 |
PDBsum entry 1s9f
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Transferase/DNA
|
PDB id
|
|
|
|
1s9f
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
References listed in PDB file
|
|
|
Key reference
|
|
|
Title
|
|
Dpo4 is hindered in extending a g.T mismatch by a reverse wobble.
|
|
|
Authors
|
|
J.Trincao,
R.E.Johnson,
W.T.Wolfle,
C.R.Escalante,
S.Prakash,
L.Prakash,
A.K.Aggarwal.
|
|
|
Ref.
|
|
Nat Struct Mol Biol, 2004,
11,
457-462.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
The ability or inability of a DNA polymerase to extend a mispair directly
affects the establishment of genomic mutations. We report here kinetic analyses
of the ability of Dpo4, a Y-family polymerase from Sulfolobus solfataricus, to
extend from all mispairs opposite a template G or T. Dpo4 is equally inefficient
at extending these mispairs, which include, surprisingly, a G.T mispair expected
to conform closely to Watson-Crick geometry. To elucidate the basis of this, we
solved the structure of Dpo4 bound to G.T-mispaired primer template in the
presence of an incoming nucleotide. As a control, we also determined the
structure of Dpo4 bound to a matched A-T base pair at the primer terminus. The
structures offer a basis for the low efficiency of Dpo4 in extending a G.T
mispair: a reverse wobble that deflects the primer 3'-OH away from the incoming
nucleotide.
|
|
|
|
|
|
|
|
Figure 1.
Figure 1. Dpo4-catalyzed extension of a G  T
primer -terminal mispair. (a) The incorporation of dGTP
opposite C following an A-T primer -terminal base pair (left) is
500-fold more efficient when compared to the incorporation of
dGTP opposite C following a G T
primer -terminal mispair (right). The incorporation of dGTP (200
 M)
was examined for the indicated time periods for each substrate.
The enzyme concentrations for the A-T base-paired and the G T
mispaired primer termini were 0.2 nM and 1.2 nM, respectively.
(b) The rate of dGTP incorporation opposite a template C
following an A-T primer -terminal base pair (left) or following
a G T
primer -terminal mispair (right) was graphed as a function of
dGTP concentration. The solid line represents the best fit to
the Michaelis-Menten equation. The steady-state parameters, k
[cat] and K [m], are listed in Table 1.
|
|
Figure 2.
Figure 2. Dpo4 in complex with matched and mismatched primer
termini. (a) An overall view of different Dpo4 -DNA
complexes: the type I structure determined by Yang and
colleagues, and the structures reported here of Dpo4 bound to a
mismatched (G T)
and the matched (A-T) primer template. Dpo4 in each complex is
colored by palm (blue), fingers (yellow) and thumb (orange)
domains, as well as PAD (green). The terminal base pair of the
DNA in each complex is red. (b) Close-up view of the
template-primer terminus in each complex, highlighting the
terminal base pair (red), the incoming nucleotide, the active
site residues (Asp7, Asp105 and Glu106) coordinating a metal ion
(yellow ball, refined as Ca^2+), residues (Tyr48, Arg51 and
Lys159) bonding to the di- or triphosphate moiety of the
incoming nucleotide, as well as residues (Val32, Ala42 and
Gly58) from the fingers domain that impinge on the templating
base. Also in red is the C3' atom at the primer terminus. The
figure was generated with MolMol32 and PovRay
(http://www.povray.org). (c) 2F [o] - F [c] electron density for
a portion of the primer and the incoming nucleotide in the G
T
complex. The map was computed with the terminal guanine (ddG)
and the incoming nucleotide (ddCTP) omitted (and followed by
simulated annealing). The terminal guanine inverts to form a
reverse wobble. Also in red is the hypothetical position for the
guanine in a standard wobble configuration (which is completely
out of density).
|
|
|
|
|
|
The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
Nat Struct Mol Biol
(2004,
11,
457-462)
copyright 2004.
|
|
|
|
|
|
|
