Biochemistry, 4th Edition P91 pdf

28.1 How Is DNA Replicated? 863
replication involves two replication forks that move in opposite directions. Bidirec-
tional replication predicts that, if radioactively labeled nucleotides are provided as
substrates for new DNA synthesis, both replication forks will become radioactively
labeled. The experiment illustrated in Figure 28.2 confirms this prediction.
Replication Requires Unwinding of the DNA Helix
Semiconservative replication depends on unwinding the DNA double helix to ex-
pose single-stranded templates to polymerase action. For a double helix to unwind,
it must either rotate about its axis (while the ends of its strands are held fixed), or
positive supercoils must be introduced, one for each turn of the helix unwound (see
Chapter 11). If the chromosome is circular, as in E. coli, only the latter alternative is
possible. Because DNA replication in E. coli proceeds at a rate approaching 1000
nucleotides per second and there are about 10 bp per helical turn, the chromosome
would accumulate 100 positive supercoils per second! In effect, the DNA would be-
come too tightly supercoiled to allow unwinding of the strands.
DNA gyrase, a Type II topoisomerase, acts to overcome the torsional stress im-
posed upon unwinding; DNA gyrase introduces negative supercoils at the expense of
ATP hydrolysis. The unwinding reaction is driven by helicases (see also Chapter 16),
a class of proteins that catalyze the ATP-dependent unwinding of DNA double he-
lices. Unlike topoisomerases that alter the linking number of dsDNA through phos-
phodiester bond breakage and reunion (see Chapter 11), helicases simply disrupt
the hydrogen bonds that hold the two strands of duplex DNA together. A helicase
molecule requires a single-stranded region for binding. It then moves along the sin-
gle strand, unwinding the double-stranded DNA in an ATP-dependent process. SSB
(single-stranded DNA-binding protein) binds to the unwound strands, preventing
their re-annealing. At least ten distinct DNA helicases involved in different aspects of
DNA and RNA metabolism have been found in E. coli alone. DnaB is the DNA heli-
case acting in E. coli DNA replication. DnaB helicase assembles as a hexameric (␣
6
)
“doughnut”-shaped protein ring, with DNA passing through its hole.

DNA Replication Is Semidiscontinuous
As shown in Figure 28.2, both parental DNA strands are replicated at each advancing
replication fork. The enzyme that carries out DNA replication is DNA polymerase.
A template is something whose edge is shaped
in a particular way so that it can serve as a guide
in making a similar object with a corresponding
contour.
Emerging
p
rogeny DNA
AT
GC
TA
A
GC
AT
GC
AT
GC
CG
A
A
GC
AT
GC
GC
GCCG
AT
AT
AT

GC
AT
AT
T
GC
AT
AT
GC
AT
AT
GC
Old OldNew
New
New
Old Old
FIGURE 28.1 DNA replication:Strand separation fol-
lowed by the copying of each strand.
(a)
Labeled DNA
Labeled DNA
Unidirectional
replication
Bidirectional
replication
(b)
FIGURE 28.2 Bidirectional replication. (a) Comparison of
labeling during unidirectional versus bidirectional repli-
cation. (b) An autoradiogram of E. coli chromosome
replication in the presence of radioactive thymidine
confirms bidirectional replication.

(Photo courtesy of David
M. Prescott, University of Colorado.)
864 Chapter 28 DNA Metabolism: Replication, Recombination, and Repair
This enzyme uses single-stranded DNA (ssDNA) as a template and makes a comple-
mentary strand by polymerizing deoxynucleotides in the order specified by their base
pairing with bases in the template. DNA polymerases synthesize DNA only in a 5Ј→3Ј
direction, reading the antiparallel template strand in a 3Ј→5Ј sense. A dilemma arises:
How does DNA polymerase copy the parent strand that runs in the 5Ј→3Ј direction
at the replication fork? It turns out that replication is semidiscontinuous (Figure 28.3): As
the DNA helix is unwound during its replication, the 3Ј→5Ј strand (as defined by the
direction that the replication fork is moving) can be copied continuously by DNA
polymerase synthesizing in the 5Ј→3Ј direction behind the replication fork. The
other parental strand is copied only when a sufficient stretch of its sequence has been
exposed for DNA polymerase to read it in the 3Ј→5Ј sense. Thus, one parental strand
is copied continuously to give a newly synthesized copy, called the leading strand, at
each replication fork. The other parental strand is copied in an intermittent, or dis-
continuous, mode to yield a set of fragments 1000 to 2000 nucleotides in length,
called the Okazaki fragments (Figure 28.3a). These fragments are then joined to
form an intact lagging strand. Because both strands are synthesized in concert by a
dimeric DNA polymerase situated at the replication fork, the 5Ј→3Ј parental strand
must wrap around in trombone fashion so that the unit of dimeric DNA polymerase
replicating it can move along it in the 3Ј→5Ј direction (Figure 28.3b). Overall, each
of the two DNA duplexes produced in DNA replication contains one “old” and one
“new” DNA strand, and half of the new strand was formed by leading strand synthesis
and the other half by lagging strand synthesis.
The Lagging Strand Is Formed from Okazaki Fragments
In 1968, Tuneko and Reiji Okazaki provided biochemical verification of the semi-
discontinuous pattern of DNA replication just described. The Okazakis exposed a
rapidly dividing E. coli culture to
3

H-labeled thymidine for 30 seconds, quickly col-
lected the cells, and found that half of the label incorporated into nucleic acid ap-
peared in short ssDNA chains just 1000 to 2000 nucleotides in length. (The other
half of the radioactivity was recovered in very large DNA molecules.) Subsequent ex-
periments demonstrated that with time, the newly synthesized short ssDNA Okazaki
fragments became covalently joined to form longer polynucleotide chains, in ac-
cord with a semidiscontinuous mode of replication. The generality of this mode of
(a)
3Ј
5Ј
3Ј
5Ј
3Ј
5Ј
5Ј
3Ј
Leading strand
Parental strands
Movement of
replication fork
Lagging strand
(b)
3Ј
5Ј
3Ј
5Ј
5Ј
3Ј
Okazaki fragments
Parental strands

Movement of
replication fork
Lagging strand
Dimeric DNA polymerase
3Ј
Leading strand
Okazaki fragments
FIGURE 28.3 The semidiscontinuous model for DNA replication. Newly synthesized DNA is shown as red.
(a) Leading and lagging strand synthesis. (b) Synthesis of both strands carried out by a dimeric DNA poly-
merase situated at the replication fork. Because DNA polymerase must read the template strand in the 3Ј→5Ј
direction, the 5Ј→3Ј parental strand must wrap around in trombone fashion.
28.2 What Are the Properties of DNA Polymerases? 865
replication has been corroborated with electron micrographs of DNA undergoing
replication in eukaryotic cells.
28.2 What Are the Properties of DNA Polymerases?
The enzymes that replicate DNA are called DNA polymerases. All DNA polymerases,
whether from prokaryotic or eukaryotic sources, share the following properties:
1. The incoming base is selected within the DNA polymerase active site, as deter-
mined by Watson–Crick geometric interactions with the corresponding base in
the template strand.
2. Chain growth is in the 5Ј→3Ј direction and is antiparallel to the template strand.
3. DNA polymerases cannot initiate DNA synthesis de novo—all require a primer
oligonucleotide with a free 3Ј-OH to build upon.
Despite these commonalities, DNA replication in bacterial cells is simpler than in
eukaryotes and thus will be considered first.
E. coli Cells Have Several Different DNA Polymerases
Table 28.1 compares the properties of the principal DNA polymerases in E. coli.
These enzymes are nicknamed pol and numbered I through V in order of their
discovery. DNA polymerases I, II, and V function principally in DNA repair; DNA
polymerase III is the chief DNA-replicating enzyme of E. coli. Only 40 or so copies of

this enzyme are present per cell.
The First DNA Polymerase Discovered Was E. coli DNA Polymerase I
In 1957, Arthur Kornberg and his colleagues discovered the first DNA polymerase,
DNA polymerase I. DNA polymerase I catalyzed the synthesis of DNA in vitro if
provided with all four deoxynucleoside-5Ј-triphosphates (dATP, dTTP, dCTP,
dGTP), a template DNA strand to copy, and a primer. A primer is essential because
DNA polymerases can elongate only preexisting chains; they cannot join two
deoxyribonucleoside-5Ј-phosphates together to make the initial phosphodiester
bond. The primer base pairs with the template DNA, forming a short, double-
stranded region. This primer must possess a free 3Ј-OH end to which an incoming
deoxynucleoside monophosphate is added. One of the four dNTPs is selected as
substrate, pyrophosphate (PP
i
) is released, and the dNMP is linked to the 3Ј-OH of
the primer chain through formation of a phosphoester bond (Figure 28.4). The
deoxynucleotide selected as substrate is chosen through its geometric fit with the
template base to form a Watson–Crick base pair. As DNA polymerase I catalyzes the
successive addition of deoxynucleotide units to the 3Ј-end of the primer, the chain
is elongated in the 5Ј→3Ј direction, forming a polynucleotide sequence that is
antiparallel and complementary to the template. DNA polymerase I can proceed
Property Pol I Pol II Pol III (core)*
Mass (kD) 103 88 130(␣), 27.5(⑀), 8.6(␪)
Molecules/cell 400 40
Turnover number
†
20 40 1000
Polymerization 5Ј⎯→3Ј Yes Yes Yes
Exonuclease 3Ј⎯→5Ј Yes Yes Yes
Exonuclease 5Ј⎯→3Ј Yes No No
*␣-, ⑀-, and ␪-subunits.

†
Nucleotides polymerized at 37°C/second/molecule of enzyme.
TABLE 28.1
Properties of the DNA Polymerases of E. coli
866 Chapter 28 DNA Metabolism: Replication, Recombination, and Repair
along the template strand, synthesizing a complementary strand of 3 to 200 bases
before it “falls off” (dissociates from) the template. The degree to which the en-
zyme remains associated with the template through successive cycles of nucleotide
addition is referred to as its processivity. As DNA polymerases go, DNA polymerase
I is a modestly processive enzyme. Arthur Kornberg was awarded the Nobel Prize
in Physiology or Medicine in 1959 for his discovery of this DNA polymerase. DNA
polymerase I is the best characterized of these enzymes.
E. coli DNA Polymerase I Has Three Active Sites on Its Single
Polypeptide Chain
In addition to its 5Ј→3Ј polymerase activity, E. coli DNA polymerase I has two other
catalytic functions: a 3Ј→5Ј exonuclease (3Ј-exonuclease) activity and a 5Ј→3Ј exonu-
clease (5Ј-exonuclease) activity. The three distinct catalytic activities of DNA polym-
erase I reside in separate active sites in the enzyme.
E. coli DNA Polymerase I Is Its Own Proofreader and Editor
The exonuclease activities of E. coli DNA polymerase I are functions that enhance the
accuracy of DNA replication. The 3Ј-exonuclease activity removes nucleotides from
the 3Ј-end of the growing chain (Figure 28.5), an action that negates the action of the
polymerase activity. Its purpose, however, is to remove incorrect (mismatched) bases.
Primer strand
CH
2
O
O
–
O

O
O
P
O
–
–
OO
O
P
O
–
P
OH
Base
O
Base
OH
O
Base
3
Ј
5
Ј
Base
CH
2
O
5Ј
3Ј 2Ј
4Ј

5Ј
3Ј
4Ј
5Ј
Template strand
FIGURE 28.4 The chain elongation reaction catalyzed by
DNA polymerase.The 3Ј-OH carries out a nucleophilic
attack on the ␣-phosphoryl group of the incoming
dNTP to form a phosphoester bond, and PP
i
is released.
The subsequent hydrolysis of PP
i
by inorganic pyro-
phosphatase renders the reaction effectively irreversible.
G
C
T
A
G
T
T
T
A
C
A
A
C
G
C

A
T
G
A
G
C
G
G
C
T
T
Mismatched
bases
5Ј
3Ј
Template
DNA
p
olymerase I
3
Ј
Exonuclease
hydrolysis site
5
Ј
3'
FIGURE 28.5 The 3Ј→5Ј exonuclease activity of DNA
polymerase I removes nucleotides from the 3Ј-end of
the growing DNA chain.
28.2 What Are the Properties of DNA Polymerases? 867

Although the 3Ј-exonuclease works slowly compared to the polymerase, the poly-
merase cannot elongate an improperly base-paired primer terminus. Thus, the rela-
tively slow 3Ј-exonuclease has time to act and remove the mispaired nucleotide. There-
fore, the polymerase active site is a proofreader, and the 3Ј-exonuclease activity is an
editor. This check on the accuracy of base pairing enhances the overall precision of the
process.
The 5Ј-exonuclease of DNA polymerase I acts upon duplex DNA, degrading it
from the 5Ј-end by releasing mononucleotides and oligonucleotides. It can remove
distorted (mispaired) segments lying in the path of the advancing polymerase. Its
biological roles depend on the ability of DNA polymerase I to bind at nicks (single-
stranded breaks) in dsDNA and move in the 5Ј→3Ј direction, removing successive
nucleotides with its 5Ј-exonucleolytic activity. (This overall process is known as nick
translation, because the nick is translated [that is, moved] down the DNA.) This
5Ј-exonuclease activity plays an important role in primer removal during DNA repli-
cation, as we shall soon see. DNA polymerase I is also involved in DNA repair
processes (see Section 28.8).
E. coli DNA Polymerase III Holoenzyme Replicates the E. coli
Chromosome
In its holoenzyme form, DNA polymerase III is the enzyme responsible for replica-
tion of the E. coli chromosome. The simplest form of DNA polymerase III showing
any DNA-synthesizing activity in vitro, “core” DNA polymerase III, is 165 kD in size
and consists of three polypeptides: ␣ (130 kD), ⑀ (27.5 kD), and ␪ (8.6 kD). In vivo,
core DNA polymerase III functions as part of a multisubunit complex, the DNA
polymerase III holoenzyme, which is composed of ten different kinds of subunits
(Table 28.2). The various auxiliary subunits increase both the polymerase activity of
the core enzyme and its processivity. DNA polymerase III holoenzyme synthesizes
DNA strands at a speed of nearly 1 kb/sec. DNA polymerase III holoenzyme is
organized in the following way: Two core (␣⑀␪) DNA polymerase III units and one
␥-complex are attached to DnaB helicase via two ␶-subunits to form a structure
known as DNA polymerase III*. In turn, each core polymerase within DNA po-

lymerase III* binds to a ␤-subunit dimer to create DNA polymerase III holoenzyme,
a 17-subunit
((␣⑀␪)
2
2␤
2
␶
2
␥␦␦Ј␹␺) complex (Figure 28.6). The ␥-complex is respon-
sible for assembly of the DNA polymerase III holoenzyme complex onto DNA. The
␥-complex of the holoenzyme acts as a clamp loader by catalyzing the ATP-
dependent transfer of a pair of ␤-subunits to each strand of the DNA template.
Each ␤-subunit dimer forms a closed ring around a DNA strand and acts as a tight
clamp that can slide along the DNA (Figure 28.7). Each ␤
2
-sliding clamp tethers a
Subunit Mass (kD) Function
␣ 130 Polymerase
⑀ 27.5 3Ј-Exonuclease
␪ 8.6 ⑀-subunit stabilization
␶ 71 DNA template binding; core enzyme dimerization
␤ 41 Sliding clamp, processivity
␥ 47.5 Part of the ␥-complex*
␦ 39 Part of the ␥-complex*
␦Ј 37 Part of the ␥-complex*
␹ 17 Interaction with SSB and the ␥-complex
␺ 15 Interaction with ␹ and the ␥-complex
*Subunits ␶, ␥, ␦, ␦Ј, ␹, and ␺ form the so-called ␥-complex responsible for adding ␤-subunits (the sliding clamp) to DNA
and anchoring the sliding clamp to the two core DNA polymerase III structures.The ␥-complex is referred to as the clamp
loader.

TABLE 28.2
Subunits of E. coli DNA Polymerase III Holoenzyme
868 Chapter 28 DNA Metabolism: Replication, Recombination, and Repair
core polymerase to the template, accounting for the great processivity of the DNA
polymerase holoenzyme. This complex can replicate an entire strand of the E. coli
genome (more than 4.6 megabases) without dissociating. Compare this to the pro-
cessivity of DNA polymerase I, which is only 20!
The core polymerase synthesizing the lagging strand must release from the DNA
template when synthesis of an Okazaki fragment is completed and rejoin the template
at the next RNA primer to begin synthesis of the next Okazaki fragment. The
␶-subunit serves as a “processivity switch” that accomplishes this purpose. The ␶-subunit
is usually “off ” and is turned “on” only on the lagging strand and only when synthesis
of an Okazaki fragment is completed. When activated, ␶ ejects the ␤
2
-sliding clamp
bound to the lagging strand core polymerase. Almost immediately, the lagging strand
core polymerase is reloaded onto a new ␤
2
-sliding clamp at the 3Ј-end of next RNA
primer, and synthesis of the next Okazaki fragment commences.
A DNA Polymerase III Holoenzyme Sits at Each Replication Fork
We now can present a snapshot of the enzymatic apparatus assembled at a replication
fork (Figure 28.8 and Table 28.3). DNA gyrase (topoisomerase) and DnaB helicase
unwind the DNA double helix, and the unwound, single-stranded regions of DNA are
RNA primer
DnaB
helicase
Primase
␶
␹␹

␥
␦Ј␦
␶
dsDNA
Direction of
polymerase
movement
Leading
strand
␣⑀␪ core
␤
␤
3Ј
5Ј
dsDNA
3Ј
5Ј
Lagging
strand
␣⑀␪ core
␤
␤
Direction of
polymerase
movement
RNA
primer
Okazaki
fragment
Primase

FIGURE 28.6 DNA polymerase III holoenzyme is a
dimeric polymerase. One unit of polymerase synthesizes
the leading strand, and the other synthesizes the lag-
ging strand. Because DNA synthesis always proceeds in
the 5Ј→3Ј direction as the template strand is read in
the 3Ј→5Ј direction, lagging-strand synthesis must take
place on a looped-out template. Lagging-strand synthe-
sis requires repeated priming. Primase bound to the
DnaB helicase carries out this function, periodically
forming new RNA primers on the lagging strand. All
single-stranded regions of DNA are coated with SSB
(not shown).
(a)
(b)
ᮤ
FIGURE 28.7 (a) Ribbon diagram of the ␤-subunit dimer of the DNA polymerase III holoenzyme on B-DNA,
viewed down the axis of the DNA. One monomer of the ␤-subunit dimer is colored blue and the other yellow.
The centrally located DNA is multicolored.(b) Space-filling model of the ␤-subunit dimer of the DNA polymerase
III holoenzyme on B-DNA.The hole formed by the ␤-subunits (diameter Ϸ 3.5 nm) is large enough to easily
accommodate DNA (diameter Ϸ 2.5 nm) with no steric repulsion (pdb id ϭ 2POL).The rest of polymerase III
holoenzyme (“core”polymerase ϩ ␥-complex) associates with this sliding clamp to form the replicative poly-
merase (not shown).
28.2 What Are the Properties of DNA Polymerases? 869
maintained through interaction with SSB. Primase (DnaG) synthesizes an RNA
primer on the lagging strand; the leading strand, which needs priming only once, was
primed when replication was initiated. The lagging strand template is looped around,
and each replicative DNA polymerase moves 5Ј→3Ј relative to its strand, copying tem-
plate and synthesizing a new DNA strand. Each replicative polymerase is tethered to
the DNA by its ␤-subunit sliding clamp. The DNA polymerase III ␥-complex periodi-
cally unclamps and then reclamps ␤-subunits on the lagging strand as the primer for

each new Okazaki fragment is encountered. Downstream on the lagging strand, DNA
polymerase I excises the RNA primer and replaces it with DNA, and DNA ligase seals
the remaining nick.
DNA Ligase Seals the Nicks Between Okazaki Fragments
DNA ligase (see Chapter 12) seals nicks in double-stranded DNA where a 3Ј-OH and
a 5Ј-phosphate are juxtaposed. This enzyme is responsible for joining Okazaki frag-
ments together to make the lagging strand a covalently contiguous polynucleotide
chain.
DNA Replication Terminates at the Ter Region
Located diametrically opposite from oriC on the E. coli circular map is a terminus re-
gion, the Ter, or t, locus. The oppositely moving replication forks meet here, and repli-
cation is terminated. The Ter region contains a number of short DNA sequences, with
DNA polymerase I
DNA ligase
5Ј
3Ј
5Ј
3Ј
Old Okazaki
fragment
Primer
Lagging strand template
DNA gyrase
5Ј
3Ј
Primase
Helicase
Okazaki
fragment
Primer

Leading strand
template
SSB
Newly synthesized
leading strand
Dimeric replicative
DNA polymerase
␤-Subunit
“sliding clamp”
Primer
FIGURE 28.8 General features of a replication fork.The
DNA duplex is unwound by the action of DNA gyrase
and helicase, and the single strands are coated with SSB
(ssDNA-binding protein). Primase periodically primes
synthesis on the lagging strand. Each half of the dimeric
replicative polymerase is a “core”polymerase bound to
its template strand by a ␤-subunit sliding clamp. DNA
polymerase I and DNA ligase act downstream on the
lagging strand to remove RNA primers, replace them
with DNA, and ligate the Okazaki fragments.
Protein Function
DNA gyrase Unwinding DNA
SSB Single-stranded DNA binding
DnaA Initiation factor; origin-binding protein
DnaB 5Ј⎯→3Ј helicase (DNA unwinding)
DnaC DnaB chaperone; loading DnaB on DNA
Primase (DnaG) Synthesis of RNA primer
DNA polymerase III holoenzyme Elongation (DNA synthesis)
DNA polymerase I Excises RNA primer, fills in with DNA
DNA ligase Covalently links Okazaki fragments

Tus Termination
TABLE 28.3
Proteins Involved in DNA Replication in E. coli
870 Chapter 28 DNA Metabolism: Replication, Recombination, and Repair
a consensus core element 5Ј-GTGTGTTGT. These Ter sequences act as terminators;
clusters of three or four Ter sequences are organized into two sets inversely oriented
with respect to one another. One set blocks the clockwise-moving replication fork, and
its inverted counterpart blocks the counterclockwise-moving replication fork. Termi-
nation requires binding of a specific replication termination protein, Tus protein, to
Ter. Tus protein is a contrahelicase. That is, Tus protein prevents the DNA duplex from
unwinding by blocking progression of the replication fork and inhibiting the ATP-
dependent DnaB helicase activity. Final synthesis of both duplexes is completed.
DNA Polymerases Are Immobilized in Replication Factories
Most drawings of DNA replication (such as Figure 28.8) suggest that the DNA
polymerases are tracking along the DNA, like locomotives along train tracks, syn-
thesizing DNA as they go. Experimental evidence, however, favors the view that the
DNA polymerases are immobilized, either via attachment to the cell membrane in
prokaryotic cells or to the nuclear matrix in eukaryotic cells. All the associated pro-
teins of DNA replication, as well as proteins necessary to hold DNA polymerase at
its fixed location, constitute replication factories. The DNA is then fed through the
DNA polymerases within the replication factory, much like tape is fed past the heads
of a tape player, with all four strands of newly replicated DNA looping out from this
fixed structure (Figure 28.9).
28.3 Why Are There So Many DNA Polymerases?
Cells Have Different Versions of DNA Polymerase,
Each for a Particular Purpose
A host of different DNA polymerases have been discovered, and even simple bacte-
ria such as Escherichia coli have more than one. Based on sequence homology, poly-
merases can be grouped into seven different families. The families differ in terms of
A DEEPER LOOK

A Mechanism for All Polymerases
Thomas A. Steitz of Yale University has suggested that biosynthesis
of nucleic acids proceeds by an enzymatic mechanism that is uni-
versal among polymerases. His suggestion is based on structural
studies indicating that DNA polymerases use a “two-metal-ion”
mechanism to catalyze nucleotide addition during elongation of
a growing polynucleotide chain (see accompanying figure). The in-
coming nucleotide has two Mg
2ϩ
ions coordinated to its phosphate
groups, and these metal ions interact with two aspartate residues
that are highly conserved in DNA (and RNA) polymerases. These
residues in phage T7 DNA polymerase are D705 and D882. One
metal ion, designated A, interacts with the O atom of the free
3Ј-OH group on the polynucleotide chain, lowering its affinity for
its hydrogen. This interaction promotes nucleophilic attack of the
3Ј-O on the phosphorus atom in the ␣-phosphate of the incoming
nucleotide. The second metal ion (B in the figure) assists depar-
ture of the product pyrophosphate group from the incoming nu-
cleotide. Together, the two metal ions stabilize the pentacovalent
transition state on the ␣-phosphorus atom.
Adapted from Steitz, T., 1998. A mechanism for all polymerases. Nature
391:231–232. (See also Doublié, S., et al., 1998. Crystal structure of bacte-
riophage T7 DNA replication complex at 2.2 Å resolution. Nature 391:
251–258; and Kiefer, J. R., et al., 1998. Visualizing DNA replication in a
catalytically active Bacillus DNA polymerase crystal. Nature 391:304–307.)
–
–
–
O

O
O
O
O
O
O
O
O
O
O
–
O
O
–
O
O
–
O
O
O
C
C
C
BaseЈ
Base
Base
BaseЈ
Primer
B
D882

A
Me
2+
Me
2+
P
P
P
D705
␣
C
Template
OH
dNTP
␤
␥
. . . C
Replication
factory
FIGURE 28.9 A replication factory “fixed”to a cellular sub-
structure extrudes loops of newly synthesized DNA as
parental DNA duplex is fed in from the sides.Parental
DNA strands are green; newly synthesized strands are
blue; small circles indicate origins of replication.
28.4 How Is DNA Replicated in Eukaryotic Cells? 871
the biological function served by family members. For example, family A includes
DNA polymerases involved in DNA repair in bacteria; family B polymerases include
the eukaryotic DNA polymerases predominantly involved in replication of chromo-
somal DNA; family C has the bacterial chromosomal DNA-replicating enzymes;
members of families X and Y act in DNA repair pathways; and RT designates the

DNA polymerases of retroviruses (such as HIV) and the telomerases that renew the
ends of eukaryotic chromosomes. RT polymerases are novel in that they use RNA as
the template.
The Common Architecture of DNA Polymerases
Despite sequence variation, the various DNA polymerase structures more or less fol-
low a common architectural pattern that is reminiscent of a right hand, with distinct
structural domains referred to as fingers, palm, and thumb (Figure 28.10). The ac-
tive site of the polymerase, where deoxynucleotide addition to the growing chain is
catalyzed, is located in the crevice within the palm domain that lies between the fin-
gers and thumb domains. The fingers domain acts in deoxynucleotide recognition
and binding, and the thumb is responsible for DNA binding, in the following man-
ner: When the DNA polymerase binds to template-primer duplex DNA, its thumb
domain closes around the DNA so that the DNA is bound in a groove formed by the
thumb and palm. A dNTP substrate is then selected by the polymerase, and dNTP
binding induces a conformational change in the fingers, which now rotate toward
the polymerase active site in the palm. Catalysis ensues and a dNMP is added to the
3Ј-end of the growing primer strand; pyrophosphate is released, and the polymerase
translocates one base farther along the template strand. In essence, all DNA polym-
erases are molecular motors that synthesize DNA, using dNTP substrates to add
dNMP units to the primer strand, as they move along the template strand, reading
its base sequence.
28.4 How Is DNA Replicated in Eukaryotic Cells?
DNA replication in eukaryotic cells shows strong parallels with prokaryotic DNA
replication, but it is vastly more complex. First, eukaryotic DNA is organized into
chromosomes which are compartmentalized within the nucleus. Furthermore,
these chromosomes must be duplicated with high fidelity once (and only once!)
each cell cycle. For example, in a dividing human cell, a carefully choreographed
replication of 6 billion bp of DNA distributed among 46 chromosomes occurs. The
C-terminus
Fingers

Palm
Thumb
Polymerase
active site
Exonuclease active site
Exonuclease
domain
N-terminal
domain
5Ј
5Ј
3Ј
FIGURE 28.10 A structural paradigm for DNA poly-
merases, bacteriophage RB69 DNA polymerase.Ternary
complex formed between the RB69 DNA polymerase,
DNA, and dNTP.The N-terminal domain of the protein
(residues 1–108 and 340–382) is in yellow, the exonucle-
ase domain (residues 109–339) is in red, the palm
(residues 383–468 and 573–729) is magenta, the fingers
(residues 469–572) are blue, and the thumb (residues
730–903) is green.The DNA is given in stick representa-
tion, with the primer in gold and the template in blue-
gray. A dNTP substrate (red) is shown at the active site, as
are the two Ca
2ϩ
ions (light blue spheres). Note also the
calcium ion (blue sphere) at the exonuclease active site.
(Adapted from Figure 1 in Franklin, M. C., Wang, J., and Steitz,T. A.,
2001. Structure of the replicating complex of a Pol ␣ family DNA
polymerase. Cell 105:657–667. Courtesy of Thomas A. Steitz.)

872 Chapter 28 DNA Metabolism: Replication, Recombination, and Repair
events associated with cell growth and division in eukaryotic cells fall into a general
sequence having four distinct phases: M, G
1
, S, and G
2
(Figure 28.11). Eukaryotic
cells have solved the problem of replicating their enormous genomes in the few
hours allotted to the S phase by initiating DNA replication at multiple origins of
replication distributed along each chromosome. Depending on the organism and
cell type, replication origins are DNA regions 0.5 to 2 kbp in size that occur every
3 to 300 kbp (for example, an average human chromosome has several hundred
replication origins). Since eukaryotic DNA replication proceeds concomitantly
throughout the genome, each eukaryotic chromosome must contain many units of
replication, called replicons.
The Cell Cycle Controls the Timing of DNA Replication
Checkpoints, Cyclins, and CDKs Progression through the cell cycle is regulated
through a series of checkpoints that control whether the cell continues into the
next phase. These checkpoints are situated to ensure that all the necessary steps
in each phase of the cycle have been satisfactorily completed before the next
phase is entered. If conditions for advancement to the next phase are not met,
the cycle is arrested until they are. Checkpoints depend on cyclins and cyclin-
dependent protein kinases (CDKs). Cyclin is the name given to a class of proteins
synthesized at one phase of the cell cycle and degraded at another. Thus, cyclins
appear and then disappear at specific times during the cell cycle. Cyclins are
larger than the small CDK protein kinase subunits to which they bind. The vari-
ous CDKs are inactive unless complexed with their specific cyclin partners. In
turn, these CDKs control events at each phase of the cycle by targeting specific
proteins for phosphorylation. Destruction of the phase-specific cyclin at the end
of the phase inactivates the CDK.

Initiation of Replication Eukaryotic cells initiate DNA replication at multiple ori-
gins, and two replication forks arise from each origin. The two replication forks
then move away from each other in opposite directions. Initiation of replication de-
pends on the origin recognition complex, or ORC, a protein complex that binds to
replication origins. Indeed, eukaryotic replication origins are defined as nucleotide
sequences that bind ORC. Stable maintenance of the eukaryotic genome demands
that DNA replication occurs only once per cell cycle. This demand is met by divid-
ing initiation of DNA replication into two steps: (1) the licensing of replication ori-
gins during late M or early G
1
, and (2) the activation of replication at the origins
during S phase through the action of two protein kinases, Cdc7-Dbf4 and S-CDK
(the S-phase cyclin-dependent protein kinase).
Licensing involves the highly regulated assembly of prereplication complexes
(pre-RCs) on origins of replication. Early in G
1
(just after M), the ORC (a hetero-
hexameric complex of Orc1-6) serves as a “landing pad” for proteins essential to
replication control. Binding of these proteins to ORC establishes a pre-RC, but only
within this window of opportunity during G
1
. Yeast, a simple eukaryote, provides an
informative model: ORC binds to origins and recruits Cdc6 (in its phosphorylated
form, Cdc6p), Cdt1, and the MCM proteins (Figure 28.12). Cdc6 and Cdt1 are the
replicator activator proteins. Cdc6 is degraded following replication initiation,
thereby precluding the possibility for errant replication initiation events until after
mitosis, when Cdc6 accumulates again. MCM proteins are also known as replication
licensing factors, because they “license,” or permit, DNA replication to occur. The
MCM proteins assemble as hexameric helicases that render the chromosomes com-
petent for replication. Two MCM complexes are active within each origin, one for

each replication fork. The pre-RC therefore consists of Cdc6, Cdt1, the MCM com-
plexes, and other proteins.
DNA replication is the defining characteristic of the S phase of the cell cycle. The
switch from G
1
to S is triggered by phosphorylation events carried out by S-CDK and
Cdc7-Dbf4. Phosphorylation of the MCM proteins and binding of Cdc45 activates
the helicase activity of MCM (Figure 28.12). Phosphorylation of Sld2 and Sld3, a pair
S
DNA
replication
and growth
G
2
Growth and
preparation
for cell
division
G
1
Rapid
growth
and
metabolic
activity
M
Mitosis
FIGURE 28.11 The eukaryotic cell cycle.The stages of mi-
tosis and cell division define the M phase (M for mitosis).
G

1
(G for gap, not growth) is typically the longest part of
the cell cycle; G
1
is characterized by rapid growth and
metabolic activity.Cells that are quiescent, that is, not
growing and dividing (such as neurons), are said to be in
G
0
.The S phase is the time of DNA synthesis. S is fol-
lowed by G
2
, a relatively short period of growth when
the cell prepares for cell division. Cell cycle times vary
from less than 24 hours (rapidly dividing cells such as
the epithelial cells lining the mouth and gut) to hun-
dreds of days.