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2: All DNA Replication Takes Place in a Semiconservative Manner
DNA Replication and Recombination
(a) Conservative replication
(b) Dispersive replication
(c) Semiconservative replication
9.1 Three proposed models of replication are conservative replication, dispersive
replication, and semiconservative replication.
25% of the molecules would consist entirely of the original
DNA and 75% would consist entirely of new DNA. With each
additional round of replication, the proportion of molecules
with new DNA would increase, although the number of molecules with the original DNA would remain constant.
Dispersive replication would always produce hybrid molecules, containing some original and some new DNA, but the
proportion of new DNA within the molecules would increase
with each replication event. In contrast, with semiconservative replication, one round of replication would produce two
hybrid molecules, each consisting of half original DNA and
half new DNA. After a second round of replication, half the
molecules would be hybrid, and the other half would consist
of new DNA only. Additional rounds of replication would
produce more and more molecules consisting entirely of new
DNA, and a few hybrid molecules would persist.
Meselson and Stahl distinguished between the heavy
N-laden DNA and the light 14N-containing DNA with
the use of equilibrium density gradient centrifugation
(Figure 9.2). In this technique, a centrifuge tube is filled with
A centrifuge tube is filled
with a heavy salt solution
and DNA fragments.
Meselson and Stahl’s Experiment
To determine which of the three models of replication
applied to E. coli cells, Matthew Meselson and Franklin Stahl
needed a way to distinguish old and new DNA. They did so
by using two isotopes of nitrogen, 14N (the common form)
and 15N (a rare, heavy form). Meselson and Stahl grew a culture of E. coli in a medium that contained 15N as the sole
nitrogen source; after many generations, all the E. coli cells
had 15N incorporated into the purine and pyrimidine bases
of DNA (see Figure 8.8). Meselson and Stahl took a sample
of these bacteria, switched the rest of the bacteria to a
medium that contained only 14N, and then took additional
samples of bacteria over the next few cellular generations. In
each sample, the bacterial DNA that was synthesized before
the change in medium contained 15N and was relatively
heavy, whereas any DNA synthesized after the switch contained 14N and was relatively light.
It is then spun in a centrifuge
at high speeds for several days.
A density gradient develops
within the tube. Heavy DNA
(with 15N) will move toward the
bottom; light DNA (with 14N)
will remain closer to the top.
9.2 Meselson and Stahl used equilibrium density gradient
centrifugation to distinguish between heavy, 15N-laden DNA
and lighter, 14N-laden DNA.
Question: Which model of DNA replication—conservative, dispersive, or semiconservative—applies to E. coli ?
DNA from bacteria that had been
grown on medium containing 15N
appeared as a single band.
After one round of replication,
the DNA appeared as a single
band at intermediate weight.
After a second round of replication,
DNA appeared as two bands, one light
and the other intermediate in weight.
Samples taken after additional
rounds of replication appeared
as two bands, as in part c.
Conclusion: DNA replication in E.coli is semiconservative.
9.3 Meselson and Stahl demonstrated that DNA replication is semiconservative.
a heavy salt solution and a substance of which the density is
to be measured—in this case, DNA fragments. The tube is
then spun in a centrifuge at high speeds. After several days of
spinning, a gradient of density develops within the tube,
with high density at the bottom and low density at the top.
The density of the DNA fragments matches that of the salt:
light molecules rise and heavy molecules sink.
Meselson and Stahl found that DNA from bacteria
grown only on medium containing 15N produced a single
band at the position expected of DNA containing only 15N
(Figure 9.3a). DNA from bacteria transferred to the medium
with 14N and allowed one round of replication also produced
a single band but at a position intermediate between that
expected of DNA containing only 15N and that expected of
DNA containing only 14N (Figure 9.3b). This result is inconsistent with the conservative replication model, which predicts one heavy band (the original DNA molecules) and one
light band (the new DNA molecules). A single band of intermediate density is predicted by both the semiconservative
and the dispersive models.
To distinguish between these two models, Meselson and
Stahl grew the bacteria in medium containing 14N for a
second generation. After a second round of replication in
medium with 14N, two bands of equal intensity appeared,
one in the intermediate position and the other at the position expected of DNA containing only 14N (Figure 9.3c). All
samples taken after additional rounds of replication produced two bands, and the band representing light DNA
became progressively stronger (Figure 9.3d). Meselson and
Stahl’s results were exactly as expected for semiconservative
replication and are incompatible with those predicated for
both conservative and dispersive replication.
Replication is semiconservative: each DNA strand serves as a
template for the synthesis of a new DNA molecule. Meselson and
Stahl convincingly demonstrated that replication in E. coli is
✔ Concept Check 1
How many bands of DNA would be expected in Meselson and
Stahl’s experiment after two rounds of conservative replication?
DNA Replication and Recombination
4 Eventually two circular DNA
molecules are produced.
1 Double-stranded DNA
unwinds at the
2 …producing single-stranded
templates for the synthesis of
new DNA. A replication
bubble forms, usually having
a replication fork at each end.
3 The forks proceed
around the circle.
Conclusion: The products of theta
replication are two circular DNA molecules.
9.4 Theta replication is a type of replication common in E. coli and other organisms possessing
circular DNA. [Electron micrographs from Bernard Hirt, L’Institut Suisse de Recherche Expérimentale sur le Cancer.]
Modes of Replication
After Meselson and Stahl’s work, investigators confirmed that
other organisms also use semiconservative replication. No evidence was found for conservative or dispersive replication.
There are, however, several different ways in which semiconservative replication can take place, differing principally in the
nature of the template DNA—whether it is linear or circular.
Individual units of replication are called replicons, each
of which contains a replication origin. Replication starts at
the origin and continues until the entire replicon has been
replicated. Bacterial chromosomes have a single replication
origin, whereas eukaryotic chromosomes contain many.
A common type of replication that takes place in circular DNA, such as that found in E. coli and other bacteria, is
called theta replication (Figure 9.4) because it generates a
structure that resembles the Greek letter theta (). In theta
replication, double-stranded DNA begins to unwind at the
replication origin, producing single-stranded nucleotide
strands that then serve as templates on which new DNA can
be synthesized. The unwinding of the double helix generates
a loop, termed a replication bubble. Unwinding may be at
one or both ends of the bubble, making it progressively
larger. DNA replication on both of the template strands is
simultaneous with unwinding. The point of unwinding,
where the two single nucleotide strands separate from the
double-stranded DNA helix, is called a replication fork.
If there are two replication forks, one at each end of the
replication bubble, the forks proceed outward in both directions in a process called bidirectional replication, simultaneously unwinding and replicating the DNA until they
eventually meet. If a single replication fork is present, it proceeds around the entire circle to produce two complete circular DNA molecules, each consisting of one old and one
new nucleotide strand.
Circular DNA molecules that undergo theta replication
have a single origin of replication. Because of the limited size
of these DNA molecules, replication starting from one origin
can traverse the entire chromosome in a reasonable amount
of time. The large linear chromosomes in eukaryotic cells,
however, contain far too much DNA to be replicated speedily
from a single origin. Replication takes place on eukaryotic
chromosomes simultaneously from thousands of origins.
Typical eukaryotic replicons are from 20,000 to 300,000
base pairs in length. At each replication origin, the DNA
1 Each chromosome contains
unwinds and produces a replication bubble. Replication
takes place on both strands at each end of the bubble, with
the two replication forks spreading outward. Eventually, the
replication forks of adjacent replicons run into each other,
and the replicons fuse to form long stretches of newly synthesized DNA (Figure 9.5). Replication and fusion of all the
replicons leads to two identical DNA molecules. Important
features of theta replication and linear eukaryotic replication
are summarized in Table 9.1.
2 At each origin, the DNA unwinds,
producing a replication bubble.
3 DNA synthesis takes place on both
strands at each end of the bubble as
the replication forks proceed outward.
Requirements of Replication
Although the process of replication includes many components, they can be combined into three major groups:
1. a template consisting of single-stranded DNA,
2. raw materials (substrates) to be assembled into a new
nucleotide strand, and
3. enzymes and other proteins that “read” the template and
assemble the substrates into a DNA molecule.
Because of the semiconservative nature of DNA replication, a double-stranded DNA molecule must unwind to
expose the bases that act as a template for the assembly of
new polynucleotide strands, which are complementary and
antiparallel to the template strands. The raw materials from
which new DNA molecules are synthesized are deoxyribonucleoside triphosphates (dNTPs), each consisting of a
deoxyribose sugar and a base (a nucleoside) attached to three
phosphate groups (Figure 9.6a). In DNA synthesis,
nucleotides are added to the 3Ј-hydroxyl (3¿ -OH) group of
the growing nucleotide strand (Figure 9.6b). The 3¿ -OH
group of the last nucleotide on the strand attacks the 5¿ phosphate group of the incoming dNTP. Two phosphate
groups are cleaved from the incoming dNTP, and a phosphodiester bond is created between the two nucleotides.
DNA synthesis does not happen spontaneously. Rather,
it requires a host of enzymes and proteins that function in a
coordinated manner. We will examine this complex array of
proteins and enzymes as we consider the replication process
in more detail.
4 Eventually, the forks of adjacent
bubbles run into each other and
the segments of DNA fuse,…
5 …producing two identical
linear DNA molecules.
Conclusion: The products of eukaryotic DNA replication are
two linear DNA molecules.
9.5 Linear DNA replication takes place in eukaryotic
Characteristics of theta and linear eukaryotic replication
Two circular molecules
Two linear molecules
1 New DNA is synthesized
2 In replication, the
3’-OH group of the
last nucleotide on
the strand attacks the
5’-phosphate group of
the incoming dNTP.
4 A phosphodiester
between the two
9.6 New DNA is synthesized
The newly synthesized strand is
complementary and antiparallel
to the template strand; the
two strands are held together
by hydrogen bonds
(represented by red dotted
lines) between the bases.
3 Two phosphates
are cleaved off.
DNA synthesis requires a single-stranded DNA template, deoxyribonucleoside triphosphates, a growing nucleotide strand, and a
group of enzymes and proteins.
Direction of Replication
In DNA synthesis, new nucleotides are joined one at a time
to the 3¿ end of the newly synthesized strand. DNA polymerases, the enzymes that synthesize DNA, can add
nucleotides only to the 3¿ end of the growing strand (not the
5¿ end), and so new DNA strands always elongate in the same
5¿ -to-3¿ direction (5¿ : 3¿ ). Because the two single-stranded
5 …and phosphate
ions are released.
DNA templates are antiparallel and strand elongation is
always 5¿ : 3¿ , if synthesis on one template proceeds from,
say, right to left, then synthesis on the other template must
proceed in the opposite direction, from left to right (Figure
9.7). As DNA unwinds during replication, the antiparallel
nature of the two DNA strands means that one template is
exposed in the 5¿ : 3¿ direction and the other template is
exposed in the 3¿ : 5¿ direction (see Figure 9.7); so how can
synthesis take place simultaneously on both strands at
As the DNA unwinds, the template strand that is exposed
in the 3¿ : 5¿ direction (the lower strand in Figures 9.7 and
9.8) allows the new strand to be synthesized continuously, in
the 5¿ : 3¿ direction. This new strand, which undergoes
continuous replication, is called the leading strand.
3 …DNA synthesis proceeds from
right to left on one strand…
Direction of synthesis
1 Because two template
strands are antiparallel…
2 …and DNA synthesis
is always 5’
Direction of synthesis
9.7 DNA synthesis takes place in opposite
directions on the two DNA template strands.
DNA replication at a single replication fork begins when
a double-stranded DNA molecule unwinds to provide
two single-strand templates.
4 …and from left to right
on the other strand.
The other template strand is exposed in the 5¿ : 3¿
direction (the upper strand in Figures 9.7 and 9.8). After a
short length of the DNA has been unwound, synthesis must
proceed 5¿ : 3¿ ; that is, in the direction opposite that of
unwinding (Figure 9.8). Because only a short length of DNA
needs to be unwound before synthesis on this strand gets
started, the replication machinery soon runs out of template.
By that time, more DNA has unwound, providing new template at the 5¿ end of the new strand. DNA synthesis must
start anew at the replication fork and proceed in the direction opposite that of the movement of the fork until it runs
into the previously replicated segment of DNA. This process
is repeated again and again, and so synthesis of this strand is
in short, discontinuous bursts. The newly made strand that
undergoes discontinuous replication is called the lagging
The short lengths of DNA produced by discontinuous
replication of the lagging strand are called Okazaki fragments, after Reiji Okazaki, who discovered them. In bacterial cells, each Okazaki fragment ranges in length from about
1000 to 2000 nucleotides; in eukaryotic cells, they are about
100 to 200 nucleotides long. Okazaki fragments on the lagging strand are linked together to create a continuous new
All DNA synthesis is 5¿ : 3¿ , meaning that new nucleotides are
always added to the 3¿ end of the growing nucleotide strand. At
each replication fork, synthesis of the leading strand proceeds continuously and that of the lagging strand proceeds discontinuously.
✔ Concept Check 2
Discontinuous replication is a result of which property of DNA?
1 On the lower template strand, DNA synthesis
proceeds continuously in the 5’
direction, the same as that of unwinding.
9.3 The Replication of DNA
Requires a Large Number
of Enzymes and Proteins
2 On the upper template strand,
DNA synthesis begins at the fork and
proceeds in the direction opposite that of
unwinding; so it soon runs out of template.
Replication takes place in four stages: initiation, unwinding,
elongation, and termination.
3 DNA synthesis starts again on the
upper strand, at the fork, each time
proceeding away from the fork.
4 DNA synthesis on this strand is
discontinuous; short fragments of DNA
produced by discontinuous synthesis
are called Okazaki fragments.
9.8 DNA synthesis is continuous on one template strand
of DNA and discontinuous on the other.
Bacterial DNA Replication
The following discussion of the process of replication will
focus on bacterial systems, where replication has been most
thoroughly studied and is best understood. Although many
aspects of replication in eukaryotic cells are similar to those
in prokaryotic cells, there are some important differences.
We will compare bacterial and eukaryotic replication later in
Initiation The circular chromosome of E. coli has a single
d. Five-carbon sugar
c. Antiparallel nucleotide strands
b. Charged phosphate group
a. Complementary bases
replication origin (oriC). The minimal sequence required for
oriC to function consists of 245 bp that contain several
critical sites. An initiator protein (known as DnaA in E. coli)
binds to oriC and causes a short section of DNA to unwind.
This unwinding allows helicase and other single-strandbinding proteins to attach to the polynucleotide strand
Unwinding Because DNA synthesis requires a singlestranded template and because double-stranded DNA
must be unwound before DNA synthesis can take place,
DNA Replication and Recombination
1 Initiator proteins bind
to oriC, the origin
2 …causing a short stretch
of DNA to unwind.
3 The unwinding allows
helicase and other
proteins to attach to
the single-stranded DNA.
9.9 E. coli DNA replication begins when initiator proteins
the cell relies on several proteins and enzymes to accomplish the unwinding. A DNA helicase breaks the hydrogen
bonds that exist between the bases of the two nucleotide
strands of a DNA molecule. Helicase cannot initiate the
unwinding of double-stranded DNA; the initiator protein
first separates DNA strands at the origin, providing a short
stretch of single-stranded DNA to which a helicase binds.
Helicase binds to the lagging-strand template at each
replication fork and moves in the 5¿ : 3¿ direction along
this strand, thus also moving the replication fork
After DNA has been unwound by helicase, the singlestranded nucleotide chains have a tendency to form hydrogen bonds and reanneal (stick back together). Secondary
structures also may form between complementary
nucleotides on the same strand. To stabilize the singlestranded DNA long enough for replication to take place,
single-strand-binding proteins (SSBs) attach tightly to the
exposed single-stranded DNA (see Figure 9.10). Unlike
many DNA-binding proteins, SSBs are indifferent to base
sequence: they will bind to any single-stranded DNA. Singlestrand-binding proteins form tetramers (groups of four);
each tetramer covers from 35 to 65 nucleotides.
Another protein essential for the unwinding process is
the enzyme DNA gyrase, a topoisomerase. As discussed in
Chapter 8, topoisomerases control the supercoiling of DNA.
In replication, DNA gyrase reduces the torsional strain
(torque) that builds up ahead of the replication fork as a
result of unwinding (see Figure 9.10). It reduces torque by
making a double-stranded break in one segment of the
DNA helix, passing another segment of the helix through
the break, and then resealing the broken ends of the DNA.
bind to oriC, the origin of replication.
1 DNA helicase binds to the lagging-strand template at
each replication fork and moves in the 5’
direction along this strand, breaking hydrogen bonds
and moving the replication fork.
proteins stabilize the
exposed singlestranded DNA.
3 DNA gyrase relieves
strain ahead of the
9.10 DNA helicase unwinds DNA
by binding to the lagging-strand
template at each replication fork
and moving in the 5¿ : 3¿