 |
PDBsum entry 1wn7
|
|
|
|
References listed in PDB file
|
 |
|
Key reference
|
|
|
Title
|
|
Structural mechanism for coordination of proofreading and polymerase activities in archaeal DNA polymerases.
|
|
|
Authors
|
|
T.Kuroita,
H.Matsumura,
N.Yokota,
M.Kitabayashi,
H.Hashimoto,
T.Inoue,
T.Imanaka,
Y.Kai.
|
|
|
Ref.
|
|
J Mol Biol, 2005,
351,
291-298.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
A novel mechanism for controlling the proofreading and polymerase activities of
archaeal DNA polymerases was studied. The 3'-5'exonuclease (proofreading)
activity and PCR performance of the family B DNA polymerase from Thermococcus
kodakaraensis KOD1 (previously Pyrococcus kodakaraensis KOD1) were altered
efficiently by mutation of a "unique loop" in the exonuclease domain.
Interestingly, eight different H147 mutants showed considerable variations in
respect to their 3'-5'exonuclease activity, from 9% to 276%, as against that of
the wild-type (WT) enzyme. We determined the 2.75A crystal structure of the
H147E mutant of KOD DNA polymerase that shows 30% of the 3'-5'exonuclease
activity, excellent PCR performance and WT-like fidelity. The structural data
indicate that the properties of the H147E mutant were altered by a
conformational change of the Editing-cleft caused by an interaction between the
unique loop and the Thumb domain. Our data suggest that electrostatic and
hydrophobic interactions between the unique loop of the exonuclease domain and
the tip of the Thumb domain are essential for determining the properties of
these DNA polymerases.
|
|
|
|
|
|
|
|
Figure 1.
Figure 1. Overall structure of KOD DNA polymerase. The
structure is composed of domains and subdomains, namely the
N-terminal domain (N-ter, violet), exonuclease domain (Exo,
gray) including the unique loop (blue), polymerase domain (Pol)
including the Palm (brown) and Fingers (green) subdomains, and
the Thumb domain (red), including the Thumb-1 and Thumb-2
subdomains and disordered regions (dotted lines). The orange
region represents the E-cleft. The positions of two amino acids
in the exonuclease domain are shown as colored balls (red,
AA147; light blue, AA142).
|
|
Figure 2.
Figure 2. Amino acid sequence alignments of proposed
structurally corresponding residues of KOD DNA polymerases
(138-152, exonuclease domain; and 682-697, Thumb domain).
Acidic, basic and hydrophobic residues are shown in red, blue
and green, respectively. The boxed amino acid residues comprise
the loop structure in the exonuclease domain. The underlined
amino acid residues are the core sequence of the Exo I motif.
The abbreviations used are as follows: KOD, Thermococcus
kodakaraensis DNA polymerase; Tgo, Thermococcus gorgonarius DNA
polymerase; Tli, Thermococcus litoralis DNA polymerase;
9°N-7, Thermococcus sp. 9°N-7 DNA polymerase; Tag,
Thermococcus aggregans DNA polymerase; Pfu, Pyrococcus furiosus
DNA polymerase; RB69, bacteriophage RB69 DNA polymerase; T7,
bacteriophage T7 DNA polymerase.
|
|
|
|
|
|
The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2005,
351,
291-298)
copyright 2005.
|
|
|
Secondary reference #1
|
|
|
Title
|
|
Crystal structure of DNA polymerase from hyperthermophilic archaeon pyrococcus kodakaraensis kod1.
|
|
|
Authors
|
|
H.Hashimoto,
M.Nishioka,
S.Fujiwara,
M.Takagi,
T.Imanaka,
T.Inoue,
Y.Kai.
|
|
|
Ref.
|
|
J Mol Biol, 2001,
306,
469-477.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Figure 1.
Figure 1. (a) Overall structure of
KOD DNA polymerase. The struc-
ture is composed of domains and
subdomains, which are N-terminal
(N-ter, violet), Exonuclease (Exo,
blue), Polymerase (Pol) domain
including the Palm (brown) and
Fingers (green) subdomains and
the Thumb domain (red), including
the Thumb-1 and Thumb-2 subdo-
mains. Conserved carboxylate
residues in Polymerase and Exonu-
clease active site are shown by ball-
and-stick models. (b) Confor-
mational comparison of Thumb
domains among three archaeal
DNA polymerases. Red, KOD
DNA polymerase; blue, Tgo DNA
polymerase; and green, 9°N-7 DNA
polymerase. The comparison shows
that the Thumb domain of KOD
DNA polymerase displays the most
``opened'' conformation.
|
|
Figure 4.
Figure 4. Molecular surface with electrostatic potential
map around the forked-point. The red and blue surfaces
are acidic and basic regions, respectively. Domains and
subdomains are labeled with orange letters. Polymerase
and Exonuclease active sites are labeled with P and E,
respectively. The b-hairpin is labeled with b.
|
|
|
|
|
|
The above figures are
reproduced from the cited reference
with permission from Elsevier
|
|
|
|
|
|
|
