Biology Reference
In-Depth Information
dNTP as well as in the processivity and translocation of
the polymerase. When a polymerase binds to the DNA
substrate to form a binary complex, it adopts an
“open” conformation. Upon binding the correct
incoming dNTP to form a ternary complex, the fingers
subdomain rotates so that the protein achieves a “closed”
conformation. In the closed conformation, residues of
the fingers subdomain help to constrain the active site
containing the nascent base pair (i.e., the pair comprised
of the template base and the incoming dNTP). Only the
four correct Watson
Kinetic and Chemical Mechanisms
of DNA Polymerization
A general mechanism that applies to high-fidelity
DNA polymerases involved in chromosomal DNA
synthesis is provided in
Figure 5.4
.
87
e
89
As illustrated,
this intricate biological process occurs via a multiplica-
tive mechanism in which the polymerase binds DNA
prior to the binding of dNTP. The first point for gener-
ating high catalytic efficiency and polymerization
fidelity occurs through the binding of dNTP to the poly-
merase:DNA complex (step 2). The binding affinity for
a nucleotide opposite its correct pairing partner is gener-
ally designated as a K
d
value. In general, the K
d
value for
binding a correct nucleotide is ~10
m
M while K
d
values
for incorrect nucleotides are typically at least 10-fold
higher. This reduction in binding affinity provides
a key control point for maintaining fidelity during
DNA synthesis. After the polymerase binds a correct
dNTP, the enzyme:DNA complex undergoes a confor-
mational change (step 3) that is proposed to align the
incoming dNTP into a precise geometrical orientation
that allows efficient phosphoryl transfer (step 4). This
conformational change step reflects an “induced-fit”
mechanism and influences fidelity by further imposing
discrimination against nucleotidemisinsertion.
90
The con-
formational change step is consistent with structural
evidence for the transition of the polymerase from an
“open” conformation in the binary complex to the
“closed” conformation in the ternary complex. Inherent
in this transition is movement of the fingers subdomain
which rotates and helps constrain the active site contain-
ing the nascent base pair. In most high-fidelity polymer-
ases, a misaligned intermediate caused by the binding of
an incorrect dNTP alters the geometry of the polymer-
ase's active site such that the rate constant for the confor-
mational change step is reduced significantly. This
Crick base pairs will fit properly in
the active site of classical polymerases when it is in the
closed conformation. Only in this conformation do the
3
0
-oxygen atom of the primer terminus, the
a
-phosphate
of the incoming nucleotide, and the two Mg
2
þ
ions exist
in the proper positions for efficient catalysis. Structures
of classical DNA polymerases bound to incorrect
incoming dNTPs in ternary complexes have been diffi-
cult to obtain presumably because of the unstable nature
of such complexes. However, several structures approx-
imating this complex have been determined.
78,79
In these
structures, the polymerases adopt a “partially open”
conformation which is not capable of performing effi-
cient phosphoryl transfer.
Subtle differences in these structural features have
a large impact on the biological function of these
enzymes. For example, DNA polymerases involved in
chromosomal replication tend to have long, extended
fingers
80-82
whereas DNA polymerases that specifically
replicate DNA lesions have shorter fingers.
83-85
It
appears that the difference in the size of the fingers influ-
ences the fidelity and processivity of these polymerases.
In fact, there is evidence that specialized DNA polymer-
ases need shorter fingers to accommodate structurally
diverse DNA lesions whereas the longer fingers found
on replicative DNA polymerase are important for
higher replication fidelity and enhanced processivity.
86
e
FIGURE 5.4
Kinetic mechanism for DNA poly-
merases. Individual steps along the pathway for
DNA polymerization are numbered and identified as
described in the text. Abbreviations: E
dNTP
2
Conformational
change
E:DNA
n
DNA binding
E:DNA
n
:dNTP
¼
polymerase;
3
DNA
n
¼
DNA substrate; E'
¼
conformational change
dNTP binding
1
in DNA polymerase; PP
i
¼
inorganic pyrophosphate;
E':DNA
n
:dNTP
DNA
n
þ
1
¼
DNA product (DNA extended by one
nucleobase).
Processive
DNA synthesis
8
Phosphoryl
transfer
E + DNA
n
4
7
E':DNA
n+1
:PP
i
PP
i
5
Enzyme
dissociation
6
Conformational
change
E:DNA
n+1
E:DNA
n+1
:PP
i
Pyrophosphate
release
