section 24.2
Enzymology of DNA Replication
551
S u b s t r a t e
P r o d u c t
(a)
5'
3’
3'
5'
3’
5'
3'
5'
(b )
(C)
(d)
(e)
(0
3'
5'
J
N ic k ■>.
3 'T5'
0
.-R N A
3-
5
'
r
5' —
N i c k - ^
- - R N A
3 i5 '
T
— 3'
_
.
«/
3' —
— 5'
3'
5-
3'
5'
3'
5'
3'
5'
3'
5'
5’
3-
5'
3'
5'
N o e x o n u c l e o ly ti c a c tiv ity
F I G U R E 2 4 - 7
Several substrates and products alter activity of the polymerase I 5'
3' exonuclease. The 5' terminus attacked by the
enzyme is shown by the small straight arrow, (a) 5'-Terminal nucleotides are removed from each end of the molecule,
(b) A gap is enlarged by cleavage at a 5' terminus, (c) A gap is formed by removal of a 5'-terminal nucleotide at a nick.
The gap is then enlarged as in (b). (d) RNA (colored line) at the boundary of a gap is removed by cleavage at the
5' terminus of the RNA. (e) A gap is formed from a nick at a DNA-RNA boundary by removal of 5'-P-terminal
ribonucleotides, (f) A non-hydrogen-bonded 5' terminus is resistant to the exonuclease; there is an endonucleolytic
activity.
Polymerase HI
Polymerase I plays an essential role in the replication
process in
E. coli,
but it is not responsible for the over-
all polymerization of the replicating strands. The enzyme
that accomplishes this is a less abundant enzyme, poly-
merase III (pol III). (A DNA polymerase II has also been
isolated from
E. coli,
but it probably plays no role in DNA
synthesis.) Pol III catalyzes the same polymerization re-
action as pot I but has certain distinguishing features. It
is a very complex enzyme and is associated with eight
other proteins to form the pol III holoenzyme. (The term
holoenzyme
refers to an enzyme that contains several dif-
ferent subunits and retains some activity even when one
or more subunits is missing.) Pol III is similar to pol I in
that it has a requirement for a template and a primer but its
substrate specificity is much more limited. For a template
pol III cannot act at a nick nor can it unwind a helix and
carry out strand displacement. The latter deficiency means
that an auxiliary system is needed to unwind the helix
ahead of a replication fork. Pol III, like pol I, possesses
a 3' -> 5' exonuclease activity, which performs the major
editing function in DNA replication. Polymerase III also
has a 3' exonuclease activity, but this activity does not seem
to play a role in replication.
Pol I and pol III holoenzyme are both essential for
E. co li
replication. The need for two polymerases seems
to be characteristic of all cellular organisms but not all
viruses, e.g.,
E. coli.
Phage T
4
synthesizes its own DNA
polymerase, which is capable of carrying out all functions
necessary for synthesizing phage DNA.
In the usual polymerization reaction, the activation en-
ergy for phosphodiester bond formation comes from cleav-
ing of the triphosphate. Since DNA ligase can use a
monophosphate, another source of energy is needed. This
energy is obtained by hydrolyzing either ATP or NAD;
the energy source depends on the organism from which
the DNA ligase is obtained.
Ligases have two major functions: the sealing of single-
strand breaks produced randomly in DNA molecules by
nucleases and the joining of fragments during a par-
ticular stage of replication.
DNA ligases
are enzymes
that can form a phosphodiester bond at a single-strand
break in DNA, a reaction between a 3'-OH group and a
5'-monophosphate. These groups must be termini of ad-
jacent base-paired deoxynucleotides (Figure 24-4).
Bacteria usually contain a single species of ligase. Mam-
malian cells possess two DNA ligases (I and II) present
in very small amounts compared with bacteria. Both
