Lewin Benjamin (ed.) Genes IX
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Replication fork 2
Origin
Replication fork
1
terE,D,A
terC,B
terminate
terminate
fork 1 tork2
ilt*ijFil .1*.."i4 The
primosome
is required
to
restart
a statled
repUcation fork
after the DNA has
been reoaired.
ment,
whereas the nearest
lagging
strand is ini-
tiated 50 tol00
bp before reachingter.
The result
of this inhibition
is
to
halt
move-
ment of the replication
fork
and
(presumably)
to cause disassembly
of the replication
appara-
tus. F:*:-,!Sii i*"ii*
reminds
us that Tus
stops the
movement
of a replication
fork in
only one
direction. The crystal
structure
of a Ttrs-ter com-
plex
shows
that the Tus
protein
binds to INA
asymmetrically; a-helices
of the
protein pro-
trude around
the double helix
at the end that
blocks the replication fork.
Presumably
a fork
proceeding
in the
opposite
direction can dis-
place
Tus
and thus continue.
A difficulty in
understanding the function
of.the
system in
vivo
is that it
appears to be
dispensable, because
mutations if.the ter
sites or intus
are not lethal.
Summary
DNA
synthesis occurs by
semidiscontinuous
replication,
in which the leading
strand
of
DNA
growing
5'-)'is
extended continuously,
but the
lagging
strand that
grows
overall in the oppo-
site J'-5' direction is made
as short
Okazaki
fragments,
each synthesized
5'-3'. The leading
strand and
each Okazaki fragment
of the lagging
strand initiate
with an RNA
primer
that is
F€r.:ti,q* i:,it.31.1 Reptication termini
in E. coLi are located
beyond the
point
at which the
replication
forks
actualty
meet.
I'{{;ilftil 1*""}* Tus binds to fer
asymmetricatty and btocks
reptication
in onty one directjon.
extended by
DNA
polymerase. Bacteria and
eukaryotes each
possess
more than
one DNA
polymerase
activity. DNA
polymerase III
synthe-
sizes both
lagging and
leading strands
inE. coli.
Many
proteins
are
required for
DNA
polymerase
III
action and several
constitute
part
of the
repli-
some 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
(o)
subunit.
Processivity
of the
core
complex
is maintained
by the
B
clamp, which
forms a ring
around DNA.
The
clamp is loaded onto
DNA by the
clamp Ioader
complex. Clamp/clamp
loader
pairs
with simi-
lar
structural
features are
widely found in both
prokaryotic
and
eukaryotic
replication
systems.
The looping model
for the
replication
fork
proposes
that, as one
half of
the dimer advances
DNA
accessible
DNA blocked
Replication
proceeds
Replication
terminates
-+
+<*
18.18 Summary
453
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.
Ihe transition from completion of one Okazaki
fragment
to the start of the
next requires the
Iagging
strand catalytic
subunit to dissociate
from
DNA and then
reattach
to a
p
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
peri-
odically with
DnaG, which
provides
the
pri-
mase
that synthesizes RNA.
Phage t+ codes for a replication apparatus
consisting of seven
proteins:
DNA
polymerase.
helicase,
single-strand
binding
protein, prim-
ing
activities, and accessory
proteins.
Similar
functions
are required in other replication sys-
tems, including a HeLa cell system that repli-
cates SV40 DNA. Different
enzymes-DNA
polymerase
o and DNA
polymerase
6-initiate
and elonsate the new
strands of
DNA.
fhe
<ix priming
event also requires DnaB,
DnaC,
and DnaT. PriA is the component that
defines the
primosome
assembly
site
(pas)
for
qX
replicons; it
displaces SSB from DNA in an
action that involves cleavage
of
ATP. PriB and
PriC are
additional components of the
primo-
some.
The
importance of the
primosome
for the
bacterial cell is that it is used to restart replica-
tion at forks that stall when thev
encounter
damased DNA.
Tile
common mode of origin activation
involves
an initial limited melting
of the dou-
ble helix, followed
by more
general
unwinding
to create
single strands. Several
proteins
act
sequentially
at the E. coli origin. Replication is
initiated
at oriC in E. coli when DnaA
binds to a
series of 9
bp
repeats. This
is followed by bind-
ing
to a series of l3
bp
repeats,
where it uses
hydrolysis
of ATP to
generate
the energy to sep-
arate the DNA
strands.
The
prepriming
com-
plex
of DnaC-DnaB
displaces DnaA. DnaC is
released in
a
reaction
that depends
on
ATP
hydrolysis;
DnaB is
joined
by the replicase
enzyme, and replication
is initiated by two forks
that set out in
opposite directions.
Similar events
occur at the lambda
origin, where
phage pro-
teins O and P
are the counterparts
of bacterial
proteins
DnaA
and DnaC, respectively.
In SV40
replication.
several of these activities
are com-
rined in
the functions
of
T
antigen.
The availability
of DnaA at the origin is an
important
component
of the system that deter-
mines
when replication cycles
should initiate.
CHAPTER 18 DNA
Reotication
Following
initiation of replication, DnaA
hydrolyzes
its AIP under the stimulus of the
p
sliding clamp, thereby
generating
an inactive
form
of
the
protein.
In addition, oriCmust com-
pete
with Ihe dat site
for
binding
DnaA.
Several sites
that are methylated by the
Dam methylase are
present
in the E.
coli origin,
including those of the l3-mer binding sites for
DnaA.
The
origin
remains hemimethylated and
is in a sequestered state
for
-10
minutes follow-
ing initiation
of
a replication cycle. During this
period
it
is associated with the membrane and
reinitiation of replication
is repressed.
The
pro-
tein SeqA
is involved in sequestration and may
interact with
DnaA.
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456
CHAPTER 18 DNA
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