254 Chapter 5: DNA Replication, Repair, and Recombination

more elaborate controls. For example, the orderly maintenance of di erent cell
types and tissues in animals and plants requires that DNA replication be tightly
regulated. Moreover, eukaryotic DNA replication must be coordinated with the
elaborate process of mitosis, as we discuss in Chapter 17.
As we see in the next section, the eukaryotic replication machinery has the
added complication of having to replicate through nucleosomes, the repeating
structural unit of chromosomes discussed in Chapter 4. Nucleosomes are spaced
at intervals of about 200 nucleotide pairs along the DNA, which, as we will see,
explains why new Okazaki fragments are synthesized on the lagging strand at
intervals of 100–200 nucleotides in eukaryotes, instead of 1000–2000 nucleotides
as in bacteria. Nucleosomes may also act as barriers that slow down the move-
ment of DNA polymerase molecules, which may be why eukaryotic replication
forks move only about one-tenth as fast as bacterial replication forks.

Summary
DNA replication takes place at a Y-shaped structure called a replication fork. A
self-correcting DNA polymerase enzyme catalyzes nucleotide polymerization in a
5ʹ-to-3ʹ direction, copying a DNA template strand with remarkable delity. Since
the two strands of a DNA double helix are antiparallel, this 5ʹ-to-3ʹ DNA synthesis replication origin
can take place continuously on only one of the strands at a replication fork (the
leading strand). On the lagging strand, short DNA fragments must be made by a
“backstitching” process. Because the self-correcting DNA polymerase cannot start
a new chain, these lagging-strand DNA fragments are primed by short RNA primer LOCAL OPENING
OF DNA HELIX
molecules that are subsequently erased and replaced with DNA.
DNA replication requires the cooperation of many proteins. ese include (1)
DNA polymerase and DNA primase to catalyze nucleoside triphosphate polymer-
ization; (2) DNA helicases and single-strand DNA-binding (SSB) proteins to help in
opening up the DNA helix so that it can be copied; (3) DNA ligase and an enzyme
that degrades RNA primers to seal together the discontinuously synthesized lagging- RNA PRIMER
SYNTHESIS
strand DNA fragments; and (4) DNA topoisomerases to help to relieve helical wind-
ing and DNA tangling problems. Many of these proteins associate with each other
at a replication fork to form a highly e cient “replication machine,” through which
the activities and spatial movements of the individual components are coordinated.
LEADING-STRAND
DNA SYNTHESIS
THE INITIATION AND COMPLETION OF DNA BEGINS

REPLICATION IN CHROMOSOMES
We have seen how a set of replication proteins rapidly and accurately generates
two daughter DNA double helices behind a replication fork. But how is this rep-
RNA PRIMERS START
lication machinery assembled in the rst place, and how are replication forks LAGGING-STRAND
created on an intact, double-strand DNA molecule? In this section, we discuss SYNTHESIS
how cells initiate DNA replication and how they carefully regulate this process to lagging strand leading strand
ensure that it takes place not only at the proper positions on the chromosome but of fork 1 of fork 2
also at the appropriate time in the life of the cell. We also discuss a few of the spe-
cial problems that the replication machinery in eukaryotic cells must overcome.
ese include the need to replicate the enormously long DNA molecules found in
eukaryotic chromosomes, as well as the di culty of copying DNA molecules that leading strand lagging strand
are tightly complexed with histones in nucleosomes. of fork 1 of fork 2

FORK 1 FORK 2

DNA Synthesis Begins at Replication Origins

Figure 5–23 A replication bubble formed
As discussed previously, the DNA double helix is normally very stable: the two by replication-fork initiation. This diagram
DNA strands are locked together rmly by many hydrogen bonds formed between outlines the major steps in the initiation of
the bases on each strand. To begin DNA replication, the double helix must rst replication forks at replication origins. The
structure formed at the last step, in which
be opened up and the two strands separated to expose unpaired bases. As we
both strands of the parental DNA helix have
shall see, the process of DNA replication is begun by special initiator proteins that been separated from each other and serve
bind to double-strand DNA and pry the two strands apart, breaking the hydrogen as templates for DNA synthesis, is called a
bonds between the bases. replication bubble.
THE INITIATION AND COMPLETION OF DNA REPLICATION IN CHROMOSOMES 255

Figure 5–24 DNA replication of a bacterial genome. It takes E. coli replication

about 30 minutes to duplicate its genome of 4.6 × 106 nucleotide pairs. For origin
simplicity, no Okazaki fragments are shown on the lagging strand. What
happens as the two replication forks approach each other and collide at the
end of the replication cycle is not well understood, although the replication
machines are disassembled as part of the process.

e positions at which the DNA helix is rst opened are called replication ori- replication
begins
gins (Figure 5–23). In simple cells like those of bacteria or yeast, origins are spec-
i ed by DNA sequences several hundred nucleotide pairs in length. is DNA
contains both short sequences that attract initiator proteins and stretches of DNA
that are especially easy to open. We saw in Figure 4–4 that an A-T base pair is held
together by fewer hydrogen bonds than a G-C base pair. erefore, DNA rich in
A-T base pairs is relatively easy to pull apart, and regions of DNA enriched in A-T
base pairs are typically found at replication origins.
Although the basic process of replication-fork initiation depicted in Figure
5–23 is fundamentally the same for bacteria and eukaryotes, the detailed way in
which this process is performed and regulated di ers between these two groups
of organisms. We rst consider the simpler and better-understood case in bacte-
ria and then turn to the more complex situation found in yeasts, mammals, and
other eukaryotes.

Bacterial Chromosomes Typically Have a Single Origin of DNA replication

completed
Replication
e genome of E. coli is contained in a single circular DNA molecule of 4.6 × 106
nucleotide pairs. DNA replication begins at a single origin of replication, and the
two replication forks assembled there proceed (at approximately 1000 nucleotides
per second) in opposite directions until they meet up roughly halfway around the
chromosome (Figure 5–24). e only point at which E. coli can control DNA rep-
lication is initiation: once the forks have been assembled at the origin, they syn- 2 circular daughter DNA molecules
thesize DNA at relatively constant speed until replication is nished. erefore,
it is not surprising that the initiation of DNA replication is highly regulated. e
process begins when initiator proteins (in their ATP-bound state) bind in multiple
copies to speci c DNA sites located at the replication origin, wrapping the DNA
around the proteins to form a large protein–DNA complex that destabilizes the
adjacent double helix. is complex then attracts two DNA helicases, each bound
to a helicase loader, and these are placed around adjacent DNA single strands
whose bases have been exposed by the assembly of the initiator protein–DNA
complex. e helicase loader is analogous to the clamp loader we encountered
above; it has the additional job of keeping the helicase in an inactive form until it
is properly loaded onto a nascent replication fork. Once the helicases are loaded,
the loaders dissociate and the helicases begin to unwind DNA, exposing enough
single-strand DNA for DNA primase to synthesize the rst RNA primers (Figure
5–25). is quickly leads to the assembly of remaining proteins to create two rep-
lication forks, with replication machines that move, with respect to the replication
origin, in opposite directions. ey continue to synthesize DNA until all of the
DNA template downstream of each fork has been replicated.
In E. coli, the interaction of the initiator protein with the replication origin is
carefully regulated, with initiation occurring only when su cient nutrients are
available for the bacterium to complete an entire round of replication. Initiation is
also controlled to ensure that only one round of DNA replication occurs for each
cell division. After replication is initiated, the initiator protein is inactivated by
hydrolysis of its bound ATP molecule, and the origin of replication experiences a
“refractory period.” e refractory period is caused by a delay in the methylation
of newly incorporated A nucleotides in the origin (Figure 5–26). Initiation cannot
occur again until the A’s are methylated and the initiator protein is restored to its
ATP-bound state.
256 Chapter 5: DNA Replication, Repair, and Recombination

replication origin parental Figure 5–25 The proteins that initiate

DNA helix DNA replication in bacteria. The
mechanism shown was established
AT-rich sequence BINDING OF INITIATOR by studies in vitro with mixtures of
PROTEINS TO highly purified proteins. For E. coli DNA
REPLICATION ORIGIN replication, the major initiator protein, the
AND DESTABILIZATION helicase, and the primase are the dnaA,
initiator proteins OF AT-RICH SEQUENCE
dnaB, and dnaG proteins, respectively.
In the first step, several molecules of
the initiator protein bind to specific DNA
DNA helicase bound to sequences at the replication origin and
helicase-loading protein
LOADING OF DNA destabilize the double helix by forming a
HELICASES compact structure in which the DNA is
tightly wrapped around the protein. Next,
two helicases are brought in by helicase-
loading proteins (the dnaC proteins),
which inhibit the helicases until they are
properly loaded at the replication origin.
helicase-
loading ACTIVATION OF HELICASES Helicase-loading proteins prevent the
protein replicative DNA helices from inappropriately
entering other single-strand stretches of
DNA in the bacterial genome. Aided by
single-strand binding protein (not shown),
LOADING OF DNA PRIMASE the loaded helicases open up the DNA,
thereby enabling primases to enter and
synthesize initial primers. In subsequent
steps, two complete replication forks are
assembled at the origin and move off in
DNA primase
opposite directions. The initiator proteins
RNA PRIMER SYNTHESIS
are displaced as the left-hand fork moves
DNA polymerase ENABLES DNA POLYMERASES through them (not shown).
begins leading-strand TO START NEW CHAINS
synthesis

RNA LOADING OF TWO ADDITIONAL

primer DNA POLYMERASES
LAGGING-STRAND
SYNTHESIS BEGINS

TWO REPLICATION FORKS

MOVING IN OPPOSITE DIRECTIONS

Eukaryotic Chromosomes Contain Multiple Origins of Replication

We have seen how two replication forks begin at a single replication origin in bac-
teria and proceed in opposite directions, moving away from the origin until all of
the DNA in the single circular chromosome is replicated. e bacterial genome is
su ciently small for these two replication forks to duplicate the genome in about
30 minutes. Because of the much greater size of most eukaryotic chromosomes, a
di erent strategy is required to allow their replication in a timely manner.
A method for determining the general pattern of eukaryotic chromosome
replication was developed in the early 1960s. Human cells growing in culture are
labeled for a short time with 3H-thymidine so that the DNA synthesized during
this period becomes highly radioactive. e cells are then gently lysed, and the
DNA is stretched on the surface of a glass slide coated with a photographic emul-
sion. Development of the emulsion reveals the pattern of labeled DNA through
a technique known as autoradiography. e time allotted for radioactive label-
ing is chosen to allow each replication fork to move several micrometers along
the DNA, so that the replicated DNA can be detected in the light microscope as
lines of silver grains, even though the DNA molecule itself is too thin to be visible.
THE INITIATION AND COMPLETION OF DNA REPLICATION IN CHROMOSOMES 257

fully Figure 5–26 Methylation of the E. coli

methylated hemimethylated origins are replication origin creates a refractory
origin resistant to initiation period for DNA initiation. DNA
methylation occurs at GATC sequences,
11 of which are found in the origin of
replication (spanning approximately 250
nucleotide pairs). In its hemimethylated
state, the origin of replication is bound
by an inhibitor protein (Seq A, not
initiation occurs if sufficient origins become fully shown), which blocks the ability of the
resources are available to complete methylated, making them
a round of DNA replication again competent for initiation initiator proteins to unwind the origin
DNA. Eventually (about 15 minutes after
replication is initiated), the hemimethylated
origins become fully methylated by a DNA
In this way, both the rate and the direction of replication-fork movement can methylase enzyme; Seq A then dissociates.
A single enzyme, the Dam methylase,
be determined (Figure 5–27). From the rate at which tracks of replicated DNA is responsible for methylating all E. coli
increase in length with increasing labeling time, the eukaryotic replication forks GATC sequences. A lag in methylation after
are estimated to travel at about 50 nucleotides per second. is is approximately the replication of GATC sequences is also
twentyfold slower than the rate at which bacterial replication forks move, possibly used by the E. coli mismatch proofreading
system to distinguish the newly synthesized
re ecting the increased di culty of replicating DNA that is packaged tightly in
DNA strand from the parental DNA strand;
chromatin. in that case, the relevant GATC sequences
An average-size human chromosome contains a single linear DNA molecule are scattered throughout the chromosome,
of about 150 million nucleotide pairs. It would take 0.02 seconds/nucleotide × 150 and they are not bound by Seq A.
× 106 nucleotides = 3.0 × 106 seconds (about 35 days) to replicate such a DNA mol-
ecule from end to end with a single replication fork moving at a rate of 50 nucleo-
tides per second. As expected, therefore, the autoradiographic experiments just
described reveal that many forks, belonging to separate replication bubbles, are
moving simultaneously on each eukaryotic chromosome.
Much faster and more sophisticated methods now exist for monitoring DNA
replication initiation and tracking the movement of DNA replication forks across
whole genomes. One approach uses DNA microarrays—grids the size of a post-
age stamp studded with hundreds of thousands of fragments of known DNA
sequence. As we will see in detail in Chapter 8, each di erent DNA fragment is
placed at a unique position on the microarray, and whole genomes can thereby
be represented in an orderly manner. If a DNA sample from a group of replicating
Figure 5–27 The experiments that
cells is broken up and hybridized to a microarray representing that organism’s demonstrated the pattern in which
genome, the amount of each DNA sequence can be determined. Because a seg- replication forks are formed and move
ment of a genome that has been replicated will contain twice as much DNA as on eukaryotic chromosomes. The new
an unreplicated segment, replication-fork initiation and fork movement can be DNA made in human cells in culture
was labeled briefly with a pulse of highly
accurately monitored across an entire genome (Figure 5–28).
radioactive thymidine ( 3H-thymidine).
Experiments of this type have shown the following: (1) Approximately 30,000– (A) In this experiment, the cells were
50,000 origins of replication are used each time a human cell divides. (2) e lysed, and the DNA was stretched out
human genome has many more (perhaps tenfold more) potential origins than on a glass slide that was subsequently
this, and di erent cell types use di erent sets of origins. is may allow a cell to covered with a photographic emulsion.
After several months, the emulsion was
coordinate its active origins with other features of its chromosomes such as which developed, revealing a line of silver grains
over the radioactive DNA. The brown DNA
50 µm in this figure is shown only to help with
the interpretation of the autoradiograph;
DNA
the unlabeled DNA is invisible in such
replication origin experiments. (B) This experiment was the
same except that a further incubation in
LABEL WITH 3H-THYMIDINE unlabeled medium allowed additional DNA,
FOR 10 MINUTES with a lower level of radioactivity, to be
replicated. The pairs of dark tracks in (B)
were found to have silver grains tapering
(A)
off in opposite directions, demonstrating
silver grains bidirectional fork movement from a central
ADD UNLABELED MEDIUM FOR replication origin where a replication bubble
10 MINUTES TO REDUCE LEVELS OF forms (see Figure 5–23). A replication fork
NEWLY INCORPORATED 3H-THYMIDINE
is thought to stop only when it encounters
a replication fork moving in the opposite
(B) direction or when it reaches the end of the
chromosome; in this way, all the DNA is
replication bubble replication bubble eventually replicated.
258 Chapter 5: DNA Replication, Repair, and Recombination

culture of cells Figure 5–28 Use of DNA microarrays

arrested before DNA to monitor the formation and progress
replication begins of replication forks. For this experiment,
a population of cells is synchronized so
that they all begin replication at the same
allow time. DNA is collected and hybridized
replication to the microarray; DNA that has been
to begin replicated once gives a hybridization
signal (dark green squares) twice as high
0 min 5 min 10 min 20 min
as that of unreplicated DNA (light green
squares). The spots on these microarrays
represent consecutive sequences along a
segment of a chromosome arranged left
to right, top to bottom. Only 81 spots are
shown here, but the actual arrays contain
fragment DNA, separate strands, and fluorescently label
hundreds of thousands of sequences
that span an entire genome. As can be
seen, replication begins at an origin and
proceeds bidirectionally. For simplicity, only
one origin is shown here. In human cells,
replication begins at 30,000–50,000 origins
located throughout the genome. Using
this approach it is possible to observe the
formation and progress of every replication
fork across a genome.
NO REPLICATION REPLICATION REPLICATION DNA FULLY
BEGINS AT CONTINUES REPLICATED
ORIGIN

genes are being expressed. e excess origins also provide “backups” in case a pri-
mary origin fails. (3) As in bacteria, replication forks are formed in pairs and create
a replication bubble as they move in opposite directions away from a common
point of origin, stopping only when they collide head-on with a replication fork
moving in the opposite direction or when they reach a chromosome end. In this
way, many replication forks operate independently on each chromosome and yet
form two complete daughter DNA helices.

In Eukaryotes, DNA Replication Takes Place During Only One Part +

of the Cell Cycle
When growing rapidly, bacteria replicate their DNA nearly continuously. In con- M
trast, DNA replication in most eukaryotic cells occurs only during a speci c part of
G2
the cell-division cycle, called the DNA synthesis phase or S phase (Figure 5–29). In
a mammalian cell, the S phase typically lasts for about 8 hours; in simpler eukary-
otic cells such as yeasts, the S phase can be as short as 40 minutes. By its end, each
chromosome has been replicated to produce two complete copies, which remain G1
joined together at their centromeres until the M phase (M for mitosis), which soon
follows. In Chapter 17, we describe the control system that runs the cell cycle, and
we explain why entry into each phase of the cycle requires the cell to have suc-
cessfully completed the previous phase. S
In the following sections, we explore how chromosome replication is coordi-
nated within the S phase of the cell cycle.

Different Regions on the Same Chromosome Replicate at Distinct

Times in S Phase
Figure 5–29 The four successive phases
In mammalian cells, the replication of DNA in the region between one replica- of a standard eukaryotic cell cycle.
tion origin and the next should normally require only about an hour to complete, During the G1, S, and G2 phases, the
given the rate at which a replication fork moves and the largest distances mea- cell grows continuously. During M phase
sured between replication origins. Yet S phase usually lasts for about 8 hours in growth stops, the nucleus divides, and
a mammalian cell. is implies that the replication origins are not all activated the cell divides in two. DNA replication is
confined to the part of the cell cycle known
simultaneously; indeed, replication origins are activated in clusters of about 50 as S phase. G1 is the gap between
adjacent replication origins, each of which is replicated during only a small part of M phase and S phase; G2 is the gap
the total S-phase interval. between S phase and M phase.
THE INITIATION AND COMPLETION OF DNA REPLICATION IN CHROMOSOMES 259

It seems that the order in which replication origins are activated depends, in
part, on the chromatin structure in which the origins reside. We saw in Chap-
ter 4 that heterochromatin is a particularly condensed state of chromatin, while
euchromatin, where most transcription occurs, has a less condensed conforma-
tion. Heterochromatin tends to be replicated very late in S phase, suggesting that
the timing of replication is related to the packing of the DNA in chromatin.
Once initiated, however, replication forks seem to move at comparable rates
throughout S phase, so the extent of chromosome condensation seems to in u-
ence the time at which replication forks are initiated, rather than their speed once
formed.

A Large Multisubunit Complex Binds to Eukaryotic Origins of

Replication
Having seen that a eukaryotic chromosome is replicated using many origins of
replication, each of which “ res” at a characteristic time in S phase of the cell
cycle, we turn to the nature of these origins of replication. We saw earlier in this
chapter that replication origins have been precisely de ned in bacteria as speci c
DNA sequences that attract initiator proteins, which then assemble the DNA rep-
lication machinery. We shall see that this is the case for the single-cell budding
yeast S. cerevisiae, but it appears not to be strictly true for most other eukaryotes.
For budding yeast, the location of every origin of replication on each chromo-
some has been determined. e particular chromosome shown in Figure 5–30—
chromosome III from S. cerevisiae—is one of the smallest chromosomes known,
with a length less than 1/100 that of a typical human chromosome. Its major ori-
gins are spaced an average of 30,000 nucleotide pairs apart, but only a subset of
these origins is used by a given cell. Nonetheless, this chromosome can be repli-
cated in about 15 minutes.
e minimal DNA sequence required for directing DNA replication initiation
in S. cerevisiae has been determined by taking a segment of DNA that spans an
origin of replication and testing smaller and smaller DNA fragments for their abil-
ity to function as origins. Most DNA sequences that can serve as an origin of rep-
lication are found to contain (1) a binding site for a large, multisubunit initiator
protein called ORC, for origin recognition complex; (2) a stretch of DNA that is
rich in As and Ts and therefore easy to melt; and (3) at least one binding site for
proteins that facilitate ORC binding, probably by adjusting chromatin structure.
In bacteria, once the initiator protein is properly bound to the single origin
of replication, the assembly of the replication forks seems to follow more or less
automatically. In eukaryotes, the situation is signi cantly di erent because of a
profound problem eukaryotes have in replicating chromosomes: with so many
places to begin replication, how is the process regulated to ensure that all the DNA
is copied once and only once?
e answer lies in the sequential manner in which the replicative helicase is
rst loaded onto origins and is then activated to initiate DNA replication. is
matter is discussed in detail in Chapter 17, where we consider the machinery that
underlies the cell-division cycle. In brief, during G1 phase, the replicative heli-
cases are loaded onto DNA next to ORC to create a prereplicative complex. en,
upon passage from G1 phase to S phase, specialized protein kinases come into
play to activate the helicases. e resulting opening of the double helix allows the
loading of the remaining replication proteins, including the DNA polymerases.
Figure 5–30 The origins of DNA
replication on chromosome III of the
yeast S. cerevisiae. This chromosome,
one of the smallest eukaryotic
CHROMOSOME III origins of replication chromosomes known, carries a total of
180 genes. As indicated, it contains 18
replication origins, although they are used
telomere centromere telomere with different frequencies. Those in red
0 100 200 300 are typically used in less than 10% of cell
divisions, while those in green are used
nucleotide pairs (thousands) about 90% of the time.
260 Chapter 5: DNA Replication, Repair, and Recombination

Cdc6 ORC (origin recognition complex) Figure 5–31 DNA replication initiation in
eukaryotes. This mechanism ensures that
DNA
each origin of replication is activated only
once per cell cycle. An origin of replication
origin
can be used only if a prereplicative
Cdt1
complex forms there in G1 phase. At the
beginning of S phase, specialized kinases
G1 phosphorylate Mcm and ORC, activating
the former and inactivating the latter. A new
+
Mcm helicase prereplicative complex cannot form at the
origin until the cell progresses to the next
G1 phase, when the bound ORC has been
dephosphorylated. Note that the eukaryotic
Mcm helicase moves along the leading-
strand template, whereas the bacterial
prereplicative complex
helicase moves along the lagging-strand
template (see Figure 5–25). As the forks
PHOSPHORYLATION begin to move, ORC is displaced, and new
OF Mcm AND ORC
ORCs rapidly bind to the newly replicated
origins.
HELICASES ACTIVATED; ORC DISPLACED
RECRUITMENT OF DNA POLYMERASE
AND OTHER REPLICATION PROTEINS;
ORC REBINDS; DNA SYNTHESIS BEGINS

S P

P
P

P
COMPLETION OF
DNA REPLICATION

G2

e protein kinases that trigger DNA replication simultaneously prevent

assembly of new prereplicative complexes until the next M phase resets the entire
cycle (for details, see pp. 974–975). ey do this, in part, by phosphorylating ORC,
rendering it unable to accept new helicases. is strategy provides a single win-
dow of opportunity for prereplicative complexes to form (G1 phase, when kinase
activity is low) and a second window for them to be activated and subsequently
disassembled (S phase, when kinase activity is high). Because these two phases of
the cell cycle are mutually exclusive and occur in a prescribed order, each origin
of replication can re once and only once during each cell cycle.

Features of the Human Genome That Specify Origins of

Replication Remain to Be Discovered
Compared with the situation in budding yeast, the determinants of replication
origins in other eukaryotes have been di cult to discover. It has been possible to
identify speci c human DNA sequences, each several thousand nucleotide pairs
in length, that are su cient to serve as replication origins. ese origins continue
to function when moved to a di erent chromosomal region by recombinant DNA
methods, as long as they are placed in a region where the chromatin is relatively
uncondensed. However, comparisons of such DNA sequences have not revealed
speci c DNA sequences that mark origins of replication.
THE INITIATION AND COMPLETION OF DNA REPLICATION IN CHROMOSOMES 261

Despite this, a human ORC that is very similar to the yeast ORC binds to ori-
gins of replication and initiates DNA replication in humans. Many of the other
proteins that function in the initiation process in yeast likewise have central roles
in humans. It therefore seems likely that the yeast and human initiation mecha-
nisms are similar in outline, but chromatin structure, transcriptional activity, or
some property of the genome other than a speci c DNA sequence has the cen-
tral role in attracting ORC and specifying mammalian origins of replication. ese
ideas could also help to explain how a given mammalian cell chooses which of the
many possible origins to use when it replicates its genome and how this choice
could di er from cell to cell. Clearly, we have a great deal to discover about the
fundamental process of DNA replication initiation.

New Nucleosomes Are Assembled Behind the Replication Fork

Several additional aspects of DNA replication are speci c to eukaryotes. As dis-
cussed in Chapter 4, eukaryotic chromosomes are composed of roughly equal
mixtures of DNA and protein. Chromosome duplication therefore requires not
only the replication of DNA, but also the synthesis and assembly of new chro-
mosomal proteins onto the DNA behind each replication fork. Although we are
far from understanding this process in detail, we are beginning to learn how the
fundamental unit of chromatin packaging, the nucleosome, is duplicated. e cell
requires a large amount of new histone protein, approximately equal in mass to
the newly synthesized DNA, to make the new nucleosomes in each cell cycle. For
this reason, most eukaryotic organisms possess multiple copies of the gene for
each histone. Vertebrate cells, for example, have about 20 repeated gene sets, most
containing the genes that encode all ve histones (H1, H2A, H2B, H3, and H4).
Unlike most proteins, which are made continuously, histones are synthesized
mainly in S phase, when the level of histone mRNA increases about ftyfold as
a result of both increased transcription and decreased mRNA degradation. e
major histone mRNAs are degraded within minutes when DNA synthesis stops at
the end of S phase. e mechanism depends on special properties of the 3ʹ ends
of these mRNAs, as discussed in Chapter 7. In contrast, the histone proteins them-
selves are remarkably stable and may survive for the entire life of a cell. e tight
linkage between DNA synthesis and histone synthesis appears to re ect a feed-
back mechanism that monitors the level of free histone to ensure that the amount
of histone made exactly matches the amount of new DNA synthesized.
As a replication fork advances, it must pass through the parental nucleosomes.
In the cell, e cient replication requires chromatin remodeling complexes (dis-
cussed in Chapter 4) to destabilize the DNA–histone interfaces. Aided by such
complexes, replication forks can transit even highly condensed chromatin e -
ciently.
As a replication fork passes through chromatin, the histones are transiently
displaced leaving about 600 nucleotide pairs of non-nucleosomal DNA in its
wake. e reestablishment of nucleosomes behind a moving fork occurs in an
intriguing way. When a nucleosome is traversed by a replication fork, the histone
octamer appears to be broken into an H3-H4 tetramer and two H2A-H2B dimers
(discussed in Chapter 4). e H3-H4 tetramer remains loosely associated with
DNA and is distributed at random to one or the other daughter duplex, but the
H2A-H2B dimers are released completely from DNA. Freshly made H3-H4 tetram-
ers are added to the newly synthesized DNA to ll in the “spaces,” and H2A-H2B
dimers—half of which are old and half new—are then added at random to com-
plete the nucleosomes (Figure 5–32). e formation of new nucleosomes behind
a replication fork has an important consequence for the process of DNA replica-
tion itself. As DNA polymerase δ discontinuously synthesizes the lagging strand
(see pp. 253–254), the length of each Okazaki fragment is determined by the point
at which DNA polymerase δ is blocked by a newly formed nucleosome. is tight
coupling between nucleosome duplication and DNA replication explains why the
length of Okazaki fragments in eukaryotes (~200 nucleotides) is approximately
the same as the nucleosome repeat length.
262 Chapter 5: DNA Replication, Repair, and Recombination

NAP1 loading H2A-H2B dimer Figure 5–32 Formation of nucleosomes

H2A-H2B dimer behind a replication fork. Parental
H3-H4 tetramers are distributed at random
to the daughter DNA molecules, with
roughly equal numbers inherited by each
sliding clamp daughter. In contrast, H2A-H2B dimers
are released from the DNA as the
replication fork replication fork passes. This release
begins just in front of the replication fork
parental and is facilitated by chromatin remodeling
H3-H4 tetramer
complexes that move with the fork.
Histone chaperones (NAP1 and
newly synthesized
CAF1) restore the full complement of
H3-H4 tetramer
histones to daughter molecules using both
parental chromatin parental and newly synthesized histones.
Although some daughter nucleosomes
contain only parental histones or only newly
H2A-H2B dimer synthesized histones, most are hybrids of
displaced in front
of replication fork old and new. For simplicity, the DNA double
helix shown as a single red line. (Adapted
CAF1 loading from J.D. Watson et al., Molecular Biology
H3-H4 tetramer of the Gene, 5th ed. Cold Spring Harbor:
Cold Spring Harbor Laboratory Press,
2004.)
e orderly and rapid addition of new H3-H4 tetramers and H2A-H2B dimers
behind a replication fork requires histone chaperones (also called chromatin
assembly factors). ese multisubunit complexes bind the highly basic histones
and release them for assembly only in the appropriate context. e histone chap-
erones, along with their cargoes, are directed to newly replicated DNA through
a speci c interaction with the eukaryotic sliding clamp called PCNA (see Figure
5–32B). ese clamps are left behind moving replication forks and remain on the
DNA long enough for the histone chaperones to complete their tasks.

Telomerase Replicates the Ends of Chromosomes

We saw earlier that synthesis of the lagging strand at a replication fork must occur
discontinuously through a backstitching mechanism that produces short DNA
fragments. is mechanism encounters a special problem when the replication
fork reaches an end of a linear chromosome. e nal RNA primer synthesized
on the lagging-strand template cannot be replaced by DNA because there is no
3ʹ-OH end available for the repair polymerase. Without a mechanism to deal with
this problem, DNA would be lost from the ends of all chromosomes each time a
cell divides.
Bacteria solve this “end-replication” problem by having circular DNA mole-
cules as chromosomes (see Figure 5–24). Eukaryotes solve it in a di erent way:
they have specialized nucleotide sequences at the ends of their chromosomes
that are incorporated into structures called telomeres (discussed in Chapter 4).
Telomeres contain many tandem repeats of a short sequence that is similar in
organisms as diverse as protozoa, fungi, plants, and mammals. In humans, the
sequence of the repeat unit is GGGTTA, and it is repeated roughly a thousand
times at each telomere.
Telomere DNA sequences are recognized by sequence-speci c DNA-bind-
ing proteins that attract an enzyme, called telomerase, that replenishes these
sequences each time a cell divides. Telomerase recognizes the tip of an existing
telomere DNA repeat sequence and elongates it in the 5ʹ-to-3ʹ direction, using
an RNA template that is a component of the enzyme itself to synthesize new cop-
ies of the repeat (Figure 5–33). e enzymatic portion of telomerase resembles
other reverse transcriptases, proteins that synthesize DNA using an RNA template,
although, in this case, the telomerase RNA also contributes functional groups to
make the catalysis more e cient. After extension of the parental DNA strand by
telomerase, replication of the lagging strand at the chromosome end can be com-
pleted by the conventional DNA polymerases, using these extensions as a tem-
plate to synthesize the complementary strand (Figure 5–34).
THE INITIATION AND COMPLETION OF DNA REPLICATION IN CHROMOSOMES 263

Figure 5–33 Structure of a portion of

remainder of telomerase. Telomerase is a large protein–
telomerase protein
telomerase RNA
“fingers“ RNA complex. The RNA (blue) contains
a templating sequence for synthesizing
new DNA telomere repeats. The synthesis
reaction itself is carried out by the reverse
region of
transcriptase domain of the protein, shown
telomerase RNA
used as template in green. A reverse transcriptase is a
special form of polymerase enzyme that
uses an RNA template to make a DNA
strand; telomerase is unique in carrying
its own RNA template with it. Telomerase
also has several additional protein domains
(not shown) that are needed to assemble
the enzyme at the ends of chromosomes.
3′ (Modified from J. Lingner and T.R. Cech,
Curr. Opin. Genet. Dev. 8:226–232, 1998.
“palm“—active site
With permission from Elsevier.)
of telomerase
protein
5′
newly
rest of synthesized “thumb“
chromosome telomere DNA

Telomeres Are Packaged Into Specialized Structures That Protect

the Ends of Chromosomes
e ends of chromosomes present cells with an additional problem. As we will
see in the next part of this chapter, when a chromosome is accidently broken,
the break is rapidly repaired (see Figure 5–45). Telomeres must clearly be distin-
guished from these accidental breaks; otherwise the cell will attempt to “repair”
telomeres, causing chromosome fusions and other genetic abnormalities. Telo-
meres have several features to prevent this from happening.
A specialized nuclease chews back the 5ʹ end of a telomere leaving a protrud-
ing single-strand end. is protruding end—in combination with the GGGTTA
repeats in telomeres—attracts a group of proteins that form a protective chromo-
some cap known as shelterin. In particular, shelterin “hides” telomeres from the
cell’s damage detectors that continually monitor DNA. When human telomeres
are arti cially cross-linked and viewed by electron microscopy, structures known
as “t-loops” are observed in which the protruding end of the telomere loops back
and tucks itself into the duplex DNA of the telomere repeat sequence (Figure
5–35). It is believed that t-loops are regulated by shelterin and provide additional
protection for the ends of chromosomes.
parent strand
3′
TTGGGGTTGGGGTTGGGGTTG
AACCCC
5′ incomplete, newly synthesized lagging strand
TELOMERASE
BINDS
3′
TTGGGGTTGGGGTTGGGGTTG direction of
AACCCC ACCCCAAC telomere Figure 5–34 Telomere replication. Shown
5′ 3′ 5′ synthesis here are the reactions that synthesize
TELOMERASE
the repeating sequences that form the
EXTENDS 3′ END telomerase with bound RNA template
(RNA-templated ends of the chromosomes (telomeres) of
DNA synthesis) diverse eukaryotic organisms. The 3ʹ end
3′ of the parental DNA strand is extended
TTGGGGTTGGGGTTGGGGTTGGGGTTGGGGTTG by RNA-templated DNA synthesis; this
AACCCC ACCCCAAC
5′ 5′
allows the incomplete daughter DNA
3′
COMPLETION OF strand that is paired with it to be extended
LAGGING STRAND in its 5ʹ direction. This incomplete, lagging
BY DNA POLYMERASE strand is presumed to be completed by
(DNA-templated
DNA synthesis) DNA polymerase α, which carries a DNA
3′ primase as one of its subunits (Movie 5.6).
TTGGGGTTGGGGTTGGGGTTGGGGTTGGGGTTG The telomere sequence illustrated is that
AACCCC CCCCAACCCCAACCCC
5′ of the ciliate Tetrahymena, in which these
DNA polymerase reactions were first discovered.
264 Chapter 5: DNA Replication, Repair, and Recombination

3′ overhang
5′ 3′
3′ 5′
telomere
repeats

t-loop
5′
5′ 3′
3′
strand exchange by 3′ overhang
(A) (B)
1 µm

Telomere Length Is Regulated by Cells and Organisms Figure 5–35 A t-loop at the end of a
mammalian chromosome. (A) Electron
Because the processes that grow and shrink each telomere sequence are only micrograph of the DNA at the end of an
approximately balanced, a chromosome end contains a variable number of telo- interphase human chromosome. The
chromosome was fixed, deproteinated, and
meric repeats. Not surprisingly, many cells have homeostatic mechanisms that artificially thickened before viewing. The
maintain the number of these repeats within a limited range (Figure 5–36). loop seen here is approximately 15,000
In most of the dividing somatic cells of humans, however, telomeres gradually nucleotide pairs in length. (B) Structure
shorten, and it has been proposed that this provides a counting mechanism that of a t-loop. The insertion of the single-
helps prevent the unlimited proliferation of wayward cells in adult tissues. In its strand 3ʹ end into the duplex repeats is
carried out, and the structure maintained,
simplest form, this idea holds that our somatic cells start o in the embryo with a by specialized proteins. (From J.D. Griffith
full complement of telomeric repeats. ese are then eroded to di erent extents in et al., Cell 97:503–514, 1999. With
di erent cell types. Some stem cells, notably those in tissues that must be replen- permission from Elsevier.)
ished at a high rate throughout life—bone marrow or gut lining, for example—
retain full telomerase activity. However, in many other types of cells, the level of
telomerase is turned down so that the enzyme cannot quite keep up with chromo-
some duplication. Such cells lose 100–200 nucleotides from each telomere every
time they divide. After many cell generations, the descendant cells will inherit
chromosomes that lack telomere function, and, as a result of this defect, activate
a DNA-damage response causing them to withdraw permanently from the cell
cycle and cease dividing—a process called replicative cell senescence (discussed
in Chapter 17). In theory, such a mechanism could provide a safeguard against
the uncontrolled cell proliferation of abnormal cells in somatic tissues, thereby
helping to protect us from cancer.
short
long telomere telomere
chromosome end
5′ 3′ 5′ 3′
3′ 5′ 3′ 5′
telomere repeats
INCREASING NUMBER OF CELL DIVISIONS
fraction of chromosome ends

Figure 5–36 A demonstration that

yeast cells control the length of their
telomeres. In this experiment, the telomere
at one end of a particular chromosome
is artificially made either longer (left) or
shorter (right) than average. After many
cell divisions, the chromosome recovers,
showing an average telomere length and
a length distribution that is typical of the
other chromosomes in the yeast cell. A
similar feedback mechanism for controlling
telomere length has been proposed for the
increasing telomere length increasing telomere length germ-line cells of animals.
THE INITIATION AND COMPLETION OF DNA REPLICATION IN CHROMOSOMES 265

e idea that telomere length acts as a “measuring stick” to count cell divisions
and thereby regulate the lifetime of the cell lineage has been tested in several
ways. For certain types of human cells grown in tissue culture, the experimental
results support such a theory. Human broblasts normally proliferate for about 60
cell divisions in culture before undergoing replicative cell senescence. Like most
other somatic cells in humans, broblasts produce only low levels of telomerase,
and their telomeres gradually shorten each time they divide. When telomerase is
provided to the broblasts by inserting an active telomerase gene, telomere length
is maintained and many of the cells now continue to proliferate inde nitely.
It has been proposed that this type of control on cell proliferation may con-
tribute to the aging of animals like ourselves. ese ideas have been tested by
producing transgenic mice that lack telomerase entirely. e telomeres in mouse
chromosomes are about ve times longer than human telomeres, and the mice
must therefore be bred through three or more generations before their telomeres
have shrunk to the normal human length. It is therefore perhaps not surprising
that the rst generations of mice develop normally. However, the mice in later
generations develop progressively more defects in some of their highly prolifera-
tive tissues. In addition, these mice show signs of premature aging and have a
pronounced tendency to develop tumors. In these and other respects these mice
resemble humans with the genetic disease dyskeratosis congenita. Individuals
a icted with this disease carry one functional and one nonfunctional copy of the
telomerase RNA gene; they have prematurely shortened telomeres and typically
die of progressive bone marrow failure. ey also develop lung scarring and liver
cirrhosis and show abnormalities in various epidermal structures including skin,
hair follicles, and nails.
e above observations demonstrate that controlling cell proliferation by telo-
mere shortening poses a risk to an organism, because not all of the cells that begin
losing the ends of their chromosomes will stop dividing. Some apparently become
genetically unstable, but continue to divide, giving rise to variant cells that can
lead to cancer. Clearly, the use of telomere shortening as a regulating mechanism
is not foolproof and, like many mechanisms in the cell, seems to strike a balance
between bene t and risk.

Summary
e proteins that initiate DNA replication bind to DNA sequences at a replication
origin to catalyze the formation of a replication bubble with two outward-moving
replication forks. e process begins when an initiator protein–DNA complex is
formed that subsequently loads a DNA helicase onto the DNA template. Other pro-
teins are then added to form the multienzyme “replication machine” that catalyzes
DNA synthesis at each replication fork.
In bacteria and some simple eukaryotes, replication origins are speci ed by spe-
ci c DNA sequences that are only several hundred nucleotide pairs long. In other
eukaryotes, such as humans, the sequences needed to specify an origin of DNA
replication seem to be less well de ned, and the origin can span several thousand
nucleotide pairs.
Bacteria typically have a single origin of replication in a circular chromosome.
With fork speeds of up to 1000 nucleotides per second, they can replicate their
genome in less than an hour. Eukaryotic DNA replication takes place in only one
part of the cell cycle, the S phase. e replication fork in eukaryotes moves about 10
times more slowly than the bacterial replication fork, and the much longer eukary-
otic chromosomes each require many replication origins to complete their replica-
tion in an S phase, which typically lasts for 8 hours in human cells. e di erent
replication origins in these eukaryotic chromosomes are activated in a sequence,
determined in part by the structure of the chromatin, with the most condensed
regions of chromatin typically beginning their replication last. After the replication
fork has passed, chromatin structure is re-formed by the addition of new histones to
the old histones that are directly inherited by each daughter DNA molecule.
Eukaryotes solve the problem of replicating the ends of their linear chromosomes
with a specialized end structure, the telomere, maintained by a special nucleotide