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PDBsum entry 2j6t
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Transferase/DNA
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PDB id
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2j6t
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References listed in PDB file
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Key reference
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Title
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Sulfolobus solfataricus DNA polymerase dpo4 is partially inhibited by "wobble" pairing between o6-Methylguanine and cytosine, But accurate bypass is preferred.
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Authors
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R.L.Eoff,
A.Irimia,
M.Egli,
F.P.Guengerich.
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Ref.
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J Biol Chem, 2007,
282,
1456-1467.
[DOI no: ]
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PubMed id
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Abstract
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We examined the effect of a single O6-methylguanine (O6-MeG) template residue on
catalysis by a model Y family polymerase, Dpo4 from Sulfolobus solfataricus.
Mass spectral analysis of Dpo4-catalyzed extension products revealed that the
enzyme accurately bypasses O6-MeG, with C being the major product (approximately
70%) and T or A being the minor species (approximately 20% or approximately 10%,
respectively), consistent with steady-state kinetic parameters. Transient-state
kinetic experiments revealed that kpol, the maximum forward rate constant
describing polymerization, for dCTP incorporation opposite O6-MeG was
approximately 6-fold slower than observed for unmodified G, and no measurable
product was observed for dTTP incorporation in the pre-steady state. The lack of
any structural information regarding how O6-MeG paired in a polymerase active
site led us to perform x-ray crystallographic studies, which show that
"wobble" pairing occurs between C and O6-MeG. A structure containing T
opposite O6-MeG was solved, but much of the ribose and pyrimidine base density
was disordered, in accordance with a much higher Km,dTTP that drives the
difference in efficiency between C and T incorporation. The more stabilized
C:O6-MeG pairing reinforces the importance of hydrogen bonding with respect to
nucleotide selection within a geometrically tolerant polymerase active site.
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Figure 6.
FIGURE 6. Electron density and DNA duplex conformations
observed at the active site of ternary complexes. A, O^6-MeG:C;
B, O^6-MeG:T; and C, O^6-MeG:dATP. Dpo4 is shown in schematic
form (cyan). The DNA duplex is shown in ball-and-stick
representation. Calcium ions are shown as green spheres. The
electron density map (black mesh) 3F[o] - 2F[c] is contoured at
the 1  level.
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Figure 7.
FIGURE 7. Comparative C and T base pairing orientations
opposite O^6-MeG at the catalytic center of the ternary
complexes. A, the 14th nucleotide C forms a wobble base pair
with template O^6-MeG in the O^6-MeG:C structure. B, the 14th
nucleotide T is positioned in three conformations opposite
O^6-MeG, owing to the disorder observed in the region near
thymidine in the O^6-MeG:T structure. The electron density map
(black mesh) 3F[o] - 2F[c] is contoured at the 1 level.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2007,
282,
1456-1467)
copyright 2007.
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