Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter by by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morg

DNA REPLICATION MECHANISMS
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Figure 5–22 The DNA-helix-passing reaction catalyzed by DNA
topoisomerase II. Unlike type I topoisomerases, type II enzymes hydrolyze
ATP (red), which is needed to release and reset the enzyme after each cycle.
Type II topoisomerases are largely confined to proliferating cells in eukaryotes;
partly for that reason, they have been effective targets for anticancer drugs.
Some of these drugs inhibit topoisomerase II at the third step in the figure
and thereby produce high levels of double-strand breaks that kill rapidly
dividing cells. The small yellow circles represent the phosphates in the DNA
backbone that become covalently bonded to the topoisomerase (see
Figure 5–21).
two circular DNA
double helices
that are interlocked
topoisomerase II
double-strand break in the helix. These enzymes are activated by sites on chromosomes
where two double helices cross over each other such as those generated by
supercoiling in front of a replication fork (see Figure 5–20). Once a topoisomerase
II molecule binds to such a crossing site, the protein uses ATP hydrolysis to perform
the following set of reactions efficiently: (1) it breaks one double helix reversibly
to create a DNA “gate”; (2) it causes the second, nearby double helix to pass
through this opening; and (3) it then reseals the break and dissociates from the
DNA. At crossover points generated by supercoiling, passage of the double helix
through the gate occurs in the direction that will reduce supercoiling. In this way,
type II topoisomerases can relieve the overwinding tension generated in front of
a replication fork. Their reaction mechanism also allows type II DNA topoisomerases
to efficiently separate two interlocked DNA circles (Figure 5–22).
Topoisomerase II also prevents the severe DNA tangling problems that would
otherwise arise during DNA replication. This role is nicely illustrated by mutant
yeast cells that produce, in place of the normal topoisomerase II, a version that is
inactive above 37°C. When the mutant cells are warmed to this temperature, their
daughter chromosomes remain intertwined after DNA replication and are unable
to separate. The enormous usefulness of topoisomerase II for untangling chromosomes
can readily be appreciated by anyone who has struggled to remove a tangle
from a fishing line without the aid of scissors.
2 ATP
P i
P i
topoisomerase
recognizes the
entanglement and
makes a reversible
covalent attachment
to the two opposite
strands of one of the
double helices (orange)
creating a doublestrand
break and
forming a protein gate
the topoisomerase
gate opens to let the
second DNA helix pass
the gate shuts releasing
the red helix
DNA Replication Is Fundamentally Similar in Eukaryotes and
Bacteria
Much of what we know about DNA replication was first derived from studies
of purified bacterial and bacteriophage multienzyme systems capable of DNA
replication in vitro. The development of these systems in the 1970s was greatly
facilitated by the prior isolation of mutants in a variety of replication genes; these
mutants were exploited to identify and purify the corresponding replication proteins.
The first mammalian replication system that accurately replicated DNA in
vitro was described in the mid-1980s, and mutations in genes encoding nearly all
of the replication components have now been isolated and analyzed in the yeast
Saccharomyces cerevisiae. As a result, much is known about the detailed enzymology
of DNA replication in eukaryotes, and it is clear that the fundamental features
of DNA replication—including replication-fork geometry and the use of a multiprotein
replication machine—have been conserved during the long evolutionary
process that separated bacteria from eukaryotes.
There are more protein components in eukaryotic replication machines than
there are in the bacterial analogs, even though the basic functions are the same.
Thus, for example, the eukaryotic single-strand binding (SSB) protein is formed
from three subunits, whereas only a single subunit is found in bacteria. Similarly,
the eukaryotic DNA primase is incorporated into a multisubunit enzyme that also
contains a polymerase called DNA polymerase α-primase. This protein complex
begins each Okazaki fragment on the lagging strand with RNA and then extends
the RNA primer with a short length of DNA. At this point, the two main eukaryotic
replicative DNA polymerases, Polδ and Polε, come into play: Polδ completes
each Okazaki fragment on the lagging strand and Polε extends the leading strand.
The increased complexity of eukaryotic replication machinery probably reflects
2 ADP
two circular DNA double
helices that are separated
MBoC6 m5.24/5.22
reversal of
the covalent
attachment
of the topoisomerase
restores an
intact orange
double helix