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PDBsum entry 2agp
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Transferase/DNA
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PDB id
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2agp
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References listed in PDB file
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Key reference
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Title
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Fidelity of dpo4: effect of metal ions, Nucleotide selection and pyrophosphorolysis.
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Authors
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A.Vaisman,
H.Ling,
R.Woodgate,
W.Yang.
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Ref.
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EMBO J, 2005,
24,
2957-2967.
[DOI no: ]
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PubMed id
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Abstract
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We report the crystal structures of a translesion DNA polymerase, Dpo4,
complexed with a matched or mismatched incoming nucleotide and with a
pyrophosphate product after misincorporation. These structures suggest two
mechanisms by which Dpo4 may reject a wrong incoming nucleotide with its
preformed and open active site. First, a mismatched replicating base pair leads
to poor base stacking and alignment of the metal ions and as a consequence,
inhibits incorporation. By replacing Mg2+ with Mn2+, which has a relaxed
coordination requirement and tolerates misalignment, the catalytic efficiency of
misincorporation increases dramatically. Mn2+ also enhances translesion
synthesis by Dpo4. Subtle conformational changes that lead to the proper metal
ion coordination may, therefore, be a key step in catalysis. Second, the slow
release of pyrophosphate may increase the fidelity of Dpo4 by stalling mispaired
primer extension and promoting pyrophosphorolysis that reverses the
polymerization reaction. Indeed, Dpo4 has robust pyrophosphorolysis activity and
degrades the primer strand in the presence of pyrophosphate. The correct
incoming nucleotide allows DNA synthesis to overcome pyrophosphorolysis, but an
incorrect incoming nucleotide does not.
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Figure 1.
Figure 1 Ribbon diagrams of the T/dATP (A), T/dGTP-1 (B) and
T/dGTP-2 (C) and T/G (D) structures around the active site. Dpo4
is shown as ribbons. The three conserved carboxylates in the
active site, the last two base pairs of the primer/template and
the replicating base pair are shown as ball-and-stick models.
The template strand is shown in blue and the primer strand in
purple. The incoming nucleotide is shown in different colors for
each crystal structure. The metal ions are shown as green
spheres. The 2F[o]-F[c] electron density maps are contoured at 1
 level
and superimposed onto the nucleic acid portion.
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Figure 2.
Figure 2 Structural comparison of Dpo4 and T7 DNA polymerase.
(A) The replicating base pairs in three Dpo4 structures (T/dGTP,
T/dATP and Ab-2A) are shown as ball-and-stick models. The two
metal ions (A and B) are shown as green spheres. The A-metal ion
position differs in each structure. The conformation of the
triphosphate is denoted as 'chair-like' and 'goat tail-like'.
(B) Superposition of T/dATP, T/dGTP and Ab-2A structures. The C
 traces,
DNA and nucleotide substrate are shown in stick models. A
zoom-in stereo view of the finger domain with the replicating
base pair and metal ions (outlined in gray) is shown on the
right. The colors representing each structure are indicated. (C)
Superposition of the metal ion coordination in Dpo4 (Ab-2A,
yellow and brown colors) and T7 DNA polymerase (PDB: 1T7P, blue
and green colors) in a stereo view. The oxygen atoms of the
three conserved carboxylates and those involved in metal ion
coordination are highlighted in red. The metal ion coordination
is schematically drawn on the right. Red indicates ligands
conserved in both polymerases, light green in Dpo4 only and blue
in T7 only. The hypothesized 3'-OH of the primer strand is shown
in gray.
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The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
EMBO J
(2005,
24,
2957-2967)
copyright 2005.
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