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PDBsum entry 1x9m
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Transferase/electron transport/DNA
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PDB id
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1x9m
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References listed in PDB file
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Key reference
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Title
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Crystal structures of 2-Acetylaminofluorene and 2-Aminofluorene in complex with t7 DNA polymerase reveal mechanisms of mutagenesis.
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Authors
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S.Dutta,
Y.Li,
D.Johnson,
L.Dzantiev,
C.C.Richardson,
L.J.Romano,
T.Ellenberger.
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Ref.
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Proc Natl Acad Sci U S A, 2004,
101,
16186-16191.
[DOI no: ]
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PubMed id
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Abstract
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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.
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Figure 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).
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Figure 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  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.
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