Biology Reference
In-Depth Information
results of kinetic and structural studies suggest that the
physiological metal ion, Mg
2
þ
, enforces a correct tetrahe-
dral geometry in the arrangement of the oxygens present
on the phosphate groups whereas Mn
2
þ
accommodates
a variety of coordination states including square planar,
tetrahedral, and octahedral. As such, reductions in
fidelity caused by Mn
2
þ
result from the ability of this
metal ion to support phosphoryl transfer even in the
presence of misaligned intermediates.
100
After phosphoryl transfer, a second conformational
change (step 5) occurs that is required for pyrophos-
phate release (step 6). The release of pyrophosphate is
tightly linked with the ability of the DNA polymerase
to translocate along DNA to the next templating posi-
tion. Following this translocation step, the polymerase
can either remain bound to the nucleic acid and
continue primer elongation (step 8) or dissociate from
the elongated primer (step 7) and initiate DNA
synthesis on another usable primer template. The
ability to incorporate multiple nucleotides without
dissociating from DNA defines the processivity of the
polymerase. In loose terms, this can be viewed as the
ratio of (step 8/step 7). Polymerases involved in nuclear
and mitochondrial DNA synthesis are considered
highly processive as they display ratios of 100 or
greater. In contrast, specialized polymerases involved
in translesion DNA synthesis are far less processive
and display ratios closer to 1. The difference in proces-
sivity between classical and non-classical polymerases
is a consequence of their biological function. This makes
intuitive sense when one considers that high-fidelity
polymerase such as pol
d
must replicate thousands of
base pairs per binding event whereas a specialized
DNA polymerase such as pol
h
are only involved in
by-passing DNA lesions.
Exonuclease Proofreading
The last line of defense inpreventingmisincorporation
events is through the proofreading capacity of the DNA
polymerase catalyzed by its associated exonuclease
activity. Exonuclease proofreading should not be
confused with pyrophosphorolysis, the simple reversal
of the polymerization reaction. In fact, exonuclease
proofreading is far more complicated as it encompasses
translocation of the primer-terminus from the poly-
merase active site into the exonuclease active site, strand
separation of several nucleotides, positioning of the 3'-
end of the primer in the exonuclease active site, and
hydrolysis of the phosphodiester bond to excise the
terminal nucleotide (
Figure 5.6
). In addition to erasing
potentially pro-mutagenic mismatches, proofreading
also returns the polymerase to a correct primer-terminus
and allows for the renewal of “correct” DNA synthesis
without the need for polymerase dissociation and rebind-
ing. In the context of chemotherapy, this activity repre-
sents a potential mechanism of drug resistance as it
catalyzes the removal of chain-terminating nucleotides
from DNA.
Exonuclease activity requires the binding of two cata-
lytically important metals through conserved carboxylic
acid containing amino acids.
101-104
Polymerases that
contain mutations in these conserved amino acids are
incapable of excising misincorporated nucleotides and
Misinsertion
OH
OH
OH
i
ii
v
+
OH
OH
iv
iii
H
2
O
FIGURE 5.6
Exonuclease proofreading plays an important role in maintaining replicative fidelity. The minimal pathway for exonuclease
proofreading includes (
i
) movement of the mispaired primer-template from the polymerase active site into the exonuclease active site, (
ii
) strand
separation of several nucleotides, (
iii
) positioning of the 3'-end of the primer in the exonuclease active site, and (
iv
) hydrolysis of the phos-
phodiester bond to excise the terminal nucleotide. After excising potentially pro-mutagenic mismatches, the primer-template is returned to the
polymerization active site to resume DNA synthesis.
