 |
PDBsum entry 2va3
|
|
|
|
References listed in PDB file
|
 |
|
Key reference
|
|
|
Title
|
|
Structure and activity of y-Class DNA polymerase dpo4 from sulfolobus solfataricus with templates containing the hydrophobic thymine analog 2,4-Difluorotoluene.
|
|
|
Authors
|
|
A.Irimia,
R.L.Eoff,
P.S.Pallan,
F.P.Guengerich,
M.Egli.
|
|
|
Ref.
|
|
J Biol Chem, 2007,
282,
36421-36433.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
The 2,4-difluorotoluene (DFT) analog of thymine has been used extensively to
probe the relative importance of shape and hydrogen bonding for correct
nucleotide insertion by DNA polymerases. As far as high fidelity (A-class)
polymerases are concerned, shape is considered by some as key to incorporation
of A(T) opposite T(A) and G(C) opposite C(G). We have carried out a detailed
kinetic analysis of in vitro primer extension opposite DFT-containing templates
by the trans-lesion (Y-class) DNA polymerase Dpo4 from Sulfolobus solfataricus.
Although full-length product formation was observed, steady-state kinetic data
show that dATP insertion opposite DFT is greatly inhibited relative to insertion
opposite T (approximately 5,000-fold). No products were observed in the
pre-steady-state. Furthermore, it is noteworthy that Dpo4 strongly prefers dATP
opposite DFT over dGTP (approximately 200-fold) and that the polymerase is able
to extend an A:DFT but not a G:DFT pair. We present crystal structures of Dpo4
in complex with DNA duplexes containing the DFT analog, the first for any DNA
polymerase. In the structures, template-DFT is either positioned opposite
primer-A or -G at the -1 site or is unopposed by a primer base and followed by a
dGTP:A mismatch pair at the active site, representative of a -1 frameshift. The
three structures provide insight into the discrimination by Dpo4 between dATP
and dGTP opposite DFT and its inability to extend beyond a G:DFT pair. Although
hydrogen bonding is clearly important for error-free replication by this Y-class
DNA polymerase, our work demonstrates that Dpo4 also relies on shape and
electrostatics to distinguish between correct and incorrect incoming nucleotide.
|
|
|
|
|
|
|
|
Figure 2.
FIGURE 2. Dpo4-catalyzed polymerization opposite
DFT-containing template DNA. Dpo4-catalyzed (100 nM) extension
of 12/18-mer DNA (200 nM) containing DFT was allowed to proceed
in the presence of a 4 mM dNTP mix. For comparisons regarding
the time frame of full-length extensions opposite native DNA,
please see Fig. 2 of Ref. 22 and/or Fig. 1 of Ref. 24.
|
|
Figure 5.
FIGURE 5. Examples of the quality of the final electron
density. Electron density around the DNA duplex at the active
site is shown. A, Dpo4(DFT:13A); template
5'-TTCAG(DFT)AGTCCTTCCCCC-3' and primer 5'-GGGGGAAGGACTA-3',
ddCTP. B, Dpo4(dGTP); template 5'-TTCA(DFT)TAGTCCTTCCCCC-3' and
primer 5'-GGGGGAAGGACTA-3', dGTP. C, one of the Dpo4(DFT:13G)
complexes per asymmetric unit; D, second Dpo4(DFT:13G) complex;
template 5'-TTCAG(DFT)AGTCCTTCCCCC-3' and primer
5'-GGGGGAAGGACTG-3', ddCTP. The 3F[o] - 2F[c]electron density
maps (blue chicken wire) are contoured at the 1  level.
The Dpo4 protein is shown as ribbons and the DNA duplex as
sticks. The Ca^2+ ions are depicted as green spheres.
|
|
|
|
|
|
The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2007,
282,
36421-36433)
copyright 2007.
|
|
|
|
|
|
|
