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chapter 24
DNA Replication, Repair, and Mutagenesis
TABLE 24-3
Properties o f Eukaryotic DNA Polymerases
Property
Polymerase
Location in cell
a
Nucleus
P
Nucleus
7
Mitochondria
S
Nucleus
e
Nucleus
Associated primase
3' exonuclease
Sensitivity to aphidicolin
Biological activity
Yes
No
High
Replication
(lagging strand)
No
No
Low
DNA repair
No
Yes
Low
Replication
No
Yes
High
Replication
(leading strand)
No
Yes
Low
Replication
eukaryotic ligases are located in the nucleus. Ligase I is
predominant in proliferating cells and presumably plays a
role in DNA replication; ligase II predominates in resting
cells.
Eukaryotic Polymerases
Five polymerizing enzymes have been isolated from
many mammalian cells (Table 24-3). Three—pol a, pol
and pol
y
—function in replication. Pol
a
is the major poly-
merase of mammalian cells; it is found in the nuclei and
is analogous to
E. coli
pol III. It is a multisubunit enzyme
with a core (4-5 subunits) responsible for polymerization
and a holoenzyme form possessing additional subunits and
activities. It lacks the 3' —»• 5' exonuclease editing function.
An intriguing protein subunit in the holoenzyme enables
it to bind AppppA (diadenosine tetraphosphate), a small
molecule that stimulates replication of resting mammalian
cells and is hypothesized to be a growth signal. The pol
a
holoenzyme of rat liver also possesses a DNA primase
activity (see below), a feature not found in prokaryotic en-
zymes. Pol /lisa nuclear polymerase, probably analogous
in function to
E. coli
pol I. Pol
y
is found in mitochon-
dria and is responsible for replication of mitochondrial
DNA. It functions in the same way as pol III but is a single
polypeptide.
24.3 The Replication Fork
DNA replication requires not only an enzymatic mecha-
nism for adding nucleotides to the growing chains but also
a means of unwinding the parental double helix. These
are distinct processes, and the unwinding of the helix is
closely related to the initiation of synthesis of precursor
fragments.
The pol III holoenzyme cannot unwind the helix.
In order for unwinding to occur, hydrogen bonds and
hydrophobic interactions must be eliminated, which re-
quires energy. Pol I utilizes the free energy of hydroly-
sis of the triphosphate for unwinding as it synthesizes a
DNA strand in a way that other polymerases cannot; in-
stead, helix unwinding is accomplished by enzymes called
helicases.
These enzymes hydrolyze ATP and utilize the
free energy of hydrolysis for unwinding.
Unwinding of the helix by a helicase is not sufficient in
itself for advance of the replication fork. Accessory pro-
teins called
single-stranded DNA-binding proteins (SSB
proteins)
are usually needed. As a helicase advances, it
leaves in its wake two single-stranded regions: a longer
one that is copied discontinuously and a shorter one
just ahead of the leading strand. In order to prevent the
single-stranded regions from reannealing or from forming
intrastrand hydrogen bonds, the single-stranded DNA is
protected with SSB proteins (Figure 24-8). SSB proteins
bind tightly to both single-stranded DNA and to one an-
other and hence are able to cover extended regions. As the
polymerase advances, it must displace the SSB proteins
so that base pairing of the added nucleotide can occur.
Some phage replication systems utilize a single protein
that functions as both a helicase and an SSB protein; the
gene-32 protein of phage T
4
is the prototype. It binds very
tightly to single-stranded DNA and exceedingly tightly
to itself, and its binding energy is great enough to unwind
the helix.
As a replication fork moves along a circular helix, rota-
tion of the daughter molecules around one another causes
the individual polynucleotide strands of the unreplicated
portion of the molecule to become wound more tightly, i.e.,
overwound. (This may be difficult to visualize but can be
seen by taking two interwound circular strings and pulling
them apart at any point.) Thus, advance of the replica-
tion forks causes positive supercoiling (Chapter 23) of the
unreplicated portion. This supercoiling obviously cannot
increase indefinitely because soon the unreplicated por-
tion becomes coiled so tightly that further advance of the
