A dilemma arises: How does DNA polymerase copy the parent strand that runs in the 5→3 direction at the replication fork?. It turns out that replication is semidiscontinuous Figure 28.3:
Trang 1replication 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 heliheli-case 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 progeny DNA
G C
A
A T G C
A T
A
A
G C
G C
G
A T
A T G C
A T
T G C
A T A T
G C
A T A T
G C
New
New
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
Trang 2This 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 5→3 direction, reading the antiparallel template strand in a 3→5 sense A dilemma arises: How does DNA polymerase copy the parent strand that runs in the 5→3 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 3→5 strand (as defined by the direction that the replication fork is moving) can be copied continuously by DNA polymerase synthesizing in the 5→3 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 3→5 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 5→3 parental strand
must wrap around in trombone fashion so that the unit of dimeric DNA polymerase
replicating it can move along it in the 3→5 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 3H-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 fragmentsbecame covalently joined to form longer polynucleotide chains, in ac-cord with a semidiscontinuous mode of replication The generality of this mode of
(a)
3 5
3
5
3
3
Leading strand
Parental strands
Movement of replication fork Lagging strand
(b)
3 5
3
5
5
3
Okazaki fragments
Parental strands
Movement of replication fork Lagging strand
Dimeric DNA polymerase
3 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 polypoly-merase must read the template strand in the 3 →5 direction, the 5 →3 parental strand must wrap around in trombone fashion.
Trang 3replication has been corroborated with electron micrographs of DNA undergoing
replication in eukaryotic cells
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 5→3 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 3-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 IIIis 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-5-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-5-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 3-OH end to which an incoming
deoxynucleoside monophosphate is added One of the four dNTPs is selected as
substrate, pyrophosphate (PPi) is released, and the dNMP is linked to the 3-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 3-end of the primer, the chain
is elongated in the 5→3 direction, forming a polynucleotide sequence that is
antiparallel and complementary to the template DNA polymerase I can proceed
*
Trang 4along 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 5→3 polymerase activity, E coli DNA polymerase I has two other
catalytic functions: a 3 →5 exonuclease (3-exonuclease) activity and a 5→3 exonu-clease (5-exonuexonu-clease) 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 3-exonuclease activity removes nucleotides from the 3-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
CH2
O O–
O O
O P O–
O P O–
P
OH
Base
OH
O Base
3
5
Base
CH2 O
5
3 2
4
5
3
4
5
Template strand
FIGURE 28.4 The chain elongation reaction catalyzed by
DNA polymerase The 3 -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
5
3
Template
DNA polymerase I
3
Exonuclease hydrolysis site
FIGURE 28.5 The 3 →5 exonuclease activity of DNA
polymerase I removes nucleotides from the 3 -end of
the growing DNA chain.
Trang 5Although the 3-exonuclease works slowly compared to the polymerase, the
poly-merase cannot elongate an improperly base-paired primer terminus Thus, the
rela-tively slow 3-exonuclease has time to act and remove the mispaired nucleotide
There-fore, the polymerase active site is a proofreader, and the 3-exonuclease activity is an
editor This check on the accuracy of base pairing enhances the overall precision of the
process
The 5-exonuclease of DNA polymerase I acts upon duplex DNA, degrading it
from the 5-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 5→3 direction, removing successive
nucleotides with its 5-exonucleolytic activity (This overall process is known as nick
translation, because the nick is translated [that is, moved] down the DNA.) This
5-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:
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 (
␥-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 (( 222 2 )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.
clamp that can slide along the DNA (Figure 28.7) Each 2-sliding clamp tethers a
27.5 3-Exonuclease
71 DNA template binding; core enzyme dimerization
17 Interaction with SSB and the -complex
15 Interaction with and the -complex
*Subunits , , , , , 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
Trang 6core 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 3-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
dsDNA
Direction of polymerase movement
Leading strand
3
5
dsDNA
3
5
Lagging strand
polymerasemoveme nt
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 5 →3 direction as the template strand is read in
the 3 →5 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)
䊴 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 ⬇ 3.5 nm) is large enough to easily
accommodate DNA (diameter ⬇ 2.5 nm) with no steric repulsion (pdb id 2POL).The rest of polymerase III holoenzyme (“core” polymerase -complex) associates with this sliding clamp to form the replicative
poly-merase (not shown).
Trang 7maintained 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 5→3 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 3-OH and
a 5-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
5
3
5
3
Old Okazaki
fragment
Primer
Lagging strand template
DNA gyrase
5
3
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.
DNA polymerase III holoenzyme Elongation (DNA synthesis)
DNA polymerase I Excises RNA primer, fills in with DNA
Trang 8a consensus core element 5-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 termiTermi-nation 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 propro-teins 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)
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 Mg2ions 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
3-OH group on the polynucleotide chain, lowering its affinity for
its hydrogen This interaction promotes nucleophilic attack of the
3-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
C C
C
Base
Base
Base Base
Primer
B D882
A
Me 2+
Me 2+
P
P
P
D705
C
Template
OH dNTP
C
Replication factory
FIGURE 28.9A 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.
Trang 9the 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 chromochromo-somal 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 finfin-gers 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
3-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
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
5
5
3
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 Ca2ions (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.)
Trang 10events associated with cell growth and division in eukaryotic cells fall into a general sequence having four distinct phases: M, G1, S, and G2(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 licensdivid-ing of replication ori-ginsduring late M or early G1, 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 G1(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 G1 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 G1to 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
G2 Growth and preparation for cell division
G1 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 mimi-tosis).
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.