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J Biol Chem, Vol. 274, Issue 35, 25033-25041, August 27, 1999
From the Escherichia coli strains carrying
null mutations in priA are chronically induced for the SOS
response and are defective in homologous recombination, repair of UV
damaged DNA, double-strand break repair, and both induced and
constitutive stable DNA replication. This led to the proposal that PriA
directed replication fork assembly at D loops formed by the homologous
recombination machinery. The demonstration that PriA specifically
recognized and bound D loop DNA supported this hypothesis. Using DNA
footprinting as an assay, we show here that PriA also directs the
assembly of a Escherichia coli strains carrying null mutations in
priA exhibit a complex phenotype that includes constitutive
induction of the SOS response (1) and defective homologous
recombination (2, 3), repair of UV-damaged DNA (2, 3), double-strand break repair (3), and both induced and constitutive stable DNA
replication (4). The dependence of stable DNA replication on
recombination proteins and the likelihood that replication initiation
during stable DNA replication occurred at either D loops or R loops (5)
led to the proposal that all of the phenotypes elaborated in
priA null mutant strains resulted from a failure of
replication forks to assemble at these structures (2, 3).
This proposal was consistent with the ability of PriA to direct the
assembly of a Replication fork assembly in simple terms requires that all the
necessary catalytic activities, DNA helicase, primase, and DNA
polymerase, have access to a specific point on the DNA. Generally, this
occurs at an origin of replication, such as oriC in E. coli (7). A temporal imperative is imposed as well because a
protein-protein interaction between DnaB, the replication fork
helicase, and the Primosome assembly occurs in discrete steps at a PAS sequence (13): (i)
PriA recognizes and binds to the PAS, (ii) PriB joins PriA to form a
PriA-PriB-PAS DNA complex, (iii) DnaT then joins this complex to form a
triprotein complex on the PAS DNA, and (iv) DnaB is then transferred
from a DnaB-DnaC complex in solution to the PriA-PriB-DnaT-PAS DNA
complex to form a preprimosome consisting of PriA, PriB, DnaT, and DnaB
on the DNA. Complete primosome assembly occurs when DnaG adds to this
complex by virtue of a protein-protein interaction with DnaB (14). We
know that PriC is also present in the preprimosome and primosome when
they are formed on large DNAs, but we have not been able to define the
step at which it is added to the complex (15).
We have investigated primosome formation on D loops composed of
synthetic oligonucleotides. We were able to detect formation of a
stable PriA-PriB-DnaT complex by gel mobility shift analysis. However,
unlike the case where a 300-nt long ssDNA carrying the Replication Proteins--
PriA, PriB, DnaT, DnaB, and DnaC were
prepared as described by Marians (17).
DNA Substrates--
DNA substrates were prepared as described in
the accompanying article (9).
Gel Mobility Shift Analysis--
Gel mobility shift analysis was
as described in Liu et al. (18).
DNA Footprinting--
Reaction mixtures (15 µl) containing 50 mM Tris-HCl (pH 8.3 at 4 °C), 10 mM
MgCl2, 10 mM dithiothreitol, 500 µg/ml bovine serum albumin, 1 nM 5'-32P-end-labeled DNA
substrate, either 200 µM CaCl2 (for DNase I cleavage) or 200 µM ZnCl2 (for S1 nuclease
cleavage), or no, 5 µM, or 1 mM ATP as
indicated, and the primosomal proteins as indicated, were incubated at
30 °C for 10 min. Either DNase I (0.4 unit, Roche Molecular
Biochemicals) or S1 nuclease (1 unit, Amersham Pharmacia Biotech) was
then added and the incubation continued for an additional 1 min.
Reactions were terminated by the addition of EDTA to 30 mM
and 1 µg of salmon sperm DNA. DNA products were ethanol precipitated
in the presence of 0.3 M NaOAc and resuspended in 98%
formamide, 10 mM EDTA. The cleavage products were analyzed by electrophoresis at 20 watts for 2 h through 12% polyacrylamide gels (30 cm × 16 cm × 0.45 mm) (29:1,
acrylamide:bisacrylamide) containing 50% (w/v) urea using 50 mM Tris, 40 mM boric acid, 1 mM
EDTA as the electrophoresis buffer. The lower chamber was made 250 mM in NaOAc to compress the spacing between bands in the
lower part of the gel. Gels were dried, exposed to PhosphorImager screens, and autoradiographed. Maxam-Gilbert sequence ladders were
prepared as described (19).
Formation of Primosomal Protein Complexes on D Loops Detected by
Gel Mobility Shift Analysis--
We have been able to analyze each
step of primosome assembly on the
The bubble and D loop used here have been described in detail in the
accompanying report (9). The top and bottom strands are composed of
82-nt long oligonucleotides (oligos) that have a central
noncomplementary region of 42 nt. When annealed together, these two
oligos form the bubble DNA. To form the D loop DNA, a 42-nt long
invading strand is annealed to the top strand of the bubble. As
elaborated in the accompanying report (9), the bubble is composed of
oligonucleotides 1 and 2L, with the top strand being oligo 1. The
invading strand in the D loop is oligo 5S.
As demonstrated previously (8) and in the accompanying report (9), PriA
was capable of forming a stable complex with D loop, but not bubble,
DNA (Fig. 1A, lane 2). No
change was detected in the mobility of the protein-DNA complex formed
when PriB was also included in the reaction mixture (Fig. 1A,
lane 3). Similar results were observed in our analysis of
primosome assembly on the
With PAS DNA as a substrate, addition of DnaB and DnaC to the
PriA-PriB-DnaT-PAS DNA complex in the presence of 10 µM
ATP resulted in the appearance of a new complex that contained the later three proteins as well as DnaB (13). However, this was not
observed with D loop DNA (Fig. 1B, lane 8). Whereas this
could be taken as indicating that a preprimosome did not assemble on the D loop DNA, other interpretations were possible. The PAS and D loop
DNA substrates are distinct in that the PAS DNA is nominally single-stranded, with no duplex region that can be unwound, whereas the
D loop DNA has two duplex regions. Thus, even though the experiment shown in Fig. 1B was not performed in the presence of an ATP
concentration high enough to effect primosome-catalyzed DNA unwinding
(21), we considered that the structure itself might be destabilizing, preventing detection of primosome complex formation by an assay that
has slow time resolution such as gel mobility shift analysis. We
therefore turned to DNA footprinting techniques to assess the possibility of primosome assembly at D loops.
Distinct Modes of Interaction of PriA with D Loop DNA and Duplex
DNA with a 3'-Single-stranded Extension--
Because the bubble and D
loop DNA contained regions of both duplex and single-stranded DNA, we
used two different probes in the footprinting reactions, DNase I, to
detect interactions with dsDNA, and S1 nuclease, to detect interactions
with ssDNA. As shown, for example, in Figs.
2 and 6, the expected specificity was
observed: DNase I only digested regions of the D loop and bubble that,
based on the nucleotide sequence (9), should have been duplex, whereas
S1 nuclease only digested regions of these DNAs that were predicted to
be single-stranded based on the nucleotide sequence (9).
DNA substrates were used that were 5'-end-labeled on either the top or
bottom strand. As predicted from the gel mobility shift analysis, no
interaction could be detected between PriA and the bubble DNA (Fig. 2,
A and B, lanes 5, and Fig. 5). On the other hand,
PriA gave a complicated footprint on the D loop DNA (Figs. 2,
A and B, lanes 8, and 6). Both the top
and bottom strand of the left-hand flanking duplex region were
protected from DNase I digestion. Protection on the top strand started
14 nt from the 5'-end and extended into the duplex region formed by the
top strand and the invading strand. Protection on the bottom strand
started 6 nt from the 3'-end and extended for about 10 nt, but did not enter the single-stranded region of the displaced strand. This positioned PriA at the left-hand three-strand junction of the D loop.
This is consistent with the preference observed by gel mobility shift
analysis for PriA binding to three-strand junctions with
5'-single-stranded tails (9). In the duplex DNA formed by the top and
invading strands, alternating regions of protection and enhancement of
cleavage could be observed. Although not perfectly so, these
alternating regions showed a periodicity of about one turn of the helix
(Fig. 2, A, lane 5, and Fig. 6).
The addition of PriB and DnaT improved the protection of the left-hand
flanking region of the top strand and altered subtly the pattern
observed in the duplex formed by the top strand and the invading
strand. Several enhanced protections (nt T44,
G45, and G54) and a new enhanced cleavage site
(nt G35) could be observed (Fig. 2, A and
B, lanes 9). This is consistent with the ability to observe
a stable PriA-PriB-DnaT-D loop DNA complex by gel mobility shift analysis.
A similar pattern of alternating protection and enhanced cleavage was
observed when DNase I was used to probe the interaction of PriA with
the invading strand (Fig. 3, lane
2). Here the affected region was smaller (32 nt), but the regions
of protection and enhancement were reflected directly across the
helical axis from the top strand (Fig. 5). As was observed with labeled
top strand, the addition of PriB and DnaT only altered the observed
pattern slightly, resulting in increased protection at the 3'-end of
the invading strand (Fig. 3, lane 3).
In the accompanying report (9) we demonstrated two modes of PriA
binding to DNA, one in which the protein bound to duplex DNA with
3'-single-stranded extensions and one that presumably reflected binding
to a D loop where the protein bound to bent DNA at three strand
junctions. In order to determine whether the striking pattern of
alternating protection and enhancement of cleavage observed when PriA
bound the D loop was particular to one or the other mode of PriA
binding to DNA, we examined the footprint of PriA on dsDNA with a
3'-single-stranded extension (Fig.
4).
In order to facilitate analysis of interactions at the 3'-end of the
top strand, we included a 5'-single-stranded extension past the duplex
region so that DNase I cleavages at the intersection of the
3'-single-stranded tail and the dsDNA would fall in an easily readable
section of the gel. The pattern of alternating protection and
enhancement observed on both the top and invading strand of the D loop
was not evident (Fig. 4, A and B, lanes 5). Protection on the top strand of the duplex extended back about 14 nt
from the single-stranded tail. Protection on the bottom strand,
although weaker, appeared to extend back about 25 nt from the 3'-end.
No enhanced cleavages were observed at all. Probing with S1 nuclease
indicated that about 7 nt of the 3'-single-stranded tail abutting the
dsDNA was also protected by PriA binding (data not shown).
Thus, the DNA footprinting reported here supports the conclusion of the
accompanying report (9) that there are two modes of PriA binding to
DNA. Modification of the pattern of DNase I cleavages as a result of
PriA binding to the D loop was observed along 58 base pairs of dsDNA
formed by the co-helical segments of the duplex consisting of the top
and bottom strands with that of the duplex consisting of the top and
invading strands (Fig. 5). This is
roughly 200 Å, a very large distance when compared with the 36-Å
Stokes radius of PriA (21). This suggests, based on the assumption that
one PriA molecule could protect about 70 Å, that there are either
multiple molecules of PriA bound per D loop, or that PriA is wrapping
the D loop DNA about itself in some manner.
PriA is a monomer in solution (22). We have also not observed any
sigmoidicity in the binding isotherms of PriA to either PAS (13) or D
loop (9) DNA that might suggest dimerization of the protein upon
binding. However, we could detect two distinct complexes of PriA bound
to PAS DNA that differed by a factor of two in the relative amount of
PriA bound (13) and quantitative Western blotting of the protein
components in primosomes formed on Primosome Assembly on D Loop DNA Results in Unwinding of the
Left-hand Flanking Duplex Region--
Replication fork assembly at a D
loop requires that DnaB, the replication fork helicase (24), gain
access to the DNA. The experiments described in the previous section
demonstrate that an intermediate consisting of a PriA-PriB-DnaT complex
could be isolated on the D loop. We therefore continued our
investigation by adding DnaB and DnaC to the footprinting assays. The
combination of DnaB and DnaC alone, without the other primosomal
proteins, gave no visible footprint, regardless of whether the top
(Fig. 6) or bottom (Fig.
7) strand was labeled and whether DNase I
(Figs. 6A, lane 7, and 7A, lane 4, respectively)
or S1 nuclease (Figs. 6B and 7B, lanes 7) was
used as a probe. Similar results were observed if the invading strand
was labeled (data not shown).
DnaB can be transferred to ssDNA by DnaC in the presence of ATP (7).
The footprinting experiments described in this section contained 5 µM ATP, which should have been sufficient for these purposes (13). Yet, even though no SSB was present to prevent DnaB
binding, no footprint was observed. There are several possible reasons
for this. The most likely explanation is that DnaB may be binding
transiently under these circumstances, leaving the displaced strand
quickly because of hydrolysis of ATP. Another alternative is that the
DnaB is present on the DNA, but is sliding back and forth rapidly. If
this were the case, one might expect a generalized reduction in DNase I
sensitivity, but this was also not observed. Finally, it is also
possible that there is no binding of DnaB at all under these
conditions. There is insufficient evidence to decide between these
three alternatives, however, these observations are consistent with our
demonstration, reported elsewhere (16), that DnaB and DnaC alone are
insufficient to support DNA polymerase III holoenzyme-catalyzed
elongation of the invading strand.
Footprinting also failed to reveal the presence of a primosome on the D
loop. Although this was consistent with our inability to observe a
stable primosomal complex by gel mobility shift analysis, it was
inconsistent with our ability to observe primosomal
protein-dependent replication initiating at a D loop (16).
Addition of DnaB and DnaC to the PriA-PriB-DnaT-D loop DNA complex did
not result in any observable modification to the footprint, regardless
of whether the top (Fig. 6, A and B, lanes 8) or
bottom strand (Fig. 7, A, lane 5, and B, lane 8)
was labeled or whether DNase I (Figs. 6A and 7A)
or S1 nuclease (Figs. 6B and 7B) was used as a probe.
Again, there were alternative possible explanations for this
observation. We rejected the possibility that this implied that a
primosome was not forming on the D loop DNA because, as mentioned above, we could observe primosomal protein-dependent
replication on a D loop template DNA (16). Instead, we considered two
alternatives: either the oligonucleotide substrate used was not large
enough to support stable binding of a complete primosome or a
rearrangement of the disposition of the proteins involved was required
for stable primosome assembly and this rearrangement was driven by ATP
hydrolysis and thus required higher concentrations of ATP than 5 µM. Accordingly, we performed the footprinting
experiments in the presence of 1 mM ATP.
Significant changes to the footprints on the D loop could be observed
in the presence of PriA, PriB, DnaT, DnaB, DnaC, and 1 mM
ATP. On the top strand, the region between T9 and
T22 became S1 nuclease-sensitive (Figs. 6, B, lane
9, and 9), as if base pairing with the bottom strand had been
lost. Little in the way of consistent changes in the DNase I footprint
were observed. Occasionally, a generalized decrease in DNase I
sensitivity was observed. However, this was very variable
(e.g. compare Fig. 6A, lane 9, to
8A, lane 5). On the bottom
strand, new DNase I-hypersensitive sites became apparent at
C13, G14, and T15 as the
surrounding nucleotides, T10, G12, and
G17 became somewhat less sensitive to DNase I (Figs. 7,
A, lane 6, and 9). Consistent
with the increased S1 nuclease sensitivity of the left-hand end of the
top strand, a similar region, between G64 and
A74, on the bottom strand also became S1 nuclease-sensitive
(Figs. 7, B, lane 9, and 9).
We interpret the conversion of a significant portion of the left-hand
flanking duplex region of the D loop from profound S1 nuclease-insensitivity to obvious S1 nuclease-sensitivity as indicating that this region of the substrate had become unwound by the action of
the primosomal proteins. Both PriA and DnaB are helicases that could
unwind this substrate. Thus, in order to determine whether the observed
unwinding was a result of the independent action of the DNA helicases
present in the reaction mixture or a result of primosome assembly, we
examined the dependence of the unwinding reaction on the presence of
all the primosomal proteins.
Neither PriA alone (Fig. 8, A and B, lanes
2 and 7, respectively), DnaB in the presence of DnaC
(Fig. 8, A and B, lanes 3 and 8,
respectively), or the combination of PriA with DnaB and DnaC (Fig. 8,
A and B, lanes 4 and 9, respectively)
showed the changes to the DNase I and S1 nuclease footprints that were
indicative of unwinding of the left-hand duplex region and were obvious
when PriB and DnaT were also present with PriA, DnaB, and DnaC in the reaction mixture (Figs. 6, A, lane 9; B, lane 9;
7B, lane 9; 8A, lane 5; 8B, lane 10).
Thus, the observed unwinding was neither the result of either PriA or
DnaB acting independently nor the result of the two helicases acting
independently, but in a concerted manner, one after the other. Instead,
we conclude that the unwinding resulted because the helicase activities
intrinsic to a primosome that had been assembled on the D loop became
activated in the presence of 1 mM ATP. This is consistent
with the known ATP requirements for primosome helicase action (21).
PriA was discovered originally because of its requirement for
conversion of viral Currently, the term primosome now applies to any protein or
multiprotein complex that is capable of providing both DNA unwinding and Okazaki fragment-priming functions at a replication fork and the
one discussed specifically in this report is referred to as the
Genetic investigations of the phenotypes of priA null
mutations suggested a role for this protein in the cell. These strains were constitutively induced for the SOS response (1) and were defective
in homologous recombination (2, 3), both induced and constitutive
stable DNA replication (4), and the repair of UV-damaged DNA (2, 3) and
double-strand DNA breaks (3). The role of D loops and R loops as
initiating structures for the two forms of stable DNA replication (5),
and the key role of a D loop during initiation of homologous
recombination (34), which is also required to initiate repair of
double-strand breaks (35), led to the proposal that all these
phenotypes resulted from a failure, in priA null strains, of
replication forks to assemble at a D loop (2, 3). Recent biochemical
evidence has provided significant support for this hypothesis.
PriA specifically recognizes and binds D loop DNA compared with a
corresponding bubble structure (8, 9). The basis for this binding is
the ability of PriA to specifically recognize bent DNA at three-strand
junctions (9). In this report, we have provided evidence that PriA will
also catalyze assembly of a Primosome assembly on PAS sequences in ssDNA occurs in five discrete
steps resulting in, sequentially, the following protein-DNA complexes
(13): (i) PriA-PAS DNA, (ii) PriA-PriB-PAS DNA, (iii) PriA-PriB-DnaT-PAS DNA, (iv) PriA-PriB-DnaT-DnaB-PAS DNA (the preprimosome), and (v) PriA-PriB-DnaT-DnaB-DnaG-DNA (the primosome). PriC is present in the latter two complexes if they are formed on large
DNAs. We were able to demonstrate, using gel mobility shift analysis,
the formation of a PriA-PriB-DnaT-D loop DNA complex, but could not,
using this methodology, observe any of the higher order complexes.
DNA footprinting revealed that PriA bound preferentially to the
left-hand side of the D loop. This is consistent with our observation,
reported in the accompanying manuscript (9), that PriA preferred to
bind three-strand junctions with 5'-single-stranded tails. PriA binding
resulted in the modification of the DNase I digestion pattern over a
large portion of the D loop. At the three-strand junction formed by the
top, invading, and bottom strands, PriA binding reduced DNase I
activity on all three strands. This region of protection extended from
the three-strand junction, 10 base pairs into the duplex formed by the
top and invading strands and 18 base pairs in the opposite direction
into the duplex formed by the top and bottom strands. Given that steric
interference in a DNase I footprint generally leads to an overestimate
of binding site size, this is a region that could be covered by a
protein that has a Stokes radius of 36 Å (22).
Interestingly, however, the footprint was more complex. PriA binding
also generated an alternating pattern of protection from and
enhancement of cleavage by DNase I essentially throughout the length of
the duplex formed by the top and invading strands. The entire region
effected by PriA binding covered almost 200 Å of DNA, far too much to
be covered by one PriA molecule. Although the obvious explanation, that
PriA wraps a portion of the D loop about itself, is attractive, we do
not have any corroborating evidence to support it. The alternative
explanation, that multiple molecules of PriA are bound to the D loop,
cannot be ruled out at this time.
In the presence of 5 µM ATP, a concentration sufficient
to support primosome assembly (13), the addition of the other
primosomal proteins did not result in any significant changes in the
footprint. This was surprising because we expected, if a primosome was
being assembled, to observe a footprint on the displaced strand
corresponding to the loading of DnaB. However, we were uncertain as to
whether a substrate this small could support stable binding of a
complete primosome. Our previous studies utilized a 304-nt long ssDNA
that contained a PAS sequence roughly in the center of the fragment (13). When a smaller substrate was used composed solely of the sequence
of the PAS, we could only observe PriA
binding.2 Thus we searched
for functional evidence for primosome assembly.
Primosome DNA helicase activity was detected in the presence of 1 mM ATP because of the appearance of S1 nuclease-sensitive sites in both the top and bottom strands of the left-hand flanking duplex region. It was clear that this was a primosomal function because: (i) all the preprimosomal proteins were required, and (ii)
neither PriA or DnaB alone nor a combination of the two helicases was
sufficient for unwinding to occur. It seems likely that the components
of the primosome assembled on a D loop are binding to both strands of
DNA. PriA binding alone spanned each of the three strands at the
left-hand junction. The 5' The disposition of the other proteins in a primosome assembled on a D
loop is not clear. In the presence of 1 mM ATP, we observed both protection from and enhancement of DNase I cleavage in an 8-nt
region on the bottom strand in the right-hand flanking duplex region.
However, no corresponding effects were observed on the top strand.
Thus, we think it unlikely that unwinding of the right-hand flanking
duplex was occurring. Instead, we take this to indicate a rearrangement
on the DNA of some of the other primosomal proteins to accommodate
loading of DnaB.
Although to simplify the analysis, these experiments were conducted in
the absence of the single-stranded DNA-binding protein (SSB), it is
clear, because we can observe replication fork assembly at a D loop
(16), that primosome occurs on D loops in the presence of SSB as well.
It seems likely, then, that the normal substrates for PriA-directed
primosome assembly in the cell are D loops that are formed by the
recombination proteins. This is supported by our demonstration of
primosomal protein- and DNA polymerase III
holoenzyme-dependent replication fork assembly on form II
DNA templates carrying a D loop (16). Thus, one can conclude, much as
bacteriophage *
This work was supported by National Institutes of Health
Grant GM34557.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
2
K. H. Zavitz and K. J. Marians,
unpublished data.
The abbreviations used are:
PAS, primosome
assembly site;
ss, single-stranded;
ds, double-stranded;
SSB, single-stranded DNA-binding protein;
nt, nucleotide;
DNase I, deoxyribonuclease I.
PriA-directed Assembly of a Primosome on D Loop DNA*
§
Molecular Biology Program,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
X174-type primosome on D loop DNA. The ability to load
a complete primosome on D loop DNA is a step necessary for replication
fork assembly.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
X174-type primosome at specialized DNA sequences
called primosome assembly sites
(PAS)1 (6). Primosomes are an
essential part of the replication fork apparatus, providing both the
DNA unwinding function and the Okazaki fragment-priming function (7).
McGlynn et al. (8) demonstrated that PriA could indeed
recognize and bind specifically to D loops. We showed in the
accompanying report (9) that PriA exhibited two modes of specific DNA
recognition. Binding to double-stranded (ds) DNAs carrying
3'-single-stranded (ss) extensions of at least 12 nucleotides (nt)
presumably reflected the 3' 
5' DNA helicase activity of the protein
(10, 11), whereas binding to D loops reflected the ability of PriA to
bind to bent DNA at three strand junctions.
subunit of the DNA polymerase III holoenzyme must
be established for proper replication fork formation (12). This, in
turn, requires that binding of the polymerase to the primer for the
nascent leading strand occur at roughly the same time that the
primosome assembles. If these events are not coordinated properly, the
primosome may move away from the polymerase before the required
protein-protein interaction is established. If replication forks do
form at D loops, clearly primosome assembly must proceed efficiently
and rapidly so that the DnaB- interaction is established with
holoenzyme that binds to the 3'-end of the invading strand.
X174 PAS was
used as the substrate (13), we could not detect any higher order
complexes when DnaB and DnaC were added to the reaction. We therefore
used DNA footprinting with both deoxyribonuclease I (DNase I) and S1
nuclease as probes. A specific complex could be detected between PriA
and the D loop DNA, but no evidence of higher order complexes was
obtained. Unwinding of one end of the D loop, which required all the
primosomal proteins, however, could be detected as a result of changes
in the sensitivity of the DNA substrate to S1 nuclease in the presence
of ATP. Thus, we conclude that a complete preprimosome could be
assembled on D loop DNA. This is consistent with our demonstration that
replication forks can assemble and extend the invading strand in a D
loop in a primosomal protein-, single-stranded DNA-binding protein
(SSB)-, and DNA polymerase III holoenzyme-dependent manner
(16).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
X174 PAS sequence using gel
mobility shift analysis (13). We therefore used the same approach
initially in an attempt to detect a similar ordered assembly of the
primosome on D loop DNA.
X174 PAS sequence (13). In that
investigation, the presence of the PriA-PriB-PAS DNA complex was only
revealed by Western analysis. Because PriB is a small protein (20), the
mass added to the complex is insufficient to cause a detectable change
in mobility. Although we have not performed a similar Western analysis here, the fact that we can detect a PriA-PriB-DnaT complex specific for
D loop DNA (Fig. 1A, lane 3) argues that the PriA-PriB-D
loop DNA complex does form. As in the case where PAS DNA was used as a
substrate (13), formation of the PriA-PriB-DnaT-D loop DNA complex did
not require ATP (data not shown).
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Fig. 1.
Formation of primosomal protein complexes on
D loop DNA analyzed by gel mobility shift analysis. Standard DNA
binding reaction mixtures containing the indicated primosomal proteins
were analyzed by gel mobility shift analysis as described under
"Materials and Methods." A, formation of a
PriA-PriB-DnaT complex on D loop DNA. B, the addition of
DnaB and DnaC to the PriA-PriB-DnaT complex does not yield a stable
preprimosomal complex.
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Fig. 2.
DNase I footprints of PriA and PriA-PriB-DnaT
complexes on bubble and D loop DNA. Standard DNA footprinting
reaction mixtures containing either the bubble or D loop substrate
labeled on either the top (panel A) or bottom (panel
B) strand as indicated by an asterisk, and 26 nM PriA, 15 nM PriB, and 28 nM
DnaT, as indicated, were analyzed as described under "Materials and
Methods." Maxam-Gilbert sequence ladders in lanes 1-3 are
of the respective labeled oligonucleotide. Correspondence of the
Maxam-Gilbert sequence ladder to the sequence of oligos dlp 1 and 2L
are indicated on the right-hand side of panels A
and B, respectively.
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Fig. 3.
DNase I footprints of primosomal protein
complexes on D loop DNA labeled on the invading strand. Standard
DNA footprinting reaction mixtures containing 5 µM ATP
and 26 nM PriA, 15 nM PriB, 28 nM
DnaT, 32 nM DnaB, and 32 nM DnaC, as indicated,
were analyzed as described under "Materials and Methods."
pp, this reaction mixture contained PriA, PriB, DnaT, DnaB,
and DnaC. Correspondence of the Maxam-Gilbert sequence ladder to the
sequence of oligo 5S is indicated on the left-hand side of
the figure.
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Fig. 4.
PriA binding to a duplex DNA with a
3'-single-stranded extension does not elicit the alternating pattern of
enhancement and protection from DNase I cleavage observed on D loop
DNA. DNA footprinting reactions containing DNA duplexes composed
of the top and invading strand of the D loop labeled on either the top
(panel A) or invading (panel B) strand and 26 nM PriA were analyzed as described under "Materials and
Methods." Correspondence of the Maxam-Gilbert sequence ladder to the
sequences of oligos dlp1 and dlp5s is indicated on the right-hand
side of panels A and B, respectively.
View larger version (18K):
[in a new window]
Fig. 5.
Summary of the enhancement of and protection
from DNase I cleavage resulting from PriA binding to D loop
DNA.
X174 ss(c) DNA suggested a
stoichiometry of two PriA molecules per primosome (15). On the other
hand, an alternating pattern of protection from and enhancement of
DNase I cleavage is characteristically observed when dsDNA lies on a
surface, such as what occurs when DNA is wrapped around a protein.
Distinguishing between these two possibilities will require additional investigation.
View larger version (77K):
[in a new window]
Fig. 6.
Primosomal protein-catalyzed unwinding of the
left-hand side of the D loop as observed on the top strand.
Standard DNA footprinting reaction mixtures containing D loop DNA
labeled on the top strand, either 5 µM ATP (lanes
4-8) or 1 mM ATP (lane 9), 26 nM PriA, 15 nM PriB, 28 nM DnaT, 32 nM DnaB, and 32 nM DnaC, as indicated, and
either DNase I (panel A) or S1 nuclease (panel B)
were analyzed as described under "Materials and Methods."
Maxam-Gilbert (lanes 1-3) sequence ladders are of the top
strand. pp, this reaction mixture contained PriA, PriB,
DnaT, DnaB, and DnaC. Correspondence of the Maxam-Gilbert sequence
ladder to the sequence of oligo dlp1 is indicated on the
left-hand side of the panels.
View larger version (80K):
[in a new window]
Fig. 7.
Primosomal protein-catalyzed unwinding of the
left-hand side of the D loop as observed on the bottom
strand. Standard DNA footprinting reaction mixtures containing D
loop DNA labeled on the bottom strand, either 5 µM ATP
(panel A, lanes 1-5; panel B, lanes 4-8) or 1 mM ATP (panel A, lane 6; panel B, lane 9), 26 nM PriA, 15 nM PriB, 28 nM DnaT, 32 nM DnaB, and 32 nM DnaC, as indicated, and
either DNase I (panel A) or S1 nuclease (panel B)
were analyzed as described under "Materials and Methods."
Maxam-Gilbert (panel B, lanes 1-3) sequence ladders are of
the bottom strand. pp, this reaction mixture contained PriA,
PriB, DnaT, DnaB, and DnaC. Lane 6 of panel A is
from the same gel as lanes 1-5. Intervening lanes not
relevant to this figure have been removed. Curvature of the gel, which
can be seen in lanes 1-5, prevent precise alignment of
lane 6 with lanes 1-5. Correspondence of the
Maxam-Gilbert sequence ladder to the sequence of oligos dlp1 and 2L is
indicated on the left-hand side of panels A and
B, respectively.
View larger version (57K):
[in a new window]
Fig. 8.
D loop unwinding requires assembly of a
primosome. Standard DNA footprinting reaction mixtures containing
D loop substrate labeled on either the top (panel A) or
bottom (panel B) strand, 1 mM ATP, 26 nM PriA, 15 nM PriB, 28 nM DnaT, 32 nM DnaB, and 32 nM DnaC, as indicated, and
either DNase I (lanes 1-5) or S1 nuclease (lanes
6-10) were analyzed as described under "Materials and
Methods." Correspondence of the cleavage patterns to the sequence of
oligos dlp1 and dlp2L are indicated on the right-hand side
of panel A and the left-hand side of panel
B, respectively.
View larger version (18K):
[in a new window]
Fig. 9.
Summary of primosome-catalyzed D loop
unwinding.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
X174 DNA to a duplex replicative form in vitro (25, 26). Subsequent studies showed that this protein initiated the assembly of a protein-DNA complex that primed DNA synthesis of the complementary strand (6). Intensive biochemical analysis of this reaction revealed that seven proteins, now called PriA, PriB, PriC, DnaT, DnaB, DnaC, and DnaG, were involved in assembly
of this protein-DNA complex (7). On X174 DNA coated with SSB, this
complex is composed of PriA, PriB, PriC, DnaT, DnaB, and DnaG (13) and
is capable of: (i) translocation along ssDNA in either direction (21),
(ii) acting as a bidirectional DNA helicase (21), and catalyzing the
synthesis of small oligoribonucleotide primers that can be used by the
DNA polymerase III holoenzyme to initiate DNA synthesis (27). The
ability to both unwind duplex DNA and synthesize ribonucleotide primers
earned this complex the name of "primosome" (27) and illuminated an
economical solution to the requirements for unwinding the duplex
template DNA and providing primers for Okazaki fragment synthesis
during chromosomal replication.
X-type primosome (7). The X-type primosome is capable of
participating in replication fork assembly in vitro (28) and
in the replication of a number of plasmid DNAs, such as pBR322 (29).
But four (PriA, PriB, PriC, and DnaT) of the seven proteins are not
required for replication initiating at oriC (30), the bacterial chromosome origin of replication. The three X-type primosomal proteins required for chromosomal DNA replication, DnaB,
DnaC, and DnaG (30), all have very specific functions: DnaB is the
replication fork DNA helicase (24), DnaC is required for the efficient
transfer of DnaB to DNA (31), and DnaG is the primase for Okazaki
fragment synthesis (32, 33). Thus, it has been unclear as to the role,
if any, of PriA, PriB, PriC, and DnaT during chromosomal replication.
X-type primosome at a D loop. This is a
necessary step in order to effect replication fork assembly on these
recombination intermediates.
3' directionality of the DnaB helicase
activity argues that this protein is loaded onto the displaced strand.
If it were bound to either the top or invading strand, we would have
expected to observe either displacement of the invading strand or
unwinding of the right-hand flanking duplex.
evolved the P protein to steal DnaB from DnaC and
suborn it to replication of its own genome, bacteriophage X174
evolved to steal an existing cellular replication system and direct it
to its own survival by mimicking the substrate for primosome assembly.
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1.
Nurse, P.,
Zavitz, K. H.,
and Marians, K. J.
(1991)
J. Bacteriol.
173,
6686-6693 2.
Sandler, S. J.,
Samra, H. S.,
and Clark, A. J.
(1996)
Genetics
143,
5-13[Abstract]
3.
Kogoma, T.,
Cadwell, G. W.,
Barnard, K. G.,
and Asai, T.
(1996)
J. Bacteriol.
178,
1258-1264 4.
Masai, H.,
Asai, T.,
Kubota, Y.,
Arai, K.-I.,
and Kogoma, T.
(1994)
EMBO J.
13,
5338-5345[Medline]
[Order article via Infotrieve]
5.
Kogoma, T.
(1997)
Microbiol. Mol. Biol. Rev.
61,
212-238
[Abstract] 6.
Marians, K. J.
(1984)
CRC Crit. Rev. Biochem.
17,
153-215[Medline]
[Order article via Infotrieve]
7.
Marians, K. J.
(1992)
Annu. Rev. Biochem.
61,
673-719[CrossRef][Medline]
[Order article via Infotrieve]
8.
McGlynn, P.,
Al-Deib, A. A.,
Liu, J.,
Marians, K. J.,
and Lloyd, R. G.
(1997)
J. Mol. Biol.
270,
212-221[CrossRef][Medline]
[Order article via Infotrieve]
9.
Nurse, P.,
Liu, J.,
and Marians, K. J.
(1999)
J. Biol. Chem.
274,
25026-25032 10.
Lee, M. S.,
and Marians, K. J.
(1987)
Proc. Natl. Acad. Sci. U. S. A.
24,
8345-8349
11.
Lasken, R. S.,
and Kornberg, A.
(1988)
J. Biol. Chem.
263,
5512-5518 12.
Kim, S.,
Dallmann, H. G.,
McHenry, C. S.,
and Marians, K. J.
(1996)
Cell
84,
643-650[CrossRef][Medline]
[Order article via Infotrieve]
13.
Ng, J. Y.,
and Marians, K. J.
(1996)
J. Biol. Chem.
271,
15642-15648 14.
Tougu, K.,
Peng, H.,
and Marians, K. J.
(1994)
J. Biol. Chem.
269,
4675-4682 15.
Ng, J. Y.,
and Marians, K. J.
(1996)
J. Biol. Chem.
271,
15649-15655 16.
Liu, J., L.,
Xu,
Sandler, S.,
and Marians, K. J.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
3552-3555 17.
Marians, K. J.
(1995)
Methods Enzymol.
262,
507-521[Medline]
[Order article via Infotrieve]
18.
Liu, J.,
Nurse, P.,
and Marians, K. J.
(1996)
J. Biol. Chem.
271,
15656-15661 19.
Maxam, A.,
and Gilbert, W.
(1980)
Methods Enzymol.
65,
499-460[Medline]
[Order article via Infotrieve]
20.
Low, R. L.,
Shlomai, J.,
and Kornberg, A.
(1982)
J. Biol. Chem.
257,
6242-6250 21.
Lee, M. S.,
and Marians, K. J.
(1989)
J. Biol. Chem.
264,
14531-14542 22.
Greenbaum, J. H.,
and Marians, K. J.
(1984)
J. Biol. Chem.
259,
2594-2601 23.
Shlomai, J.,
and Kornberg, A.
(1980)
J. Biol. Chem.
255,
6789-6793 24.
LeBowitz, J. H.,
and McMacken, R.
(1986)
J. Biol. Chem.
261,
4738-4748 25.
Wickner, S.,
and Hurwitz, J.
(1974)
Proc. Natl. Acad. Sci. U. S. A.
71,
4120-4124 26.
Schekman, R.,
Weiner, J. H.,
Weiner, A.,
and Kornberg, A.
(1975)
J. Biol. Chem.
250,
5859-5865 27.
Arai, K.-I.,
and Kornberg, A.
(1981)
Proc. Natl. Acad. Sci. U. S. A.
78,
69-73 28.
Mok, M.,
and Marians, K. J.
(1987)
J. Biol. Chem.
262,
16644-16654 29.
Minden, J. S.,
and Marians, K. J.
(1985)
J. Biol. Chem.
260,
9316-9325 30.
Kaguni, J. M.,
and Kornberg, A.
(1984)
Cell
38,
183-190[CrossRef][Medline]
[Order article via Infotrieve]
31.
Learn, B. A.,
Um, S.-J.,
Huang, L.,
and McMacken, R.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
1154-1159 32.
Bouche, J.-P.,
Zechel, K.,
and Kornberg, A.
(1975)
J. Biol. Chem.
250,
5995-6001 33.
Wickner, S.
(1977)
Proc. Natl. Acad. Sci. U. S. A.
74,
2815-2819 34.
Smith, G. R.
(1988)
Microbiol. Rev.
52,
1-28 35.
Szostak, J. W.,
Orr-Weaver, T. L.,
Rothstein, R. J.,
and Stahl, F. W.
(1981)
Cell
33,
25-35
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