(Received for publication, October 5, 1995; and in revised form, December 20, 1995)
From the
Replication forks formed in the absence of the subunit of
the DNA polymerase III holoenzyme produce shorter leading and lagging
strands than when
is present. We show that one reason for this is
that in the absence of
, but in the presence of the
-complex,
leading-strand synthesis is no longer highly processive. In the absence
of
, the size of the leading strand becomes proportional to the
concentration of
and inversely proportional to the concentration
of the
-complex. In addition, the
in the leading-strand
complex is no longer resistant to challenge by either anti-
antibodies or poly(dA):oligo(dT). Thus,
is required to cement a
processive leading-strand complex, presumably by preventing removal of
catalyzed by the
-complex.
The replication fork of Escherichia coli is
extraordinarily processive. Two replication forks form at oriC and synthesize roughly 2.2 megabase pairs of DNA before they meet
again in the terminus region. The enzymatic machinery at the
replication fork accomplishes this task while supporting two distinct
modes of DNA synthesis. Whereas the leading strand is synthesized in a
continuous fashion that reflects the overall processivity of the
replication fork, the lagging strand is synthesized discontinuously in
short Okazaki fragments 2 kb ()in length (1) . This
issue is compounded by the fact that the mechanism responsible for
maintaining the processivity of the replicative polymerase on either
strand is the same, a complex between the
subunit and the
polymerase core of the DNA polymerase III holoenzyme (Pol III
HE)(2, 3) .
The core(4) , the catalytic
polymerase/exonuclease subassembly of the Pol III HE, is essentially a
distributive enzyme, synthesizing only a few nucleotides per primer
binding event(5) . For conversion to a processive enzyme, the
core must be clamped onto DNA by associating with the
subunit(2, 3) , a dimer that encircles double-stranded
DNA (6, 7) .
can be loaded onto the primer
terminus via the action of five other HE subunits that themselves
associate, forming the
-complex (
,
,
`,
, and
)(8) .
Using rolling circle DNA replication supported
by a tailed form II DNA template (TFII) and the X-type primosomal
proteins (PriA, PriB, PriC, DnaT, DnaB, DnaC, and DnaG), the
single-stranded DNA-binding protein (SSB), and either bona fide Pol III HE or HE reconstituted from purified subunits, we have
reconstituted the coordinated leading- and lagging-strand synthesis of
the E. coli replication
fork(9, 10, 11, 12, 13) .
These replication forks were shown to have processivities of at least
0.5 megabase, yet they also made Okazaki fragments whose average length
was about 1.8 kb(9) . This suggested that the required protein
elements for establishing processivity and triggering lagging-strand
polymerase cycling were present.
The mechanism for inducing polymerase release on the lagging strand is yet to be established firmly. We have proposed that protein-protein interactions between a primase synthesizing the primer for the next Okazaki fragment and the lagging-strand polymerase initiates termination of synthesis and keys recycling of the polymerase to the new primer(11, 13) . In the bacteriophage T4 system, Hacker and Alberts (14) have argued that the lagging-strand polymerase will dissociate spontaneously from the gene 45 protein clamp when it hits the 5`-end of the previous Okazaki fragment. Stukenberg et al.(15) have made similar arguments for the E. coli fork.
Challenge experiments have shown that both the
polymerase and helicase on the leading-strand side of the fork are
processive(16) . Our recent studies have demonstrated that a
protein-protein interaction between the subunit of the Pol III HE
and DnaB is required to mediate rapid replication fork movement. (
)We show here that
contributes in yet another way to
processivity on the leading strand by protecting
in the
leading-strand polymerase complex from being removed by the action of
the
-complex.
Figure 1:
Replication forks formed in the absence
of synthesize shorter leading and lagging strands. Standard
rolling circle replication reactions in the presence of primase and
either in the presence or absence of
as indicated were performed,
processed, and analyzed as described under ``Materials and
Methods.'' Total DNA synthesis in the reactions shown here were
186 pmol +
and 19 pmol -
of
[
-
P]dAMP incorporated into acid-insoluble
product.
The
processivity of the E. coli replication fork in vitro is reflected in its high rate of speed(9, 16) ,
the inaccessibility of on the leading strand to
challenge(16, 23) , and the insensitivity of the
length of the leading strand to the concentration of
(9) .
Forks that lacked
and free
-complex were still
processive.
We examined whether this held true in the
presence of added
-complex as well.
The length of the leading
strand synthesized by -less replication forks was dependent on the
concentration of the
subunit (Fig. 2). This is strikingly
different from the situation in the presence of
where, although
overall DNA synthesis is dependent on
, the size of the leading
strand is independent of the
concentration(9) . This is
consistent with the leading-strand
-core complex needing to form
only once for synthesis of a long continuous DNA product. This
observation suggested that multiple
s were required in the absence
of
in order to synthesize the leading strand. That is,
was
being cycled in and out of the leading-strand complex.
Figure 2:
The
concentration of affects the size of the leading strand at
-less replication forks. Standard rolling circle replication
reactions performed in the absence of
and primase and containing
the indicated concentrations of
were processed and analyzed as
described under ``Materials and Methods.'' 5.0 pmol, 5.2
pmol, 5.6 pmol, and 5.3 pmol of [
-
P]dAMP
were incorporated into acid-insoluble product in the reactions shown
from left to right. The DNA products with
electrophoretic mobilities near the 6.4-kb marker and just slower than
the 9.4-kb marker result from the addition of a limited number of
nucleotides to the TFII DNA template and to dimer TFII,
respectively.
The only
group of proteins available in these reactions that could affect
loading onto 3`-ends was the
-complex. It followed that it was the
-complex that was removing
from the leading-strand complex
as well. If this were true, then the size of the leading strand
synthesized in the absence of
should also be affected by the
concentration of the
-complex. This proved to be the case.
In
the presence of , the concentration of the
-complex had no
effect on leading-strand synthesis (Fig. 3A), whereas
in the absence of
, the size of the leading strand synthesized
decreased progressively as the concentration of
-complex increased (Fig. 3B). At the highest concentrations of
-complex tested, DNA synthesis was significantly inhibited. These
observations suggested that, in the absence of
,
was being
cycled on and off the leading strand by the action of the
-complex.
Figure 3:
The size of the leading strand synthesized
by -less replication forks is inversely proportional to the
concentration of the
-complex. Standard rolling circle replication
reactions in the absence of primase and either in the presence (A) or absence (B) of
and containing the
indicated concentration of
-complex were performed, processed, and
analyzed as described under ``Materials and Methods.'' Total
DNA synthesis in the reactions shown here were: 19.8, 37.4, 37.9, 36.1,
and 24.3 pmol in A and 3.6, 4.7, 4.9, 6.0, and 5.1 pmol in B from left to right,
respectively.
Replication forks
capable of synthesizing leading strands were formed using a -TFII
DNA complex, the preprimosomal proteins (the primosomal proteins minus
DnaG), SSB, core, and the
-complex either in the presence or
absence of
. After 1.5 min, the poly(dA):oligo(dT)
challenge was added. Forks formed in the presence of
were
completely resistant to the challenge (Fig. 4). On the other
hand, those formed in the absence of
were sensitive, as evidenced
by the sharply reduced size of the leading-strand product (Fig. 4). This demonstrated that, in the absence of
, at
least one of the normally processive enzymatic components on the
leading strand was now acting distributively. The data described above
suggested that this component was
. To assess this, we repeated
the challenge experiment using anti-
antibody.
Figure 4:
-less replication forks are
distributive.
-less replication forks were reconstituted using the
-TFII DNA complex, the preprimosomal proteins (PPP) (the
primosomal proteins in the absence of primase), SSB, core,
, and
the
-complex as indicated. 1.5 min after replication was
initiated, poly(dA):oligo(dT)
was added as indicated and
the reactions continued for another 10 min. DNA products were processed
and analyzed as described under ``Materials and
Methods.''
becomes
inaccessible to anti-
antibody once it forms an initiation complex
with core(24) . We used this observation previously to show
that the
on the leading strand was processive, i.e. it
was insensitive to the presence of the antibody (16) . We
repeated those experiments here using standard rolling circle
replication reactions reconstituted in either the absence or presence
of
(Fig. 5). In each case, replication was inhibited if
the anti-
antibody was added before initiation. However, if the
anti-
antibody was added 1.5 min after initiation, only
replication forks formed in the presence of
were resistant, those
formed in its absence were inhibited almost completely.
Figure 5:
on the leading strand of
-less
replication forks is sensitive to anti-
antibody. Standard rolling
circle replication reactions were assembled as indicated in the absence
of primase and in either the absence or presence of
. In the lanes labeled with an I, anti-
antibody was
added before
to the reaction mixtures. In the lanes labeled with an E, anti-
antibody was added 1.5 min
after initiation of DNA synthesis, as indicated under ``Materials
and Methods.'' The distinct bands that appear at about 7 and 14 kb
are inactive monomer and dimer templates that become labeled by the
addition of a few nucleotide residues.
These data
show that -less replication forks are nonprocessive because the
in the leading-strand complex has now become vulnerable to
disassembly catalyzed by the
-complex.
During DNA replication, the processivity of the replication
fork arises from two contributing sources: the DNA helicase and the
leading-strand polymerase. We have recently shown that rapid fork
movement requires a physical connection between the polymerase and the
helicase that is mediated by a -DnaB protein-protein
interaction.
In the absence of this interaction, the
polymerase follows behind the helicase at a rate equal to the slow (ca. 40 nt/s) unwinding rate of the helicase alone, whereas
upon establishing a
-DnaB contact, DnaB becomes a more effective
helicase, increasing its translocation rate by more than ten-fold. We
show here that
also contributes directly to the processivity of
the leading-strand polymerase by preventing the
-complex-catalyzed
removal of
from the leading strand.
In the absence of ,
the length of the leading strand was dependent on the concentration of
and inversely proportional to the concentration of the
-complex. In the presence of
, the concentrations of these
subunits had no effect on leading-strand synthesis(9) . This
suggested that at a
-less replication fork,
, and probably
core, were being reused to synthesize the leading strand in short
bursts. The distributive nature of the
-less replication fork was
demonstrated directly in challenge experiments. Thus, not only is
essential for maintaining a high rate of fork movement,
it
is also essential for maintaining the leading-strand
-core complex
in a form that is resistant to the action of the
-complex.
This
suggests that, at least on the leading strand, may contact
,
perhaps covering a face of the protein that is essential for
interaction with the
-complex. This is consistent with the genetic
finding that mutations in dnaX (encoding both
and
)
can be suppressed by mutations in dnaN (encoding
)(24) . Our recent studies show that the C-terminal domain
of
binds both
and
, thus placing them in proximity and
presumably permitting direct protection.
Alternatively,
contacting core (probably via
) causes a rearrangement of
the
-core complex.
It is not clear when, at a -less
replication fork,
becomes available for attack by the
-complex. In the absence of both
and the
-complex, the
leading-strand
-core assembly is processive for at least 20 kb,
following along on the DNA behind the slow moving helicase.
It has also been shown that the combination of
and core
will synthesize full-length product in a single binding event on
poly(dA):oligo(dT)(5) . Perhaps the
-less leading-strand
polymerase moves fitfully behind the helicase, occasionally stalling,
and occasionally jumping ahead, and it is while the polymerase is
stalled that
can be targeted by the
-complex for recycling.
On the other hand, in the absence of
,
may be bound less
tightly to the pol III core, occasionally dissociating and diffusing
away unidirectionally. Reassociation with the core would be rapid
because of the constraints imposed by diffusion in two-dimensional
space along the DNA fiber. It may be that
is removed by
-complex while diffusing on the DNA.
In solution,
dimerizes the core to give Pol III`(25) . If this structure is
similar at the fork, then the core-
complex on the lagging strand
should be affected by
in a similar fashion as the core-
complex on the leading strand. Thus, the lagging-strand polymerase
should be refractory to recycling. Yet, this is clearly not the case.
The lagging-strand core has been shown to cycle from the just-completed
Okazaki fragment to the next primer terminus(12, 13) .
It seems likely, then, that mechanisms exist on the lagging strand that
override the protective effect of
.
These mechanisms could take
one of many forms. Conformational rearrangements could occur upon
termination of Okazaki fragment synthesis, by whatever mechanism, that
expose the lagging-strand core- to disassembly by the
-complex. On the other hand, it may be that the protective effect
of
is specific to the leading-strand polymerase.
Stukenberg et al.(15) demonstrated that would dissociate
from core when the polymerase collided with the 5`-end of a DNA strand
bound to a template, a situation similar to the lagging-strand
polymerase colliding with the 5`-triphosphate end of the primer on the
previous Okazaki fragment. These experiments were conducted using Pol
III* and
on gapped duplex bacteriophage RF DNA.
was
present, yet disassembly of the core-
complex occurred. Thus,
does not prevent recycling. This suggests that, in solution, and
when a dimeric HE subassembly binds a primer end, the polymerase bound
to the primer cannot sense, a priori, whether it should be a
leading- or a lagging-strand polymerase. Functional asymmetry that
defines the leading- and lagging-strand polymerase may arise from a
protein-protein interaction that is established between a properly
oriented
and DnaB. Establishment of this contact literally
cements the replication fork together, activating the helicase and
ensuring rapid fork movement, as well as causing a rearrangement that
makes
refractory to the action of the
-complex.