2-aminofluorene in Complex With T7 DNA Polymerase Reveal Mechanisms of Mutagenesis

Shuchismita Dutta et al. Proc Natl Acad Sci U S A. .
Free PMC article

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 --> 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.

Figures

Fig. 1.
Fig. 1.
Schematic representation of dG-AAF and dG-AF. The anti conformation (a) is energetically favored for unmodified dG. dG-AF (b) can be in anti conformation or syn conformation. However, for dG-AAF (c), the anti conformation is strongly unfavorable because of the steric hindrance between the acetyl group (circled in green) and the sugar moiety (circled in pink).
Fig. 2.
Fig. 2.
dG-AAF adopts the syn conformation with the fluorene ring intercalated into the polymerase fingers domain. (a) dG-AAF lies outside the polymerase active site, and the fingers domain of the polymerase is in an open conformation. The T7 gene 5 protein (gray) and the thioredoxin (green) processivity factor are shown as ribbons, with the O-helix within the fingers highlighted in red. The primer strand of the DNA is in light red, the template is in yellow, and dG-AAF is in cyan and green. The region around the dG-AAF binding site is circled. (b) Enlarged view of the circled region in a. The fluorene ring (green) of dG-AAF (cyan and green) is inserted into the fingers domain between helices L, O, O1, O2, and P. (c) The simulated annealing omit electron density around the region of the syn dG-AAF of the dG-AAF-containing complex is shown in stereo, contoured at 2.5 σ above the mean value. (d) Interaction between dG-AAF and the protein. Hydrophobic side chains (gold) of the fingers form a pocket around the fluorene ring. Two charged residues (pink), Arg-566 and Asp-534, form hydrogen bonding interactions with the G base.
Fig. 3.
Fig. 3.
Distortion of the O-helix and stacking of the side chain of Tyr-530 with the primer/template DNA. In the dG-AAF/ddCTP (a) and dG-AF/ddCTP (b) complexes (Table 1), this residue (dark gray) stacks with the 3′ base of the primer and occupies the position where the base of the incoming nucleotide would bind in a catalytically active, closed complex (c). The incoming nucleotide in the closed complex is shown in green in c. An incoming nucleotide is modeled in a according to its position in the active closed ternary complex and is outlined in black.

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