section 24.3
The Replication Fork
553
Topoisomerase
Leading
S S B Proteins
Strand
Unwinding enzyme (helicase)
Replicase
RNA primers
Primase
Lagging
Strand
FIGURE 24-8
Simplified overview of a bacterial DNA replication fork. The leading strand is initiated with an RNA primer and then
elongated by a DNA polymerase, pol
III
(
r e p l i c a s e
). The lagging strand is synthesized in a series of fragments, each
initiated by
p r i m a s e
which inserts short sequences of RNA primers that will be elongated by a replicase. The RNA
fragments are removed by another DNA polymerase, pol I, and the breaks in the sugar-phosphate backbone are sealed
by
D N A l i g a s e .
replication fork is impossible. This additional coiling can
be removed by a
topoisomerase
that can introduce negative
superhelicity. In
E. coli,
the cnz.yme
DNA gyrase
produces
negative superhelicity
in nonsupercoiled covalent circles
and is also responsible for removing the positive superhe-
licity generated during replication. The evidence for this
comes from
in vivo
experiments using drugs (discussed
later) that inhibit DNA gyrase; addition of any of these
drugs to a growing bacterial culture or raising the temper-
ature of cells with a temperature-sensitive gyrase protein
inhibits DNA synthesis. Furthermore,
in vitro
replication
of circular DNA can proceed only if DNA gyrase or a
similar topoisomerase is present in the reaction mixture.
Inhibitors of DNA Replication
Inhibitors of DNA synthesis are used in the laboratory and
in treatment of bacterial, viral, and neoplastic diseases.
Successful treatment of these conditions requires careful
attention to dosage and the fine difference between drug
effectiveness and toxicity.
Inhibitors of DNA synthesis can be divided into three
main classes:
1. Those that prevent or reduce the synthesis of
precursors (bases, nucleotides),
2. Those that affect either the template or the priming
ability of the growing strand, and
3. Those that act directly on polymerases or other
enzymes needed for replication.
A variety of inhibitors bind to DNA and thereby elimi-
nate its template activity. Notable among these are the
in-
tercalating agents,
which slip in between base pairs (e.g.,
the acridines, phenanthridium derivatives), and the an-
thracyclines (daunorubicin, doxorubicin, and plicamycin).
Other agents bind to DNA covalently and cause chain
breakage (bleomycin, zinostatin) and interstrand cross-
links (alkyl sulfonates, anthramycin, mitomycin, nitro-
gen mustards). Several of these compounds are also
useful antitumor agents. A variety of platinum coordi-
nation compounds bind to DNA and inhibit its tem-
plate activity, apparently by binding to guanine. Sub-
stances that prevent extension of the growing chain
(2',3'-dideoxyribonucleosides, cordycepin) are incorpo-
rated into the growing chain, but since they lack a 3'-OH
group, further extension is not possible.
Only a few substances act directly on DNA poly-
merases; they often are effective only on one or a small
number of polymerases. For example,
acyclovir
inhibits
the DNA polymerase of herpes simplex. Aphidicolin in-
hibits pol
a,
pol
S
(but not pol
p
or pol /), many viral
polymerases, and pol I and pol II of yeast; this compound
is extremely valuable in laboratory research on DNA repli-
cation. Other components of the replication complex can
also be inhibited; for example,
2
'-dideoxyazidocytidine
is an inhibitor of bacterial primase, and coumermycin,
novobiocin, oxolinic acid, and nalidixic acid are effective
inhibitors of DNA gyrase in bacteria.
Topoisomerase I Inhibitors
A variety of antibiotics and antineoplastic drugs exert their
therapeutic effects by interaction with topoisomerase I
and disruption of DNA synthesis during phase of dividing
cells. Topoisomerase I is essential for DNA replication
and cell growth. The enzyme relieves torsional stress in
DNA by inducing reversible single-strand breaks. The in-
teraction of topoisomerase I and certain drugs produces
double-strand breaks in DNA that are irreversible and can
lead to cell death.
