(Received for publication, November 2, 1994; and in revised form, December 16, 1994)
From the
Escherichia coli DNA polymerase III holoenzyme forms a
stable initiation complex with RNA-primed template in the presence of
ATP. To determine the linear arrangement of the holoenzyme subunits
along the primer-template duplex region, we cross-linked holoenzyme to
a series of photo-reactive primers. Site-specific photo-cross-linking
revealed that the ,
, and
subunits formed ATP-dependent
contacts with the primer-template. The
polymerase catalytic
subunit covalently attached to nucleotide positions -3, -9,
and -13 upstream of the primer terminus, with the most efficient
adduct formation occurring at position -9. The
subunit
contacted the primer at positions -13, -18, and -22,
with the strongest
-primer interactions occurring at position
-18. The
subunit predominated in cross-linking at position
-22. Thus, within the initiation complex,
contacts roughly
the first 13 nucleotides upstream of the 3`-primer terminus followed by
at -18 and
at -22, and the
subunit
remains a part of the initiation complex, bridging the
and
subunits.
Analyses of the interaction of photo-activatible
primer-templates with the preinitiation complex proteins (-complex
(
-
-
`-
-
) and
subunit) revealed the
subunit within the preinitiation complex covalently attached to
primer at position -3. However, addition of core DNA polymerase
III to preinitiation complex, fully reconstituting holoenzyme resulted
in replacement of
by
at the primer terminus. These data
indicate that assembly of holoenzyme onto a primer-template can occur
in distinct stages and results in a structural rearrangement during
initiation complex formation.
The multisubunit enzyme DNA polymerase III holoenzyme
(holoenzyme) ()is the major replicative enzyme responsible
for the synthesis of the Escherichia coli chromosome. This
enzyme is comprised of at least 10 different subunits (
,
,
,
,
,
,
`,
,
, and
)
(McHenry and Kornberg, 1977; McHenry and Crow, 1979; McHenry, 1982,
1988; McHenry et al., 1986; Maki and Kornberg, 1988a;
O'Donnell and Studwell, 1990). Due to the tendency of holoenzyme
to dissociate during the purification process, three functionally
distinct subassembly forms, Pol III
, Pol III`, and core Pol
III, have been isolated. The tightly associated
,
, and
subunits comprise the simplest holoenzyme form, core DNA Pol
III. The
and
subunits possess the catalytic sites for the
polymerization and 3`
5` exonuclease activities, respectively,
while the function of the
subunit remains unclear (Slater et
al., 1994). The association of the auxiliary subunits
with core Pol III defines the subassemblies Pol III` (core Pol III plus
the
subunit) and Pol III* (holoenzyme minus the
subunit).
The effects of ATP, SSB, and salt upon DNA polymerase III are
influenced by the presence or absence of the auxiliary subunits. A
distinction between holoenzyme and its simpler subassembly forms is the
ability of holoenzyme to form a stable initiation complex with a primed
template at the expense of ATP hydrolysis. The auxiliary subunits act
to lock the core polymerase onto a primer-template in an ATP (or
dATP)-dependent reaction creating a complex capable of replicating a
natural chromosome in a rapid and highly processive manner (Wickner and
Kornberg, 1973; Wickner, 1976; Johanson and McHenry, 1982; Burgers and
Kornberg, 1982). In the absence of the auxiliary subunits, ATP is
ineffective in inducing the formation of stable complexes (Johanson and
McHenry, 1982) dramatically decreasing processivity. Both the
polymerase and 3`5` exonuclease activities are inhibited by high
salt concentrations and SSB (Fay et al., 1981, 1982; Mok and
Marians, 1987; Griep and McHenry, 1989; Reems et al., 1991) in
the absence of auxiliary subunits. Thus, the auxiliary subunits appear
to reverse these inhibitory effects in the simpler polymerase forms.
In vitro, holoenzyme can be loaded onto the primer-template
in two distinct stages. In the first stage (preinitiation complex
formation), the -complex (
,
,
`,
, and
subunits) directs the
subunit to the initiation site in an
ATP-dependent reaction (Wickner, 1976; O'Donnell, 1987). In the
second stage, an initiation complex is formed by the addition of core
DNA polymerase III to the preinitiation complex creating a complex
capable of rapid and highly processive DNA replication
(O'Donnell, 1987).
The availability of special
photo-activatible probes strategically positioned within a
primer-template provides a means to determine protein-DNA contact
points in a site-specific manner. One such system designed by Gibson
and Benkovic(1987) has been used to identify the molecular interactions
within the DNA polymerase I-primer-template complex and between T4 DNA
polymerase holoenzyme and its primer-template (Catalano et al., 1990; Capson et al., 1991) The T4 replicative
complex, also a multienzyme system, contains the polymerase gene 43 and
its associated ancillary subunits, genes 44/62 and 45. Capson and
colleagues(1991) determined that within the T4 initiation complex, gene
43 contacted position -4 upstream of the 3`-primer terminus
followed by gene 62 at -9 and gene 45 at
-14-20.
In this study we have used the same
nitrophenyl azide probes developed by Benkovic to identify E. coli DNA Pol III holoenzyme contacts with primer-template. Using a
series of seven photo-activatible primers annealed to a derivative of
M13mp19, we found that within the initiation complex, ,
, and
subunits covalently attach to the primer-template in an
ATP-dependent reaction and that a change in subunit-DNA contacts occurs
during the transition from a preinitiation to an initiation complex.
Figure 1:
Chemical steps in the preparation of
phenylazide photo-cross-linking primers. A, oligonucleotide
with trifluoroacetate-protected propylamine deoxyuridine at position
-2. B, oligonucleotide after removal of the
trifluoroacetate-protecting group. C, primer after
derivatization with N-hydroxysuccimidyl-5-azido-nitrobenzoate. D, primer after 3` extension with
[P]ddATP, placing the photo-reactive probe at
position -3.
Figure 2:
Nucleotide sequence and position of the
photo-reactive probe for each of the seven derivatized primers. Seven
different primers containing uniquely positioned photo-reactive aryl
azides were annealed to a derivative of M13 and labeled at the primer
3`-terminus with [P]ddATP using
deoxynucleotidyl transferase as described under ``Materials and
Methods.'' Each of the seven derivatized primer-templates
possesses a 3`-terminal dideoxyadenosine, a 3`-penultimate
ribonucleotide (cytidine), and a single photo-reactive probe. The final
probe position after annealing the derivatized primer to M13 and
radiolabeling the 3` terminus is indicated to the left of each primer.
The photo-reactive probe is positioned either 3, 9, 13, 18, 22, 27, or
46 (-3, -9, -13,
-18, -22, -27, and
-46, respectively) nucleotides upstream from the primer
3` terminus. U* = propylamine-deoxyuridine derivatized
with phenylnitroazide.
In the presence of ATP, the multi-subunit E. coli DNA Pol III holoenzyme (,
,
,
,
,
,
`,
,
, and
subunits) forms a highly stable
initiation complex with a primed template (Wickner and Kornberg, 1973;
Wickner, 1976; Burgers and Kornberg, 1982a, 1982b; Johanson and
McHenry, 1980, 1982). To identify the subunit-DNA interactions that
occur within the initiation complex, we mapped the linear arrangement
of the holoenzyme subunits along the DNA helix using site-specific
photo-cross-linking.
Our previous work from DNase I footprint
studies indicated that holoenzyme protects 30 nucleotides of
primer (Reems and McHenry, 1994). Based on the footprint data, we
synthesized seven different primers with a single photo-reactive aryl
azide group uniquely placed within the holoenzyme binding region (Fig. 2), adapting the synthetic methods originally developed by
Gibson and Benkovic(1987) to synthesize primers containing uniquely
positioned photo-affinity labels. This method involved the introduction
of a trifluoroacetate-protected propylamine to deoxyuridine, synthesis
of protected phosphoramidite, and insertion of the
trifluoroacetate-protected propylamine deoxyuridine phosphoramidite
into an oligonucleotide chain. Standard deblocking procedures removed
the trifluoroacetate protecting group from the propylamine which was
then derivatized with the photo-reactive agent N-hydroxy-succinimidyl-5-azido-nitrobenzoate in preparation
for the photo-cross-linking studies (Fig. 1). Each of the
primers was complementary to single-stranded circular bacteriophage
M13mp19.
To map the linear arrangement of the holoenzyme subunits along the DNA helix, we photo-irradiated holoenzyme-primer-template complexes bearing a photo-reactive group within the primer strand. Seven different derivatized primers were separately annealed to M13mp19, and the 3` terminus of each primer was radiolabeled. The photo-reactive probe position for each of the seven primers was 3, 9, 13, 18, 22, 27, or 46 nucleotides upstream from the primer 3` terminus (Fig. 2). By changing the position of the photo-reactive probe relative to the 3` terminus of the primer, photolysis of holoenzyme primer-template complexes permitted the identification of different subunit-DNA contacts along the primer-template in a linear fashion.
Photo-products were resolved on a 5-20% SDS-polyacrylamide gel
with no stacking gel. Each of the gels showed two radioactive bands
corresponding to primer-template that did not enter the gel (top of gel) or to cross-linked SSB (bottom of gel) (Fig. 3). Holoenzyme subunits that were cross-linked to the
primer were determined by the gel mobility of the cross-linked species
relative to free protein. Cross-linked photo-products exhibited gel
mobilities slightly slower than their non-cross-linked counterparts due
to the molecular weight of the covalently attached primer. In addition
to non-cross-linked molecular weight markers, the individually
cross-linked proteins and SSB were used as markers.
Figure 3: Subunit-DNA adducts formed after cross-linking E. coli DNA polymerase III holoenzyme to six different photo-reactive primer-templates. Reaction conditions were as described under ``Materials and Methods.'' Initiation complexes were formed using 3 units of holoenzyme/fmol of primer-template and 1.0 mM ATP prior to photolysis. A control reaction for each probe position was performed by photo-irradiating each primer-template in the absence of holoenzyme. The position of the photo-reactive group relative to the 3` terminus of the primer is indicated above each pair of lanes.
To monitor the
3`5` exonuclease activity of holoenzyme, we compared the
enzyme's excisional activities using primers with or without a
photo-reactive group. When holoenzyme was incubated with
primer-template in the absence of all four dNTPs, the enzyme fully
degraded derivatized primers in 90 s and non-derivatized primers in 45
s. Even though there was a 2-fold reduction in exonuclease activity
using derivatized primers (data not shown), the primer was fully
degraded within the time frame of our photo-cross-linking experiments.
Thus, the aryl azide did not significantly interfere with
holoenzyme's synthetic or proofreading activities. However, to
overcome the technical difficulties for our experiments presented by
holoenzyme's ability to rapidly degrade DNA primers, we made two
alterations in the primer to reduce its susceptibility to the
enzyme's 3`
5` exonuclease activity. First, we rendered the
primer exonuclease-resistant by positioning a penultimate
ribonucleotide and a terminal dideoxyribonucleotide residue at the 3`
terminus. These modifications reduced the exonuclease activity to
stabilize holoenzyme-DNA primer-template interactions nearly 1000-fold
(Griep et al., 1990). Second, we radiolabeled the 3` rather
than the 5` terminus of the primer, ensuring that the 3`
5`
exonuclease activity would cleave the radiolabel in the form of a
mononucleotide, thus eliminating ambiguity in cross-link assignments
since only photo-products that occurred prior to excision would be
detectable. These alterations made it possible for us to detect
photo-reactive products due only to the formation of static
holoenzyme-primer-template complexes, which is necessary in determining
the linear alignment of holoenzyme subunits relative to the
primer-template.
We then cross-linked holoenzyme to the
remaining derivatized primers (Fig. 3). Primer-template with the
aryl azide positioned at -9 resulted in one highly radioactive
band that corresponded to cross-linked . Photo-irradiation of
derivatized primer-template, aryl azide positioned at -13,
resulted in faint photo-products that corresponded to the gel positions
expected for cross-linked
and
. Holoenzyme-primer-template
complexes photo-irradiated with the aryl azide positioned at -18
resulted in only one highly radioactive photo-product whose gel
migration corresponded to cross-linked
. At position -22,
the photo-products migrated to positions expected for cross-linked
and
, and at position -27, there were no apparent
photo-products. Cross-linking at position -46 was also not
detected (data not shown).
SSB photo-cross-linked to all of the annealed primers used in these experiments. All complexes were isolated by gel filtration to eliminate free primers, but we cannot absolutely rule out the possibility that trace quantities dissociated after isolation. Presumably, SSB can interact with the duplex region, albeit weakly. Previously, we observed that SSB did not protect annealed primers from DNase I digestion. When interpreting these results it should be kept in mind that one cannot compare the cross-linking intensity for two separate proteins to estimate their binding strength. Cross-linking efficiency varies markedly between proteins and is determined by the proximity of reactive amino acids to the photoreactive group. Capson and co-workers (et al., 1991) made similar observations with the T4 gene 32 protein cross-linking to annealed photoreactive primers.
Several subassembly forms of DNA
polymerase III are known to interact with primer-template in an
ATP-independent mode. However, maximum synthetic and 3`5`
exonuclease activities for holoenzyme are achieved only when ATP is
present. Analysis of ATP requirements to confirm that the cross-linked
products were due to holoenzyme and not subassembly forms revealed that
cross-linking of the
,
, and
subunits to primer
required ATP (data not shown).
Photo-cross-linking the components of the preinitiation
complex to primer-template in the presence of both ATP and SSB resulted
in the covalent attachment of the ,
, and
(or
`)
subunits (Fig. 4A, lane 1; in the system used,
the
and
` subunits cannot be distinguished). By eliminating
ATP from the reaction, both the
and
subunit interactions
with the primer-template were precluded (Fig. 4A,
compare lanes 1 and 5), whereas
(or
`)
primer-template interactions remained essentially unchanged (Fig. 4A, compare lanes 1 and 5). The
radioactive band, designated
, between the
and
subunits was apparent even in the absence of any enzyme (Fig. 4A, lanes 4 and 8), indicating
that this photo-product was not due to cross-linked holoenzyme
subunits.
Figure 4:
Subunit-DNA adducts formed after
photo-cross-linking components of the preinitiation complex ( plus
-complex subunits) to primer-template in the presence or absence
of SSB. Reaction conditions were described under ``Materials and
Methods.'' The primer was radiolabeled at the 5` terminus. The
photo-reactive probe was positioned 2 nucleotides upstream of the 3`
terminus. Enzyme-primer-template complexes were formed with 10 units of
-complex, and/or 70 units of
subunit/fmol of primer-template
in the presence or absence of 500 µM ATP. A,
photo-products obtained after irradiating enzyme-primer-template in the
presence of 2.0 µg SSB/nmol of nucleotide. B,
photo-products obtained after irradiating enzyme-primer-template in the
absence of SSB.
Photo-cross-linking of the -complex in the absence of
the
subunit still resulted in
and
(or
`)
interactions with the primer. In the presence of ATP,
within the
-complex cross-linked to the primer and did not require the
addition of the
subunit (Fig. 4A, lane
2). In the absence of ATP,
interactions with the primer were
almost eliminated (Fig. 4A, lane 6).
Photo-cross-linking of the
subunit without the
-complex did
not result in
-DNA adduct formation (Fig. 4A, lane 3). Further, no apparent
-DNA adducts were
obtained even when using excess
and a primer containing an aryl
azide positioned at -22 (data not shown). Thus,
interactions with primer required both
-complex and ATP (Fig. 4A, compare lane 1 to lanes 4 and 7), results which are consistent with the proposal
that the
-complex transfers the
subunit to primer-template
in an ATP-dependent reaction (Wickner 1976; O'Donnell, 1987).
Surprisingly (or
`)-primer interactions were dramatically different in the absence
or presence of SSB. In the absence of SSB, the amount of
(or
`) that cross-linked to the primer was increased 5-10-fold
(compare Fig. 4, A to B). Further,
(or
`) interactions with the primer-template were essentially the same
with or without ATP (Fig. 4B, compare lanes 9 and 10 to lanes 13 and 14). These
results indicate that SSB competes with the
(or
`) subunit
for binding to the primer-template at a position 2 nucleotides upstream
of the 3` terminus of the primer. Further,
(or
`)
interactions with the primer-template occur in an ATP-independent
fashion.
Figure 5:
Photo-products generated using individual
components of DNA polymerase III holoenzyme initiation complexes.
Reaction conditions were as described under ``Materials and
Methods.'' The primer was radiolabeled at the 5` terminus. The
photo-reactive probe was located 2 nucleotides upstream of the 3`
terminus. Enzyme-primer-template complexes were formed using 3 units of
holoenzyme, 10 units of -complex, 70 units of
, 3 units of
core Pol III, and/or 3 units of Pol III`/fmol of primer-template in the
presence of 500 µM ATP. These additions resulted in
enzyme/primer molar ratios of 6:1, 18:1, and 20:1 for holoenzyme, core
pol III and pol III`, respectively.
and
complex, when
included, were present at enzyme/primer ratios of 18:1 and 86:1,
respectively. Enzyme additions are indicated above each
lane.
Figure 6:
Subunit-DNA adducts occur with
primer-template and not with primer alone. Reaction conditions were as
described under ``Materials and Methods.'' The primer was
radiolabeled at the 5` terminus. The position of the photo-reactive
probe was 9 nucleotides upstream of the 3` terminus.
Enzyme-primer-template complexes were formed using 3 units of
holoenzyme, 10 units of -complex, and 70 units of
subunit/fmol of primer-template in the presence of 500 µM ATP. Photo-products obtained after irradiating enzyme that had
been incubated with either primer alone or
primer-template.
Figure 7: Excess unlabeled primer-template competitively inhibits subunit-DNA adducts with radioactive primer-template. Reaction conditions were as described under ``Materials and Methods.'' Lane 1, excess unlabeled primer-template (P:T) (100 nM), was incubated with radioactive photo-reactive primer-template (4.0 nM) 50 mM HEPES, pH 7.5, 500 µM ATP, 10 mM magnesium acetate, 0.01% (v/v) Nonidet P-40, 80 µg/ml bovine serum albumin, and 2.0 µg SSB/nmol nucleotide (25 µl) for 5 min at 30 °C prior to the addition of subsaturating levels of holoenzyme (0.5 unit/fmol primer-template), incubated for 1 min at 30 °C, and photo-irradiated. Lanes 2 and 3, excess unlabeled single-stranded circular template strands (500 nM) or unannealed primer (500 nM) were added to labeled photo-reactive primer-template (4.0 nM). After 5 min at 30 °C, holoenzyme (0.5 unit/fmol primer-template) was added to the reaction, incubated for 1 min at 30 °C and photo-irradiated. Lane 4, positive control where no competitor was added before holoenzyme-primer-template complexes were formed. Lane 5, negative control where no competitor holoenzyme were added.
The purpose of this study was to map the linear arrangement
of the holoenzyme subunits along the duplex region of the
primer-template at the initiation site. To accomplish this goal, we
used photo-cross-linking to site-specifically identify holoenzyme
subunit contacts relative to a DNA primer. These studies revealed that
the subunit cross-linked to positions -3, -9, and
-13, with the most efficient cross-linking occurring at position
-9. The
subunit cross-linked to positions -13,
-18, and -22, with the strongest
-primer interactions
occurring at position -18. The
subunit was the predominant
protein cross-linked at position -22, while at positions
-27 and -45, essentially no holoenzyme contacts were noted.
Together these results indicate that within the initiation complex,
contacts roughly the first 13 nucleotides upstream of the
3`-primer terminus, followed by
at -18 and
at
-22 (Fig. 8).
Figure 8:
The
linear alignment of E. coli DNA polymerase III holoenzyme
subunits relative to the duplex region of the primer-template at the
initiation site. Schematic representation of the holoenzyme subunit-DNA
contacts defined by site-specific photo-cross-linking. The
subunit, possessing the polymerase catalytic site, covalently attaches
to positions -3, -9, and -13, with the most efficient
cross-linking occurring at position -9. The
subunit
cross-links to positions -13, -18, and -22, with the
strongest
-primer interactions occurring at position -18.
The
subunit is the predominant subunit covalently attached to the
primer at position -22. Thus, within the initiation complex,
contacts roughly the first 13 nucleotides upstream of the
3`-primer terminus followed by
at -18 and
at
-22.
remains part of the initiation complex connecting the
and
subunits.
We previously reported the use of
fluorescence energy transfer to map the position of the subunit
within the initiation complex (Griep and McHenry, 1992). In that study,
a donor-acceptor distance of 65 Å was determined between a donor
fluorophore located at the -3 position of the primer and an
acceptor fluorophore positioned within the
subunit at
Cys
, a distance which positions the
subunit
approximately 19 nucleotides from the donor fluorophore or 22
nucleotides upstream of the 3`-primer terminus. This distance is
consistent with the present results which show that
covalently
attaches to the primer 22 nucleotides upstream of the 3`-primer
terminus. DNase I footprint analysis (Reems and McHenry, 1994) of
holoenzyme binding to the primer-template showed that holoenzyme
contacts
30 nucleotides of primer-template; photo-cross-linking
results indicate that holoenzyme contacts at least 22 nucleotides of
primer. Since DNase I cuts occur at
4-nucleotide intervals and
would be expected to be sterically hindered from cutting immediately
adjacent to a protein on DNA, it is reasonable to find a slightly
larger region defined by DNase I footprint analysis than the site-size
defined by photo-cross-linking.
Interestingly, the T4 DNA polymerase
genes 43, 44/62, and 45, which are analogous to the E. coli DNA polymerase III ,
-complex, and
subunits,
respectively, map to similar positions. Gene 43, which is responsible
for nucleotide incorporation in the T4 DNA polymerase replication
complex, contacts position -4 upstream of the 3`-primer terminus.
Gene 62, which is part of the ATP accessory complex, contacts the
primer at position -9, and gene 45, the T4 processivity factor,
contacts the primer at positions -14 and -20 (Capson et
al., 1991). The similar structural position and order of the T4
replication machine components is consistent with their functional
equivalences, predicting that this order might be conserved among all
replication machines. To extend this prediction to eukaryotic
replication enzymes, one might expect polymerase
to interact with
the primer terminus, a component of 5-protein RFC to interact at an
adjacent position, and the PCNA sliding clamp to reside at the most
distal position in eukaryotic replication complexes. Of course, extra
interactions may be present to accommodate other interactions including
the polymerase-primase complex.
DNase I footprint analysis and
fluorescence energy transfer studies also suggested that subunit
rearrangements occur during formation of holoenzyme-primer-template
complexes (Griep and McHenry, 1992; Reems and McHenry, 1994). We,
therefore, photo-cross-linked partially reconstituted complexes in
order to isolate and analyze intermediate subunit-DNA interactions.
Photo-cross-linking preinitiation complexes to primer-template
indicated that ,
, and
subunits contact the primer at
position -2. However, with the addition of core Pol III or Pol
III` to the preinitiation complex, the
subunit replaces
,
, and
subunits at position -2. These results provide
direct evidence that the binding of core polymerase to preinitiation
complexes induces a subunit rearrangement.
Our map of the linear
alignment of the holoenzyme subunits along the DNA helix provides
structural information central to defining the functional activities of
the polymerase subunits within the initiation complex. Our data
indicating that contacts at least 13 nucleotides of the primer
upstream of the 3`-primer terminus positions this subunit, which
possesses the catalytic site for the polymerization reaction, at the
primer-template junction poised to incorporate dNTPs at the 3`hydroxyl
terminus of the primer. At positions -18 and -22,
respectively,
and
contact the primer. The
subunit,
which confers processivity to the core polymerase (Fay et al., 1981) and functions as a ``sliding clamp'' (Stukenberg et al., 1991), is situated upstream of the
and
subunits, placing the processivity factor approximately two helical
turns upstream from the 3`-primer-terminus assuming a
-form DNA
conformation. The
-complex recognizes the primer-template and
couples ATP hydrolysis to clamp
at the initiation site (Onrust et al., 1991). The positioning of
between
and
suggests a role for
in locking and linking the processivity
and elongation factors. Several laboratories have reported that the
complex functions to load
onto the
primer-template and then dissociates. Our results suggest a
participatory role for the
complex in elongation.