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chapter 24
DNA Replication, Repair, and Mutagenesis
F I G U R E 2 4 - 6
Strand displacement and nick translation on linear and circular molecules. In nick translation, a nucleotide is removed
by an exonuclease activity for each nucleotide added. The growing strand is shown in color.
This
reaction
is
called
strand
displacement
(Fig-
ure 24-6). Not all DNA polymerases can carry out strand
displacement.
Pol I has several enzymatic activities other than its abil-
ity to polymerize; one of these is important in maintaining
continuity of the daughter strand, and the other improves
the fidelity of replication. These two functions are a 5' -» 3'
exonuclease activity and a 3'
—*■
5' exonuclease activity.
Exonuclease Activities o f Polymerase I
Occasionally DNA polymerase, in error, adds a nu-
cleotide to the 3 -OH terminus that cannot hydrogen-bond
to the corresponding base in the template strand. Such a
nucleotide would clearly alter the information content of
the daughter DNA molecule, and mechanisms exist for
correcting such incorporation errors.
Once having added an incorrect nucleotide and moved
on to the next position, pol I cannot add another nucleotide
because the enzyme requires a primer that is correctly
hydrogen-bonded. Where such an impasse is encountered,
a 3' —> 5' exonuclease activity, which may be thought of
simply as pol I running backward or in the 3' —>• 5' direc-
tion, is stimulated and the unpaired base is removed. After
removal of this base, the exonuclease activity stops, poly-
merizing activity is restored, and chain growth resumes.
This exonuclease activity is called the
proofreading
or
editing function
of polymerase I.
Pol I also has a potent 5' —►
3' exonucleolytic activ-
ity (Figure 24-7). This activity is directed against a base-
paired strand and consists of stepwise removal of nu-
cleotides one by one from the 5'-P terminus. Furthermore,
the nucleotide removed can be either of the deoxyribo or
the ribo type. The 5' —»• 3' exonucleolytic activity also can
be coupled to the polymerizing activity. Recall that pol I
can add nucleotides to a 3'-OH group at a nick and displace
the downstream strand. Under certain conditions, the dis-
placement reaction does not occur and instead the 5' ->• 3'
exonuclease acts on the strand that would otherwise be
displaced, removing one downstream nucleotide for each
nucleotide added to the 3' side of the nick. Thus, the po-
sition of the nick moves along the strand; this reaction is
called
nick translation.
It is an important laboratory pro-
cedure for producing labeled DNA that can be used as
probes; simply by carrying out the reaction in the pres-
ence of radioactive or chemically labeled nucleotides, an
unlabeled DNA molecule with nicks in both strands can
be converted to a labeled molecule. DNA probes are used
for many diagnostic and forensic purposes (Chapter 23).
The main function of the 5' -»
3'
exonuclease activity is
to remove ribonucleotide primers that are used in DNA
replication.
Pol I also has a 5' —>• 3' endonuclease activity. An en-
donucleolytic cut is made between two base pairs that
follow a 5'-P-terminated segment of unpaired bases, as
shown below.
M
i
l
l
G G a C
A
A
G
A
G
T
T
C
T
This type of endonucleolytic activity is unimportant in
normal replication but is important in excision repair.
