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PDBsum entry 2jei
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References listed in PDB file
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Key reference
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Title
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Molecular basis of selectivity of nucleoside triphosphate incorporation opposite o6-Benzylguanine by sulfolobus solfataricus DNA polymerase dpo4: steady-State and pre-Steady-State kinetics and X-Ray crystallography of correct and incorrect pairing.
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Authors
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R.L.Eoff,
K.C.Angel,
M.Egli,
F.P.Guengerich.
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Ref.
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J Biol Chem, 2007,
282,
13573-13584.
[DOI no: ]
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PubMed id
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Abstract
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Previous work has shown that Sulfolobus solfataricus DNA polymerase
Dpo4-catalyzed bypass of O(6)-methylguanine (O(6)-MeG) proceeds largely in an
accurate but inefficient manner with a "wobble" base pairing between C
and O(6)-MeG (Eoff, R. L., Irimia, A., Egli, M., and Guengerich, F. P. (2007) J.
Biol. Chem. 282, 1456-1467). We considered here the bulky lesion
O(6)-benzylguanine (O(6)-BzG) in DNA and catalysis by Dpo4. Mass spectrometry
analysis of polymerization products revealed that the enzyme bypasses and
extends across from O(6)-BzG, with C the major product ( approximately 70%) and
some T and A ( approximately 15% each) incorporated opposite the lesion.
Steady-state kinetic parameters indicated that Dpo4 was 7-, 5-, and 27-fold more
efficient at C incorporation opposite O(6)-BzG than T, A, or G, respectively. In
transient state kinetic analysis, the catalytic efficiency was decreased 62-fold
for C incorporation opposite O(6)-BzG relative to unmodified DNA. Crystal
structures reveal wobble pairing between C and O(6)-BzG.
Pseudo-"Watson-Crick" pairing was observed between T and O(6)-BzG. Two
other structures illustrate a possible mechanism for the accommodation of a +1
frameshift in the Dpo4 active site. The overall effect of O(6)-BzG is to
decrease the efficiency of bypass by roughly an order of magnitude in every case
except correct bypass, where the effect is not as pronounced. By comparison,
Dpo4 is more accurate but no more efficient than model replicative polymerases,
such as bacteriophage T7(-) DNA polymerase and human immunodeficiency virus-1
reverse transcriptase in the polymerization past O(6)-MeG and O(6)-BzG.
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Figure 7.
FIGURE 7. Comparison of primer ddC orientations in the
O^6-BzG:ddC-1 and O^6-BzG:ddC-2 structures and a view of the
O^6-BzG:G active site. A, stereo view of the superimposed
(r.m.s. deviation = 0.344) O^6-BzG:ddC-1 (Dpo4 (light blue) and
nucleotides and ions (dark blue)) and O^6-BzG:ddC-2 (Dpo4, ions,
and nucleotides (orange)) structures. Dpo4 is shown in schematic
diagram form for both structures. The incoming dGTP (magenta)
from the O^6-BzG:ddC-1 structure is shown in ball and stick
form. The 14th nucleotide (dark blue, ddC) from the
O^6-BzG:ddC-1 structure forms a wobble pair with O^6-BzG, but
the 14th nucleotide (orange, ddC) from the O^6-BzG:ddC-2 ternary
complex is moved into a noninstructional conformation. B, stereo
view of the O^6-BzG:G active site. Dpo4 (light blue) is shown in
schematic diagram form. The last two primer residues, p14C and
p15G, and the O^6-BzG lesion are shown in ball and stick form
(dark blue). The 14th base in the primer, p14C, is placed in a
conformation similar to that observed in the O^6-BzG:ddC-2
structure. The calcium ions (light brown) and incoming dGTP
(magenta) are also shown.
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Figure 8.
FIGURE 8. View of the O^6-BzG:T active site superimposed on
the wobble O^6-BzG:ddC-1 pair. A, stereo view of the active site
from the O^6-BzG:T ternary complex is shown superimposed on the
O^6-BzG:ddC-1 structure (r.m.s. deviation = 0.390) with Dpo4 in
schematic diagram form (O^6-BzG:T (ruby), O^6-BzG: ddC-1 (light
blue)). The O^6-BzG:T pair is shown with cyan carbon atoms, and
the O^6-BzG:ddC-1 wobble pair is shown with dark blue carbon
atoms. The O^6-BzG:T calcium ions are red, and the O^6-BzG:ddC-1
calcium ions are dark blue. The incoming dGTP from the O^6-BzG:T
structure is shown in ball and stick form (magenta). B, the p14T
residue (cyan carbon atoms) is shifted up in the active site
relative to p14ddC-1 (dark blue carbon atoms), and the benzyl
group in the O^6-BzG:T structure is shifted slightly toward the
proximal orientation.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2007,
282,
13573-13584)
copyright 2007.
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