234 MHR • Unit 3 Molecular Genetics
nitrogen. The colony of bacteria was then transferred
to a new medium containing regular nitrogen.
After the colony had doubled in size (indicating
approximately one complete round of cell division),
Meselson and Stahl isolated the DNA from the
bacterial cells and spun it in a centrifuge. Afterward,
they observed a single band marking a density
midway between the expected densities of DNA
containing^15 N(“heavy” DNA) and DNA containing(^14) N(“regular” DNA). This indicated that the DNA
after one round of replication was a hybrid — that
is, a mixture of heavy and regular DNA. This result
ruled out the possibility of conservative replication,
since that would have resulted in one band of
heavy DNA and another of regular DNA. This left
semi-conservative and dispersive replication as
potential models. To determine which was correct,
a second round of experimentation was required.
When Meselson and Stahl left the colony on the
second medium for two generations before
extracting the DNA and spinning it in a centrifuge,
they found two distinct bands. One of these bands
appeared at the same midway point, while the
other appeared at the expected density for regular
DNA. This result was consistent with the expected
pattern for semi-conservative replication. It also
ruled out the possibility of dispersive replication,
since the random assortment of DNA fragments
would result in the appearance of only one density
band. With both conservative and dispersive
replication ruled out, semi-conservative replication
became the accepted hypothesis for DNA
replication. This hypothesis has since been
supported by further experiments and by
microscopic images of replicating DNA.
The Process of Replication
The process of DNA replication is discussed in the
following pages as a series of three basic phases. In
the initiation phase, a portion of the DNA double
helix is unwound to expose the bases for new base
pairing. In the elongation phase, two new strands
of DNA are assembled using the parental DNA as
a template. Finally, in the termination phase, the
replication process is completed and the new
DNA molecules — each composed of one strand of
parental DNA and one strand of daughter DNA —
re-form into helices. In actual practice, all of these
activities may take place simultaneously on the
same molecule of DNA.
Initiation
In bacteria, the circular DNA strand includes a
specific nucleotide sequence of about 100 to 200 base
pairs known as the replication origin. This
nucleotide sequence is recognized by a group of
enzymes that bind to the DNA at the origin and
separate the two strands to open a replication
bubble. After a replication bubble has been opened,
molecules of an enzyme called DNA polymerase
insert themselves into the space between the two
strands. Using the parent strands as a template,
the polymerase molecules begin to add nucleotides
one at a time to create a new strand that is
complementary to the existing template strand.
For most of the life cycle of the cell, DNA is a
tightly bound and stable structure. Because the
bases face into the interior of the molecule, the
helix must be unwound for the individual chains
of nucleotides to serve as templates for the
formation of new strands. The points at which the
DNA helix is unwound and new strands develop
are called replication forks. One replication fork is
found at each end of a replication bubble, as shown
in Figure 7.23.
A set of enzymes known as helicasescleave and
unravel short segments of DNA just ahead of the
replicating fork. As the helicases work their way
along the DNA, the replication forks move around
the circular DNA molecule until they meet at the
other side. At this point the two daughter DNA
molecules separate from each another.
In E. coli, replicating forks move at a rate of over
45 000 nucleotides per fork per minute, and the
entire bacterial genome is replicated in less than
an hour. Given that a eukaryotic cell contains
hundreds or thousands of times more DNA than a
bacterium, and that this DNA must be unpackaged
from its complex array of nucleosomes before it can
be replicated, you would expect the process of
replication to take much longer in a eukaryotic
cell. Nevertheless, the complete replication of the
genome of a eukaryotic cell is accomplished within
a few hours.
In the bacteria E. coli, unwinding DNA spins at a rate of
over 4500 r/min — almost twice as fast as the engine speed
of an average car cruising on an expressway.