section 24.2
Enzymology of DNA Replication
549
and catalyze formation of the phosphodiester bond.
The
substrates
for DNA
polymerases
are
the four
deoxynucleoside-5'-triphosphates (dATP, dCTP, dGTP,
and dTTP) and a single-stranded template DNA. The over-
all chemical reaction catalyzed by all DNA polymerases
is
Poly(nucleotide„)-3'-OH + dNTP
poly(nucleotide„+i )-3'-OH + PPj
in which PP; represents pyrophosphate cleaved from the
dNTP. That is, a reaction occurs between a 3'-OH group
at a terminus of a DNA strand and the phosphoryl group
(the one linked to the sugar) of an incoming nucleoside
triphosphate.
Even though the hydrolysis of the nucleoside triphos-
phates has a large negative AG, the polymerization re-
action as written still has a positive AG at concen-
trations present in a cell and in laboratory reactions
(+0.5 kcal/mol = 2.1 kJ/mol). Thus, in the absence of any
other reaction DNA polymerases would catalyze depoly-
merization rather than polymerization. Indeed, if excess
pyrophosphate and a polymerase are added to a solution
containing a partially replicating DNA molecule, the poly-
merase acts like a nuclease. In order to drive the reaction
to the right, pyrophosphate must be removed, and this is
accomplished by a potent pyrophosphatase, a widely dis-
tributed enzyme that breaks down pyrophosphate to inor-
ganic phosphate. Hydrolysis of pyrophosphate has a large
negative free energy, so essentially all of the pyrophos-
phate is rapidly removed.
No DNA polymerase can catalyze the reaction between
two free nucleotides, even if one has a 3-OH group and the
other a 5'-triphosphate. Polymerization can occur only if
the nucleotide with the 3'-OH group is hydrogen-bonded
to the template strand. Such a nucleotide is called
&
primer
(Figure 24-5). The primer can either be a single nucleotide
or the terminus of a hydrogen-bonded oligonucleotide.
When an incoming nucleotide is joined to a primer it sup-
plies another free 3'-OH group, so that the growing strand
itself is a primer. Since polymerization occurs only at the
3'-OH end, strand growth is said to proceed in the 5' —> 3'
direction. All known polymerases (both DNA and RNA)
are capable of chain growth only in the 5' -> 3' direction.
This unidirectional feature of polymerases complicates the
simultaneous replication of both strands of DNA.
Polymerization is not confined to addition of a nu-
cleotide to a growing strand in a replication fork. For
example, pol I can also add nucleotides to the 3'-OH
group at a single-strand (a nick) in a double helix. This
activity results from the ability of pol I both to recognize
a 3'-OH group anywhere in the helix and to displace the
base-paired strand ahead of the available 3'-OH group.
T e m p la te
P r o d u c t
(a)
P-5'*
HO-3'.
3--OH
5 -P
N o s y n th e s is
(b ) HO-3'.
5'-P
N o s y n th e s is
(C)
P-5'
HO-3'
3--OH
5 -P
P-5'
HO-3'
5'-P
(d)
P-5'
HO-3''
3'-OH
5 -P
N o s y n th e s is
N ic k -
(e)
P-5'---------------3'-OH
HO-3'-------------------------
P-5'---------------3'-OH
----------------------5'-P
P-5’'
H O -3'
3-O H
5'-P
d )
P-5'
HO-3'
F IG U R E 2 4 -5
3'-o h
5'-P
P-5'
HO-3'
5'-P
Effect of various templates used in DNA polymerization reactions. A free 3'-OH on a hydrogen-bonded nucleotide at
the strand terminus and a non-hydrogen-bonded nucleotide at the adjacent position on the template strand are needed for
strand growth. Newly synthesized DNA is shown in color.
