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PDBsum entry 2w9c
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Transferase/DNA
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PDB id
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2w9c
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References listed in PDB file
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Key reference
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Title
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Structure-Function relationships in miscoding by sulfolobus solfataricus DNA polymerase dpo4: guanine n2,N2-Dimethyl substitution produces inactive and miscoding polymerase complexes.
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Authors
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H.Zhang,
R.L.Eoff,
I.D.Kozekov,
C.J.Rizzo,
M.Egli,
F.P.Guengerich.
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Ref.
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J Biol Chem, 2009,
284,
17687-17699.
[DOI no: ]
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PubMed id
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Abstract
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Previous work has shown that Y-family DNA polymerases tolerate large DNA
adducts, but a substantial decrease in catalytic efficiency and fidelity occurs
during bypass of N2,N2-dimethyl (Me2)-substituted guanine (N2,N2-Me2G), in
contrast to a single methyl substitution. Therefore, it is unclear why the
addition of two methyl groups is so disruptive. The presence of N2,N2-Me2G
lowered the catalytic efficiency of the model enzyme Sulfolobus solfataricus
Dpo4 16,000-fold. Dpo4 inserted dNTPs almost at random during bypass of
N2,N2-Me2G, and much of the enzyme was kinetically trapped by an inactive
ternary complex when N2,N2-Me2G was present, as judged by a reduced burst
amplitude (5% of total enzyme) and kinetic modeling. One crystal structure of
Dpo4 with a primer having a 3'-terminal dideoxycytosine (Cdd) opposite template
N2,N2-Me2G in a post-insertion position showed Cdd folded back into the minor
groove, as a catalytically incompetent complex. A second crystal had two unique
orientations for the primer terminal Cdd as follows: (i) flipped into the minor
groove and (ii) a long pairing with N2,N2-Me2G in which one hydrogen bond exists
between the O-2 atom of Cdd and the N-1 atom of N2,N2-Me2G, with a second
water-mediated hydrogen bond between the N-3 atom of Cdd and the O-6 atom of
N2,N2-Me2G. A crystal structure of Dpo4 with dTTP opposite template N2,N2-Me2G
revealed a wobble orientation. Collectively, these results explain, in a
detailed manner, the basis for the reduced efficiency and fidelity of
Dpo4-catalyzed bypass of N2,N2-Me2G compared with mono-substituted N2-alkyl G
adducts.
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Figure 3.
Crystal structures of Dpo4 bound to N^2,N^2-Me[2]G-modified
DNA.A, superimpositions of DMG-1 (red), DMG-2 (molecule A,
cyan), and DMG-3 (molecule A, green) reveal overall similarity
in Dpo4 structure. B, representative electron density near the
active site of Dpo4 in the DMG-1 structure. The 3F[o][]− 2F[c]
map (gray mesh) is shown contoured to the 1σ level. The
F[o][]− F[c] difference maps are shown contoured to 3σ and
−3σ for positive (red mesh) and negative (green mesh)
density, respectively. The terminal C[dd] residue is flipped out
of base-stacking orientation. but the incoming dGTP forms a
Watson-Crick pair with the cytosine to the 5′-side of
N^2,N^2-Me[2]G (DMG).
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Figure 4.
Structural examination of wobble pairing with
N^2,N^2-Me[2]G-modified DNA in Dpo4.A, overall structure of DNA
and corresponding electron density observed in the active site
of DMG-2 are shown. The orientation of bases observed in
molecule A (B) and molecule B (C) of the DMG-2 structure is
shown. D, overall structure of DNA and corresponding electron
density observed in the active site of DMG-2 are shown. The
orientation of bases observed in molecule A (E) and molecule B
(F) of the DMG-2 structure is shown. In all panels, the
3F[o][]− 2F[c] map (gray mesh) for DNA bound in the DMG-2
structure is shown contoured to 1σ level (gray mesh) with the
F[o][]− F[c] difference maps shown contoured to 3σ and −3σ
for positive (red mesh) and negative (green mesh) density,
respectively.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2009,
284,
17687-17699)
copyright 2009.
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