Chapter 13
DNA
replication
13.1 Introduction
13.2 DNA polymerases are the enzymes that make DNA
13.3 DNA synthesis is semidiscontinuous
13.4 Coordinating synthesis of the lagging and leading
strands
13.5 The replication apparatus of phage T4
13.6 Creating the replication forks at an origin
13.7 Common events in priming replication at the origin
13.8 Does methylation at the origin regulate initiation?
13.9 Licensing factor controls eukaryotic rereplication
Replisome is the multiprotein structure that
assembles at the bacterial replicating fork to
undertake synthesis of DNA,Contains DNA
polymerase and other enzymes.
13.1 Introduction
DNA polymerases are enzymes that synthesize a daughter strand(s) of DNA
(under direction from a DNA template),May be involved in repair or
replication.
DNA replicase is a DNA-synthesizing enzyme required specifically for
replication.
Repair of damaged DNA can take place by repair synthesis,when a strand that
has been damaged is excised and replaced by the synthesis of a new stretch,It
can also take place by recombination reactions,when the duplex region
containing the damaged is replaced by an undamaged region from another
copy of the genome.
Replication of duplex DNA takes place by synthesis of two new strands that
are complementary to the parental strands,The parental duplex is replaced by
two identical daughter duplexes,each of which has one parental strand and one
newly synthesized strand,It is called semiconservative because the conserved
units are the single strands of the parental duplex.
13.2 DNA polymerases are the enzymes that
make DNA
Figure 13.1
Semiconservative
replication
synthesizes two new
strands of DNA.
13.2 DNA
polymerases are the
enzymes that make
DNA
Figure 13.2 Repair synthesis replaces a damaged
strand of DNA.
13.2 DNA polymerases are the
enzymes that make DNA
Figure 13.3 DNA synthesis
occurs by adding nucleotides to
the 3 -OH end of the growing
chain,so that the new chain is
synthesized in the 5 -3
direction,The precursor for DNA
synthesis is a nucleoside
triphosphate,which loses the
terminal two phosphate groups in
the reaction.
13.2 DNA polymerases
are the enzymes that
make DNA
Figure 13.4 Only one DNA polymerase is the replicase,
The others participate in repair of damaged DNA.
13.2 DNA polymerases are the
enzymes that make DNA
Nick translation describes the ability of E,coli DNA
polymerase I to use a nick as a starting point from which
one strand of a duplex DNA can be degraded and
replaced by resynthesis of new material; is used to
introduce radioactively labeled nucleotides into DNA in
vitro.
13.3 DNA polymerases have various
nuclease activities
Proofreading refers to any mechanism for correcting
errors in protein or nucleic acid synthesis that involves
scrutiny of individual units after they have been added
to the chain.
13.4 DNA polymerases control
the fidelity of replication
Figure 13.6 Bacterial DNA
polymerases scrutinize the
base pair at the end of the
growing chain and excise the
nucleotide added in the case
of a misfit.
13.4 DNA polymerases
control the fidelity of
replication
Figure 13.5 Nick translation replaces part of a pre-existing
strand of duplex DNA with newly synthesized material.
13.3 DNA polymerases have various
nuclease activities
13.3 DNA polymerases have various
nuclease activities
13.3 DNA polymerases have various
nuclease activities
Figure 13.12 There are
several methods for
providing the free 3 -
OH end that DNA
polymerases require to
initiate DNA synthesis.
13.8 Priming is
required to start
DNA synthesis
Figure 13.7 Crystal structure
of phage T7 DNA
polymerase has a right hand
structure,DNA lies across
the palm and is held by the
fingers and thumb,
Photograph kindly provided
by Charles Richardson and
Tom Ellenberger.
13.5 Some DNA
polymerases have a
common structure
Figure 13.8 The catalytic domain of a DNA polymerase has a
DNA-binding cleft created by three subdomains,The active
site is in the palm,Proofreading is provided by a separate
active site in an exonuclease domain.
13.5 Some DNA polymerases have a common structure
Lagging strand of DNA must grow overall in the 3′-5′
direction and is synthesized discontinuously in the form of
short fragments (5′-3′) that are later connected covalently.
Leading strand of DNA is synthesized continuously in the 5′-
3′ direction.
Okazaki fragments are the short stretches of 1000-2000 bases
produced during discontinuous replication; they are later
joined into a covalently intact strand.
Semidiscontinuous replication is mode in which one new
strand is synthesized continuously while the other is
synthesized discontinuously.
13.6 DNA synthesis is semidiscontinuous
Figure 13.9 The leading strand is synthesized
continuously while the lagging strand is
synthesized discontinuously.
13.6 DNA synthesis is semidiscontinuous
SSB is the single-strand protein of E,coli,a
protein that binds to single-stranded DNA.
13.7 Single-stranded DNA is needed for replication
Figure 13.22
Synthesis of
Okazaki
fragments
requires priming,
extension,
removal of RNA,
gap filling,and
nick ligation.
13.10 Coordinating synthesis of the
lagging and leading strands
Figure 13.23 DNA
ligase seals nicks
between adjacent
nucleotides by
employing an enzyme-
AMP intermediate.
13.10
Coordinating
synthesis of the
lagging and
leading strands
Figure 13.10 fX174 DNA can
be separated into single
strands by the combined
effects of 3 functions,nicking
with A protein,unwinding by
Rep,and single-strand
stabilization by SSB.
13.7 Single-stranded
DNA is needed for
replication
Figure 13.13 Initiation
requires several
enzymatic activities,
including helicases,
single-strand binding
proteins,andsynthesis
of the primer.
13.8 Priming is
required to start DNA
synthesis
Figure 13.13 Initiation
requires several
enzymatic activities,
including helicases,
single-strand binding
proteins,andsynthesis
of the primer.
13.8 Priming is
required to start DNA
synthesis
Figure 13.12
Leading and
lagging strand
polymerases
move apart.
13.8 Priming is required to start DNA synthesis
Figure 13.18 DNA
polymerase III
holoenzyme assembles
in stages,generating an
enzyme complex that
synthesizes the DNA of
both new strands.
13.10 Coordinating
synthesis of the lagging
and leading strands
Figure 13.19 The b subunit of
DNA polymerase III
holoenzyme consists of a head to
tail dimer (the two subunits are
shown in red and orange) that
forms a ring completely
surrounding a DNA duplex
(shown in the center),
Photograph kindly provided by
John Kuriyan.
13.10 Coordinating
synthesis of the
lagging and leading
strands
Figure 13.20 Each catalytic
core of Pol III synthesizes a
daughter strand,DnaB is
responsible for forward
movement at the replication
fork.
13.10 Coordinating
synthesis of the lagging and
leading strands
Figure 13.21 Core
polymerase and the b
clamp dissociate at
completion of Okazaki
fragment synthesis and
reassociate at the
beginning.
13.10 Coordinating
synthesis of the lagging
and leading strands
Figure 13.16 Tus binds to ter asymmetrically
and blocks replication in only one direction.
13.9 The primosome is needed to restart replication
Figure 13.24 Similar functions are required at all
replication forks.
13.11 The replication apparatus of phage T4
Figure 13.24 Similar functions are required at all
replication forks.
13.11 The replication apparatus of phage T4
Figure 13.25 The minimal origin is defined by the
distance between the outside members of the 13-
mer and 9-mer repeats.
13.12 Creating the replication forks at an origin
Figure 13.26 Prepriming
involves formation of a
complex by sequential
association of proteins,
leading to the separation of
DNA strands.
13.12 Creating the replication
forks at an origin
Figure 13.27 The complex at
oriC can be detected by
electron microscopy,Both
complexes were visualized
with antibodies against DnaB
protein,Photographs kindly
provided by Barbara Funnell.
13.12 Creating the replication
forks at an origin
13.13 Common events in priming replication
at the origin
Figure 13.28 Transcription initiating at PR is
required to activate the origin of lambda DNA.
Figure 13.29 The lambda origin
for replication comprises two
regions,Early events are
catalyzed by O protein,which
binds to a series of 4 sites; then
DNA is melted in the adjacent
A-T-rich region,Although the
DNA is drawn as a straight
duplex,it is actually bent at the
origin.
13.13 Common events in
priming replication at the origin
13.13 Common
events in
priming
replication at
the origin
13.14 Does methylation at the origin regulate
initiation?
Figure 13.30 Replication
of methylated DNA
gives hemimethylated
DNA,which maintains
its state at GATC sites
until the Dam
methylase restores the
fully methylated
condition.
13.14 Does methylation at the origin regulate
initiation?
Figure 13.31 Only
fully methylated
origins can initiate
replication;
hemimethylated
daughter origins
cannot be used
again until they
have been restored
to the fully
methylated state.
13.14 Does methylation at the origin regulate
initiation?
Figure 13.32 A
membrane-bound
inhibitor binds to
hemimethylated DNA
at the origin,and may
function by preventing
the binding of DnaA,It
is released when the
DNA is remethylated.
13.15 Licensing factor
controls eukaryotic
rereplication
Figure 13.33 A nucleus injected
into a Xenopus egg can
replicate only once unless the
nuclear membrane is
permeabilized to allow
subsequent replication cycles.
13.15 Licensing factor
controls eukaryotic
rereplication
Figure 13.34 Licensing
factor in the nucleus is
inactivated after replication,
A new supply of licensing
factor can enter only when
the nuclear membrane
breaks down at mitosis.
13.15 Licensing
factor controls
eukaryotic
rereplication
Figure 13.35 Proteins
at the origin control
susceptibility to
initiation.
13.16 Summary
DNA synthesis occurs by semidiscontinuous replication,in which
the leading strand of DNA growing 5 3 is extended continuously,
but the lagging strand that grows overall in the opposite 3 5
direction is made as short Okazaki fragments,eachsynthesized 5 3,
The leading strand and each Okazaki fragment of the lagging strand
initiate with an RNA primer that is extended by DNA polymerase,
Bacteria and eukaryotes each possess more than one DNA
polymerase activity,DNA polymerase III synthesizes both lagging
and leading strands in E,coli,Many proteins are required for DNA
polymerase III action and several constitute part of the replisome
within which it functions,
Figure 12.16 The
rolling circle
generates a
multimeric single-
stranded tail.
13.7 Single-stranded
DNA is needed for
replication
Primer is a short sequence (often of RNA) that
is paired with one strand of DNA and provides
a free 3′-OH end at which a DNA polymerase
starts synthesis of a deoxyribonucleotide chain.
13.8 Priming is required
to start DNA synthesis
Figure 13.11 A DNA polymerase requires a 3 -
OH end to initiate replication.
13.8 Priming is required to start DNA synthesis
Figure 12.34 Replication
of ColE1 DNA is initiated
by cleaving the primer
RNA to generate a 3 -
OH end,The primer forms
a persistent hybrid in the
origin region.
13.8 Priming is
required to start
DNA synthesis
Figure 12.16 The rolling
circle generates a multimeric
single-stranded tail.
13.8 Priming is required
to start DNA synthesis
Figure 13.5 Nick
translation replaces
part of a pre-existing
strand of duplex
DNA with newly
synthesized material.
13.8 Priming is
required to start
DNA synthesis
Figure 12.15
Adenovirus terminal
protein binds to the 5
end of DNA and
provides a C-OH end
to prime synthesis of a
new DNA strand.
13.8 Priming is
required to start
DNA synthesis
Primosome describes the complex of
proteins involved in the priming action
that initiates replication on fX-type
origins,It is also involved in restarting
stalled replication forks.
13.9 The primosome is needed to restart replication
Figure 13.14
Replication is halted
by a damaged base or
nick in DNA.
13.9 The primosome is
needed to restart
replication
Figure 14.35 An E,coli
retrieval system uses a normal
strand of DNA to replace the
gap left in a newly synthesized
strand opposite a site of
unrepaired damage.
13.9 The primosome is
needed to restart replication
Figure 13.15 The
primosome is required
to restart a stalled
replication fork after
the DNA has been
repaired.
13.9 The primosome
is needed to restart
replication
Figure 12.7 Replication
termini in E,coli are
located beyond the point
at which the replication
forks actually meet.
13.9 The primosome
is needed to restart
replication
Figure 13.17
Leading and
lagging strand
polymerases
move apart.
13.10 Coordinating synthesis of the
lagging and leading strands
Figure 13.26 Prepriming
involves formation of a
complex by sequential
association of proteins,
leading to the separation of
DNA strands.
13.13 Common events
in priming replication
at the origin
13.14 Does methylation
at the origin regulate
initiation?
Figure 12.26
Attachment of
bacterial DNA to
the membrane
could provide a
mechanism for
segregation.
13.15 Licensing
factor controls
eukaryotic
rereplication
Figure 1.10
Replication of DNA
is semiconservative.
13.15 Licensing factor
controls eukaryotic
rereplication
Figure 12.10 An ARS extends
for ~50 bp and includes a
consensus sequence (A) and
additional elements (B1-B3).
13.16 Summary
DNA synthesis occurs by semidiscontinuous replication,in which
the leading strand of DNA growing 5 3 is extended continuously,
but the lagging strand that grows overall in the opposite 3 5
direction is made as short Okazaki fragments,eachsynthesized 5 3,
The leading strand and each Okazaki fragment of the lagging strand
initiate with an RNA primer that is extended by DNA polymerase,
Bacteria and eukaryotes each possess more than one DNA
polymerase activity,DNA polymerase III synthesizes both lagging
and leading strands in E,coli,Many proteins are required for DNA
polymerase III action and several constitute part of the replisome
within which it functions,
The replisome contains an asymmetric dimer of DNA polymerase
III; each new DNA strand is synthesized by a different core
complex containing a catalytic () subunit,Processivity of the core
complex is maintained by the clamp,which forms a ring round
DNA,The looping model for the replication fork proposes that,as
one half of the dimer advances to synthesize the leading strand,
the other half of the dimer pulls DNA through as a single loop that
provides the template for the lagging strand,The transition from
completion of one Okazaki fragment to the start of the next
requires the lagging strand catalytic subunit to dissociate from
DNA and then to reattach to a clamp at the priming site for the
next Okazaki fragment.
DnaB provides the helicase activity at a
replication fork; this depends on ATP
cleavage,DnaB may function by itself in
oriC replicons to provide primosome
activity by interacting periodically with
DnaG,which provides the primase that
synthesizes RNA.
Phage T4 codes for a sizeable replication apparatus,
consisting of 7 proteins,DNA polymerase,helicase,
single-strand binding protein,priming activities,and
accessory proteins,Similar functions are required in
other replication systems,including a HeLa cell
system that replicates SV40 DNA,Different enzymes,
DNA polymerase and DNA polymerase,initiate and
elongate the new strands of DNA.
The common mode of origin activation involves an initial
limited melting of the double helix,followed by more general
unwinding to create single strands,Several proteins act
sequentially at the E,coli origin,DnaA binds to a series of 9 bp
repeats and 13 bp repeats,forming an aggregate of 20 40
monomers with DNA in which the 13 bp repeats are melted,
The helicase activity of DnaB,together with DnaC,unwinds
DNA further,Similar events occur at the lambda origin,where
phage proteins O and P are the counterparts of bacterial
proteins DnaA and DnaC,respectively,In SV40 replication,
several of these activities are combined in the functions of T
antigen.
The X priming event also requires DnaB,
DnaC,and DnaT,PriA is the component that
defines the primosome assembly site (pas) for
X replicons; it displaces SSB from DNA in an
action that involves cleavage of ATP,PriB and
PriC are additional components of the
primosome.
Several sites that are methylated by the Dam
methylase are present in the E,coli origin,
including those of the 13-mer binding sites for
DnaA,The origin remains hemimethylated and is
in a sequestered state for ~10 minutes following
initiation of a replication cycle,During this period
it is associated with the membrane,and reinitiation
of replication is repressed.
After cell division,nuclei of eukaryotic cells have a
licensing factor that is needed to initiate replication,
Its destruction after initiation of replication prevents
further replication cycles from occurring in yeast,
Licensing factor cannot be imported into the nucleus
from the cytoplasm,and can be replaced only when
the nuclear membrane breaks down during mitosis.
DNA
replication
13.1 Introduction
13.2 DNA polymerases are the enzymes that make DNA
13.3 DNA synthesis is semidiscontinuous
13.4 Coordinating synthesis of the lagging and leading
strands
13.5 The replication apparatus of phage T4
13.6 Creating the replication forks at an origin
13.7 Common events in priming replication at the origin
13.8 Does methylation at the origin regulate initiation?
13.9 Licensing factor controls eukaryotic rereplication
Replisome is the multiprotein structure that
assembles at the bacterial replicating fork to
undertake synthesis of DNA,Contains DNA
polymerase and other enzymes.
13.1 Introduction
DNA polymerases are enzymes that synthesize a daughter strand(s) of DNA
(under direction from a DNA template),May be involved in repair or
replication.
DNA replicase is a DNA-synthesizing enzyme required specifically for
replication.
Repair of damaged DNA can take place by repair synthesis,when a strand that
has been damaged is excised and replaced by the synthesis of a new stretch,It
can also take place by recombination reactions,when the duplex region
containing the damaged is replaced by an undamaged region from another
copy of the genome.
Replication of duplex DNA takes place by synthesis of two new strands that
are complementary to the parental strands,The parental duplex is replaced by
two identical daughter duplexes,each of which has one parental strand and one
newly synthesized strand,It is called semiconservative because the conserved
units are the single strands of the parental duplex.
13.2 DNA polymerases are the enzymes that
make DNA
Figure 13.1
Semiconservative
replication
synthesizes two new
strands of DNA.
13.2 DNA
polymerases are the
enzymes that make
DNA
Figure 13.2 Repair synthesis replaces a damaged
strand of DNA.
13.2 DNA polymerases are the
enzymes that make DNA
Figure 13.3 DNA synthesis
occurs by adding nucleotides to
the 3 -OH end of the growing
chain,so that the new chain is
synthesized in the 5 -3
direction,The precursor for DNA
synthesis is a nucleoside
triphosphate,which loses the
terminal two phosphate groups in
the reaction.
13.2 DNA polymerases
are the enzymes that
make DNA
Figure 13.4 Only one DNA polymerase is the replicase,
The others participate in repair of damaged DNA.
13.2 DNA polymerases are the
enzymes that make DNA
Nick translation describes the ability of E,coli DNA
polymerase I to use a nick as a starting point from which
one strand of a duplex DNA can be degraded and
replaced by resynthesis of new material; is used to
introduce radioactively labeled nucleotides into DNA in
vitro.
13.3 DNA polymerases have various
nuclease activities
Proofreading refers to any mechanism for correcting
errors in protein or nucleic acid synthesis that involves
scrutiny of individual units after they have been added
to the chain.
13.4 DNA polymerases control
the fidelity of replication
Figure 13.6 Bacterial DNA
polymerases scrutinize the
base pair at the end of the
growing chain and excise the
nucleotide added in the case
of a misfit.
13.4 DNA polymerases
control the fidelity of
replication
Figure 13.5 Nick translation replaces part of a pre-existing
strand of duplex DNA with newly synthesized material.
13.3 DNA polymerases have various
nuclease activities
13.3 DNA polymerases have various
nuclease activities
13.3 DNA polymerases have various
nuclease activities
Figure 13.12 There are
several methods for
providing the free 3 -
OH end that DNA
polymerases require to
initiate DNA synthesis.
13.8 Priming is
required to start
DNA synthesis
Figure 13.7 Crystal structure
of phage T7 DNA
polymerase has a right hand
structure,DNA lies across
the palm and is held by the
fingers and thumb,
Photograph kindly provided
by Charles Richardson and
Tom Ellenberger.
13.5 Some DNA
polymerases have a
common structure
Figure 13.8 The catalytic domain of a DNA polymerase has a
DNA-binding cleft created by three subdomains,The active
site is in the palm,Proofreading is provided by a separate
active site in an exonuclease domain.
13.5 Some DNA polymerases have a common structure
Lagging strand of DNA must grow overall in the 3′-5′
direction and is synthesized discontinuously in the form of
short fragments (5′-3′) that are later connected covalently.
Leading strand of DNA is synthesized continuously in the 5′-
3′ direction.
Okazaki fragments are the short stretches of 1000-2000 bases
produced during discontinuous replication; they are later
joined into a covalently intact strand.
Semidiscontinuous replication is mode in which one new
strand is synthesized continuously while the other is
synthesized discontinuously.
13.6 DNA synthesis is semidiscontinuous
Figure 13.9 The leading strand is synthesized
continuously while the lagging strand is
synthesized discontinuously.
13.6 DNA synthesis is semidiscontinuous
SSB is the single-strand protein of E,coli,a
protein that binds to single-stranded DNA.
13.7 Single-stranded DNA is needed for replication
Figure 13.22
Synthesis of
Okazaki
fragments
requires priming,
extension,
removal of RNA,
gap filling,and
nick ligation.
13.10 Coordinating synthesis of the
lagging and leading strands
Figure 13.23 DNA
ligase seals nicks
between adjacent
nucleotides by
employing an enzyme-
AMP intermediate.
13.10
Coordinating
synthesis of the
lagging and
leading strands
Figure 13.10 fX174 DNA can
be separated into single
strands by the combined
effects of 3 functions,nicking
with A protein,unwinding by
Rep,and single-strand
stabilization by SSB.
13.7 Single-stranded
DNA is needed for
replication
Figure 13.13 Initiation
requires several
enzymatic activities,
including helicases,
single-strand binding
proteins,andsynthesis
of the primer.
13.8 Priming is
required to start DNA
synthesis
Figure 13.13 Initiation
requires several
enzymatic activities,
including helicases,
single-strand binding
proteins,andsynthesis
of the primer.
13.8 Priming is
required to start DNA
synthesis
Figure 13.12
Leading and
lagging strand
polymerases
move apart.
13.8 Priming is required to start DNA synthesis
Figure 13.18 DNA
polymerase III
holoenzyme assembles
in stages,generating an
enzyme complex that
synthesizes the DNA of
both new strands.
13.10 Coordinating
synthesis of the lagging
and leading strands
Figure 13.19 The b subunit of
DNA polymerase III
holoenzyme consists of a head to
tail dimer (the two subunits are
shown in red and orange) that
forms a ring completely
surrounding a DNA duplex
(shown in the center),
Photograph kindly provided by
John Kuriyan.
13.10 Coordinating
synthesis of the
lagging and leading
strands
Figure 13.20 Each catalytic
core of Pol III synthesizes a
daughter strand,DnaB is
responsible for forward
movement at the replication
fork.
13.10 Coordinating
synthesis of the lagging and
leading strands
Figure 13.21 Core
polymerase and the b
clamp dissociate at
completion of Okazaki
fragment synthesis and
reassociate at the
beginning.
13.10 Coordinating
synthesis of the lagging
and leading strands
Figure 13.16 Tus binds to ter asymmetrically
and blocks replication in only one direction.
13.9 The primosome is needed to restart replication
Figure 13.24 Similar functions are required at all
replication forks.
13.11 The replication apparatus of phage T4
Figure 13.24 Similar functions are required at all
replication forks.
13.11 The replication apparatus of phage T4
Figure 13.25 The minimal origin is defined by the
distance between the outside members of the 13-
mer and 9-mer repeats.
13.12 Creating the replication forks at an origin
Figure 13.26 Prepriming
involves formation of a
complex by sequential
association of proteins,
leading to the separation of
DNA strands.
13.12 Creating the replication
forks at an origin
Figure 13.27 The complex at
oriC can be detected by
electron microscopy,Both
complexes were visualized
with antibodies against DnaB
protein,Photographs kindly
provided by Barbara Funnell.
13.12 Creating the replication
forks at an origin
13.13 Common events in priming replication
at the origin
Figure 13.28 Transcription initiating at PR is
required to activate the origin of lambda DNA.
Figure 13.29 The lambda origin
for replication comprises two
regions,Early events are
catalyzed by O protein,which
binds to a series of 4 sites; then
DNA is melted in the adjacent
A-T-rich region,Although the
DNA is drawn as a straight
duplex,it is actually bent at the
origin.
13.13 Common events in
priming replication at the origin
13.13 Common
events in
priming
replication at
the origin
13.14 Does methylation at the origin regulate
initiation?
Figure 13.30 Replication
of methylated DNA
gives hemimethylated
DNA,which maintains
its state at GATC sites
until the Dam
methylase restores the
fully methylated
condition.
13.14 Does methylation at the origin regulate
initiation?
Figure 13.31 Only
fully methylated
origins can initiate
replication;
hemimethylated
daughter origins
cannot be used
again until they
have been restored
to the fully
methylated state.
13.14 Does methylation at the origin regulate
initiation?
Figure 13.32 A
membrane-bound
inhibitor binds to
hemimethylated DNA
at the origin,and may
function by preventing
the binding of DnaA,It
is released when the
DNA is remethylated.
13.15 Licensing factor
controls eukaryotic
rereplication
Figure 13.33 A nucleus injected
into a Xenopus egg can
replicate only once unless the
nuclear membrane is
permeabilized to allow
subsequent replication cycles.
13.15 Licensing factor
controls eukaryotic
rereplication
Figure 13.34 Licensing
factor in the nucleus is
inactivated after replication,
A new supply of licensing
factor can enter only when
the nuclear membrane
breaks down at mitosis.
13.15 Licensing
factor controls
eukaryotic
rereplication
Figure 13.35 Proteins
at the origin control
susceptibility to
initiation.
13.16 Summary
DNA synthesis occurs by semidiscontinuous replication,in which
the leading strand of DNA growing 5 3 is extended continuously,
but the lagging strand that grows overall in the opposite 3 5
direction is made as short Okazaki fragments,eachsynthesized 5 3,
The leading strand and each Okazaki fragment of the lagging strand
initiate with an RNA primer that is extended by DNA polymerase,
Bacteria and eukaryotes each possess more than one DNA
polymerase activity,DNA polymerase III synthesizes both lagging
and leading strands in E,coli,Many proteins are required for DNA
polymerase III action and several constitute part of the replisome
within which it functions,
Figure 12.16 The
rolling circle
generates a
multimeric single-
stranded tail.
13.7 Single-stranded
DNA is needed for
replication
Primer is a short sequence (often of RNA) that
is paired with one strand of DNA and provides
a free 3′-OH end at which a DNA polymerase
starts synthesis of a deoxyribonucleotide chain.
13.8 Priming is required
to start DNA synthesis
Figure 13.11 A DNA polymerase requires a 3 -
OH end to initiate replication.
13.8 Priming is required to start DNA synthesis
Figure 12.34 Replication
of ColE1 DNA is initiated
by cleaving the primer
RNA to generate a 3 -
OH end,The primer forms
a persistent hybrid in the
origin region.
13.8 Priming is
required to start
DNA synthesis
Figure 12.16 The rolling
circle generates a multimeric
single-stranded tail.
13.8 Priming is required
to start DNA synthesis
Figure 13.5 Nick
translation replaces
part of a pre-existing
strand of duplex
DNA with newly
synthesized material.
13.8 Priming is
required to start
DNA synthesis
Figure 12.15
Adenovirus terminal
protein binds to the 5
end of DNA and
provides a C-OH end
to prime synthesis of a
new DNA strand.
13.8 Priming is
required to start
DNA synthesis
Primosome describes the complex of
proteins involved in the priming action
that initiates replication on fX-type
origins,It is also involved in restarting
stalled replication forks.
13.9 The primosome is needed to restart replication
Figure 13.14
Replication is halted
by a damaged base or
nick in DNA.
13.9 The primosome is
needed to restart
replication
Figure 14.35 An E,coli
retrieval system uses a normal
strand of DNA to replace the
gap left in a newly synthesized
strand opposite a site of
unrepaired damage.
13.9 The primosome is
needed to restart replication
Figure 13.15 The
primosome is required
to restart a stalled
replication fork after
the DNA has been
repaired.
13.9 The primosome
is needed to restart
replication
Figure 12.7 Replication
termini in E,coli are
located beyond the point
at which the replication
forks actually meet.
13.9 The primosome
is needed to restart
replication
Figure 13.17
Leading and
lagging strand
polymerases
move apart.
13.10 Coordinating synthesis of the
lagging and leading strands
Figure 13.26 Prepriming
involves formation of a
complex by sequential
association of proteins,
leading to the separation of
DNA strands.
13.13 Common events
in priming replication
at the origin
13.14 Does methylation
at the origin regulate
initiation?
Figure 12.26
Attachment of
bacterial DNA to
the membrane
could provide a
mechanism for
segregation.
13.15 Licensing
factor controls
eukaryotic
rereplication
Figure 1.10
Replication of DNA
is semiconservative.
13.15 Licensing factor
controls eukaryotic
rereplication
Figure 12.10 An ARS extends
for ~50 bp and includes a
consensus sequence (A) and
additional elements (B1-B3).
13.16 Summary
DNA synthesis occurs by semidiscontinuous replication,in which
the leading strand of DNA growing 5 3 is extended continuously,
but the lagging strand that grows overall in the opposite 3 5
direction is made as short Okazaki fragments,eachsynthesized 5 3,
The leading strand and each Okazaki fragment of the lagging strand
initiate with an RNA primer that is extended by DNA polymerase,
Bacteria and eukaryotes each possess more than one DNA
polymerase activity,DNA polymerase III synthesizes both lagging
and leading strands in E,coli,Many proteins are required for DNA
polymerase III action and several constitute part of the replisome
within which it functions,
The replisome contains an asymmetric dimer of DNA polymerase
III; each new DNA strand is synthesized by a different core
complex containing a catalytic () subunit,Processivity of the core
complex is maintained by the clamp,which forms a ring round
DNA,The looping model for the replication fork proposes that,as
one half of the dimer advances to synthesize the leading strand,
the other half of the dimer pulls DNA through as a single loop that
provides the template for the lagging strand,The transition from
completion of one Okazaki fragment to the start of the next
requires the lagging strand catalytic subunit to dissociate from
DNA and then to reattach to a clamp at the priming site for the
next Okazaki fragment.
DnaB provides the helicase activity at a
replication fork; this depends on ATP
cleavage,DnaB may function by itself in
oriC replicons to provide primosome
activity by interacting periodically with
DnaG,which provides the primase that
synthesizes RNA.
Phage T4 codes for a sizeable replication apparatus,
consisting of 7 proteins,DNA polymerase,helicase,
single-strand binding protein,priming activities,and
accessory proteins,Similar functions are required in
other replication systems,including a HeLa cell
system that replicates SV40 DNA,Different enzymes,
DNA polymerase and DNA polymerase,initiate and
elongate the new strands of DNA.
The common mode of origin activation involves an initial
limited melting of the double helix,followed by more general
unwinding to create single strands,Several proteins act
sequentially at the E,coli origin,DnaA binds to a series of 9 bp
repeats and 13 bp repeats,forming an aggregate of 20 40
monomers with DNA in which the 13 bp repeats are melted,
The helicase activity of DnaB,together with DnaC,unwinds
DNA further,Similar events occur at the lambda origin,where
phage proteins O and P are the counterparts of bacterial
proteins DnaA and DnaC,respectively,In SV40 replication,
several of these activities are combined in the functions of T
antigen.
The X priming event also requires DnaB,
DnaC,and DnaT,PriA is the component that
defines the primosome assembly site (pas) for
X replicons; it displaces SSB from DNA in an
action that involves cleavage of ATP,PriB and
PriC are additional components of the
primosome.
Several sites that are methylated by the Dam
methylase are present in the E,coli origin,
including those of the 13-mer binding sites for
DnaA,The origin remains hemimethylated and is
in a sequestered state for ~10 minutes following
initiation of a replication cycle,During this period
it is associated with the membrane,and reinitiation
of replication is repressed.
After cell division,nuclei of eukaryotic cells have a
licensing factor that is needed to initiate replication,
Its destruction after initiation of replication prevents
further replication cycles from occurring in yeast,
Licensing factor cannot be imported into the nucleus
from the cytoplasm,and can be replaced only when
the nuclear membrane breaks down during mitosis.
