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INTRODUCTION |
The bacteriophage T4 DNA replication complex is composed of eight
proteins that form a highly efficient machine responsible for accurate
DNA synthesis. Central to the replication process is the T4 DNA
polymerase (gene product 43 (gp43)), which catalyzes the incorporation
of nucleotides in the 5' to 3' direction and contains a 3' to 5'
exonuclease proofreading activity (1). Because the polymerase only
synthesizes DNA in the 5' to 3' direction, the lagging strand must be
synthesized discontinuously in stretches of oligonucleotides known as
Okazaki fragments. In the absence of other proteins, the processivity
of the polymerase is limited, and a trimeric sliding clamp (gp45) is
required to enhance processivity (2, 3). The toroidal sliding
clamp circumscribes DNA and is assembled onto DNA by a clamp loader
(gp44/62) (4).
A helicase is required to unwind dsDNA ahead of the polymerase. The
bacteriophage T4 replicative helicase (gp41) forms a hexameric ring
that encircles the lagging strand and unwinds the DNA duplex with a 5'
to 3' polarity by hydrolyzing either ATP or GTP (5). Single-stranded
regions of DNA exposed by the action of the helicase are covered with
single-stranded binding proteins (gp32). gp32 binding prevents
formation of DNA secondary structure and reannealing of the duplex,
thereby enhancing gp43 DNA polymerase activity (6). A helicase assembly
protein (gp59) is required for the initial loading of the helicase onto
single-stranded DNA (ssDNA)1
coated with single-stranded binding proteins (7).
The activity of the helicase is enhanced by binding of a primase (gp61)
(8, 9). The primase synthesizes pentaribonucelotide primers on the
lagging strand that are extended by the polymerase into stretches of
Okazaki fragments. The primers are later removed, and ligases join the
Okazaki fragments to form a continuous complementary strand.
Previously, the T4 replisome has been studied as four basic units: a
primosome formed from the helicase, primase, and helicase loading
protein that is responsible for unwinding of duplex DNA and synthesis
of RNA primers on the lagging strand; a holoenzyme containing the
polymerase coupled to the sliding clamp on the leading strand; a
lagging strand holoenzyme; and a single-stranded DNA-binding protein.
However, it is unlikely that these units function separately; more
likely, the activities of all four are coupled. For instance, Salinas
and Benkovic (10) demonstrated coupling between leading and lagging
strand DNA synthesis and suggested that the two polymerases are
physically linked. It also seems apparent that the movement of the
holoenzyme needs to be coupled with that of the primosome as it unwinds
double-stranded DNA during leading strand synthesis. Studies in similar
systems have demonstrated this type of interaction. The bacteriophage T7 gene 4 helicase/primase protein has been shown to directly interact
with the T7 DNA polymerase (11). Furthermore, the
subunit of the
DNA polymerase III holoenzyme in Escherichia coli contacts
the DnaB helicase, thereby increasing its unwinding activity 10-fold
(12).
Numerous studies have suggested that there is a "functional"
interaction between the holoenzyme and the primosome in the T4 replisome. Cha and Alberts (13) demonstrated that only the helicase was
needed for strand displacement synthesis by the holoenzyme; the
primase and gp32 had no effect on this rate of synthesis. These results
indicated that there was molecular communication between the helicase
and polymerase. Shrock and Alberts (14) later suggested that the
helicase and polymerase interact, through changes in the processivity
of the helicase in a reconstituted replisome. Moreover, Dong et
al. (15) showed that rapid and processive strand displacement
synthesis could be observed with only the helicase and polymerase in
the presence of a macromolecular crowding agent (polyethylene glycol).
Presumably, the crowding agent drives the helicase onto the fork, where
in turn it interacts with the polymerase to give the observed results
(15, 16). An interaction between the polymerase and helicase in the
absence of DNA, however, was not found by analytical
ultracentrifugation (16). This led to the hypothesis that the coupling
between the two proteins might be mediated by formation of a ternary
complex with DNA (16).
All of these studies provide indirect evidence of a physical
interaction between the holoenzyme and the primosome in bacteriophage T4. Potential interactions between the holoenzyme and the primase and
gp59 were not analyzed. Moreover, recent studies by the Drake and Von
Hippel laboratories (16, 17) have indicated that the leading and
lagging strand holoenzymes do not actually form a physical complex. To
further investigate the formation of complexes between the two
holoenzymes and between the holoenzyme and primosome, a number of
cross-linking and fluorescence resonance energy transfer (FRET)
experiments were conducted. These experiments demonstrate that 1) the
polymerase forms a dimer, but only in the presence of DNA or an active
replication fork, 2) a physical interaction exists between the leading
strand holoenzyme and the primosome, mediated primarily through gp59
and gp43, and 3) the polymerase displaces the gp32 single-stranded
binding protein from gp59 upon binding. These results are discussed herein.
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EXPERIMENTAL PROCEDURES |
Cross-linking between gp43 Subunits--
gp43 was purified as
described previously (18). The protein was dialyzed into 20 mM Tris, pH 7.5, 150 mM NaCl, 10% glycerol before being subjected to cross-linking. The homobifunctional, thiol-thiol cross-linker 1,6 bis-maleimidohexane (BMH) was
dissolved in N,N-dimethlyformamide to a
concentration of 10 µM. One µl of this solution was
added to 19 µl of a solution containing 1 µM gp43 in
the absence or presence of 1 µM of a partially
double-stranded DNA substrate (34/62-mer, the sequence of which is as
follows: 5'-ACT CCT TCC GCA CGT AAT TTT TGA CGC ACG TTG T and 5'-ACA
CAG ACG TAC TAT CAT GAC GCC ATC AGA CAA CGT CGT CAA AAA TTA CGT GCG GAA
GGA GT). The reaction was allowed to proceed for 3 min and was quenched
with 50 mM dithiothreitol (DTT) followed by the addition of
5 µl of SDS-PAGE buffer (150 mM Tris, pH 6.8, 4% SDS,
0.1% bromphenol blue, 30% glycerol). The samples were separated by SDS-PAGE, transferred to a nitrocellulose membrane (micron), and subjected to a Western blot using an anti-gp43 antibody. The membrane was blocked for 2 h at 25 °C with 3% bovine serum albumin in
20 mM Tris, pH 7.6, 140 mM NaCl, and 0.1%
Tween 20 (TBST) and then incubated with a 1:3000 dilution of the
anti-gp43 antibody in 3% bovine serum albumin in TBST for 10 h at
4 °C. The membrane was then washed with TBST (twice, 10 min each)
followed by incubation with a 1:5000 dilution of anti-rabbit IgG
antibody linked to horseradish peroxidase (Amersham Biosciences) in 3%
bovine serum albumin in TBST. The membrane was again washed twice for
10 min each with TBST and developed with a luminol/hydrogen peroxide
mixture (Pierce).
This cross-linking procedure was repeated on an active replication
system using minicircle DNA as described in the literature (10). In
short, a 70-base minicircle was annealed to a 109-mer (partially
complementary oligonucleotide providing a 40-base 5' single-stranded
tail that serves as a lagging strand). gp43, gp45, and gp44/62 (at a
final concentration of 0.4 µM) were mixed with the DNA
substrate (final concentration of 0.2 µM) in the presence of ATP (3 mM final). gp41, gp61, and gp59 (1.2 µM final concentration) were then added, and replication
was initiated by the addition of dNTPs (0.8 mM final
concentration), rNTPs (0.8 mM final concentration), and ATP
(3 mM final concentration). After 1 min, BMH was added (to
a final concentration of 5 µM), and the reaction was
allowed to proceed for an additional 5 min. Cross-linking was quenched by the addition of 10 mM DTT, and the products were
subjected to a Western blot as described above.
The site of thiol-thiol cross-linking was mapped using AMCA-TMEA, a
cross-linker containing a fluorescent probe (Fig. 2A), which
was synthesized as previously published (19). gp43 (300 µl of a 30 µM solution) was cross-linked in the presence of the 34/62 partially duplex DNA (3 µl of a 3 mM solution) by
mixing it with 30 µl of a 3 mM solution of AMCA-TMEA for
10 min. The reaction was quenched by the addition of 10 µl 1 M DTT. The DNA was removed from gp43 by applying the
mixture to a mono-Q fast protein liquid chromatography column.
Cross-linked, dimeric gp43 was then separated from monomeric gp43 by a
Superdex-200 HPLC column. The dimeric gp43 was digested with trypsin
and applied to a C-18 column as previously described (20). The
cross-linked peptides were isolated by collecting the fraction that
absorbed at 345 nm (the absorbance of the AMCA probe). This fraction
was dried in a speed vac concentrator and analyzed by
matrix-assisted laser desorption/ionization (MALDI) mass spectrometry
as described previously (19).
Labeling of Proteins with a Trifunctional
Cross-linker--
gp41, gp59, and gp61 were purified as previously
published (19, 21, 22). Before labeling, each protein was dialyzed into
20 mM Tris, pH 7.5, 150 mM NaCl, 10% glycerol.
gp41, gp59, and gp61 each contain one free, solvent-exposed cysteine
residue: Cys-316, Cys-42, and Cys-144, respectively. The trifunctional cross-linker shown in Fig. 3A was dissolved in
N,N-dimethlyformamide to a concentration of 500 µM, and 5 µl of this solution was added to 50 µl of a
5 µM solution of either gp41, gp59, or gp61. The proteins
were labeled for 12 h at 4 °C in the dark to ensure complete labeling of the cysteine residues by the cross-linker. Excess label was
removed by applying the mixture to a 1-ml Sephadex G-25 spin column.
Another form of gp41, gp41 containing a cysteine added to the C
terminus as previously described (19) (gp41c), was also labeled in this manner.
gp43 was labeled with the trifunctional, amine-reactive probe
Sulfo-SBED (Pierce). The protein (500 µl of a 5 µM
solution) was dialyzed into 20 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol and mixed with 50 µl of a 1 mM solution of Sulfo-SBED (dissolved in
N,N-dimethlyformamide). The reaction was allowed
to proceed for 4 h and quenched by the addition of 20 mM Tris, pH 7.5 (final concentration). The protein was then
dialyzed into 20 mM Tris, pH 7.5, 150 mM NaCl,
10% glycerol.
Cross-linking between the Primosome and gp43--
gp41, gp61, or
gp59 labeled with the trifunctional cross-linker (at a final
concentration of 3 µM) was mixed with gp43 (final concentration of 0.5 µM) in the presence of the other two
primosome proteins (3 µM each) and a forked DNA substrate
(0.5 µM) in a final volume of 20 µl. gp43 labeled with
Sulfo-SBED was cross-linked to primosome proteins following the same procedure.
Two forked DNA substrates were used in these experiments. The first was
composed of a 34-mer primer annealed to a 62-mer leading strand (with
the sequence given above) and a partially complementary lagging strand
of 73 nucleotides (with the sequence
5'-T38GGGTGGGAGGGAGTGGGATGATAGTACGTCTGTGT). The second DNA substrate contained a shorter lagging strand region (34/62/36-mer), where the length of the single-stranded lagging strand
was 18 nucleotides (the sequence of the 36-mer was
5'-TGGGTGGGAGGGAGTGGGATGATAGTACGTCTGTGT). Cross-linking
was initiated by exposure to an ultraviolet lamp for 7 min followed by
the addition of 5 µl of SDS-PAGE buffer (20). In the lanes where
reduction of the disulfide was desired, DTT at a final concentration of
100 mM was added. The proteins were immediately separated
by SDS-PAGE, blotted onto a nitrocellulose membrane (Micron
Separations), and probed with streptavidin-horseradish peroxidase
(SA-HRP), as described previously (20).
Labeling of Proteins with Fluorophores--
Accessible cysteine
residues at gp32(Cys-166), gp59(Cys-42), gp41(Cys-316), and
gp61(Cys-144) were labeled with
7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM), Oregon
Green 488 maleimide (OG), or tetramethylrhodamine 5'-maleimide (TMR) as
required. Unlabeled proteins were dialyzed in labeling buffer (25 mM HEPES, pH 7.3, 150 mM NaCl, and 10% glycerol) and labeled with a 5-fold excess of the appropriate fluorescent dye for 1-4 h at 4 °C. The labeled protein was then dialyzed in labeling buffer containing 10 mM
-mercaptoethanol and frozen in aliquots at
70 °C.
Alternatively, the N terminus of gp43 was specifically labeled in a
lower pH buffer using Oregon Green 488 carboxylic acid, succinimidyl
ester, 5-isomer (OGN). Unlabeled gp43 was dialyzed in labeling buffer
at pH 6.8 and then labeled with a 3-fold excess of OGN for 4 h at
4 °C. gp43(OGN) was then dialyzed in labeling buffer at pH 7.5 and
frozen in aliquots at
70 °C. All labeling processes were performed
in less than 30 h to minimize any reduction in wild-type activity.
The activity of the labeled proteins was confirmed using conventional
activity assays. gp59(CPM) and gp41(OG) were analyzed using the ATPase
assay (23), and no significant difference was found in the rate of ATP
hydrolysis from that of the wild-type gp59 and gp41 combination. The
priming activity of gp61(TMR) was tested using the priming assay (22)
or minicircle assay (10). Labeling of gp61 resulted in a 15% loss in
priming activity and a 25% loss in synthesis of lagging strand
products. gp43(OGN) was tested using with the ATPase assay (24) and the
strand displacement assay (25). The addition of gp43(OGN) in the ATPase
assay shut down ATP hydrolysis to 75% that of unlabeled gp43,
suggesting an interaction with gp45 causing the sequestering of the
holoenzyme onto DNA. The strand displacement assay was conducted in a
benchtop format to check the ability of gp43(OGN) to displace a 36-mer from the 34/62/36-mer DNA substrate (26) and extend the 34-mer to a
62-mer. Extension of a 5'-32P]-labeled 34-mer
oligonucleotide results in full-length 62-mer 32P-labeled
products. The ratio of strand-displaced full-length products to
unextended 34-mer was used to analyze the polymerization property of
gp43(OGN). gp43(OGN) displayed 70% strand displacement activity to
that of unlabeled gp43.
Steady-state FRET Experiments--
Steady-state fluorescence
experiments were performed on an ISA FluoroMax-2 spectrofluorometer at
25 °C. Primosome proteins were mixed to a final concentration of 1.2 µM each in the presence of 0.2 µM gp43 and
0.2 µM 32/64/73-mer DNA. CPM was excited at 390 nm, and
FRET between the CPM and OG pair was observed by monitoring donor
quenching and acceptor sensitization over the wavelength range from 430 to 530 nm. Slits were adjusted to between 1 and 3 nm to keep the
spectrum on scale and were then fixed. Excitation of OG at 475 nm
produced a FRET signal between the OG and TMR pair, and emission was
monitored between 500 and 620 nm. The distance between the probes was
determined from the energy transfer values from donor quenching
(Equation 1) according to the Förster equation (27) (Equation 2),
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(Eq. 1)
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(Eq. 2)
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where ET is the transfer efficiency of the
FRET process and IAD and ID are
the fluorescent intensities of the donor in the presence and absence,
respectively, of the acceptor. RO is the
Förster distance at which the transfer efficiency is 50%, and
R is the distance between the donor and acceptor.
RO was calculated as described previously (25) using
Equation 3 (28) and determined to be 48 Å for the gp59(CPM) and
gp43(OGN) pair,
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(Eq. 3)
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where
D is the quantum yield of the donor,
2 is the orientation factor (2/3),
is the refractive index of the medium (1.4), and J is the
overlap integral between the fluorescence spectrum of the donor and the
absorption spectrum of the acceptor.
Stopped Flow Fluorescence--
Stopped flow fluorescence
experiments were performed on an Applied Photophysics SX.18MV stopped
flow reaction analyzer using fluorescence mode at a temperature of
25 °C. Excitation of CPM was performed at 390 nm. Emission of OG was
measured using a 515-nm band pass filter. The changes in fluorescence
were observed upon stopped flow mixing of syringe A (gp59(CPM), gp41,
gp61 (each at a final concentration of 1.2 µM), gp32 (1.4 µM), DNA (32/64/73-mer, 0.2 µM)) and
syringe B (gp43(OGN) (0.2 µM)). Syringe A was initially mixed versus reaction buffer to obtain a background
fluorescence value. Syringe B was then replaced with gp43(OGN) and
mixed versus syringe A to obtain the experimental values. A
minimum of five experimental traces were recorded and averaged on two
separate occasions. The fluorescence data were fit to a double
exponential using the supplied software from the manufacturer.
Simulation of the stopped flow FRET traces were performed using
KinTekSim version 2.3 (29) using the mechanism shown in Fig.
6B to fit the individual rate constants for each step. The
observed rate constants from the stopped flow FRET traces were
optimized and varied to determine the importance of the values of the
individual rates to the fit of the simulation.
 |
RESULTS |
Cross-linking Studies to Analyze gp43-gp43 Interactions--
Using
a two-hybrid approach, Salinas and Benkovic (10) identified a region of
gp43 that was critical for dimerization. This region was composed of
amino acids 400-601 and corresponds to an extension of the finger
domain in the three-dimensional structure of the protein. This region
contains two cysteine residues (Cys-507 and Cys-542) that may be close
to the interface of interaction of the polymerases. As such, BMH, a
thiol-thiol cross-linker, was employed to analyze this potential locus
of interaction between the two proteins. gp43 was mixed with BMH in the
presence or absence of a partially double-stranded piece of DNA
(34/62-mer), and the products were separated by SDS-PAGE and subjected
to a Western blot using an anti-gp43 antibody. No higher molecular
weight species than gp43 were observed in the absence of DNA. However,
when DNA was present, gp43 was cross-linked to a dimer (Fig.
1A, lane
3), indicating that conformational changes induced by DNA
binding allowed dimerization of the gp43 monomers through the
associations of two gp43·DNA complexes.

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Fig. 1.
Cross-linking of gp43 on a primer template
DNA substrate. A, gp43 was cross-linked with the
thiol-thiol cross-linker BMH in the presence or absence of partially
duplex DNA as described under "Experimental Procedures." The
cross-linked species were separated by SDS-PAGE and subjected to a
Western blot using an anti-gp43 antibody. Lane 1 corresponds to gp43 in the absence of cross-linker and DNA. gp43 was
mixed with BMH in the absence (lane 2) or
presence (lane 3) of DNA. B, the
cross-linking experiment was repeated on a rolling minicircle as
replication was occurring. gp43 was mixed with all of the replisome
proteins, dNTPs, rNTPs, and BMH in the absence (lane
1) or presence (lane 2) of minicircle
DNA.
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To ensure that the results of this experiment were physiologically
relevant, a rolling circle experiment was conducted as described
previously (10). All of the replisome proteins were assembled in the
presence of a minicircle DNA, dNTPs, and rNTPs as described under
"Experimental Procedures." After DNA replication had been
initiated, BMH was added to the mixture. The reaction was quenched, and
the products were separated by SDS-PAGE and subjected to Western blot
using an anti-gp43 antibody. Under conditions where active replication
was occurring, a cross-link band corresponding to a gp43 dimer was
observed (Fig. 1B, lane 2).
The site of cross-linking between the gp43 subunits was then mapped.
gp43 was cross-linked with the thiol-thiol cross-linker AMCA-TMEA as
described under "Experimental Procedures" (Fig.
2A). Cross-linked dimeric gp43
was separated from uncross-linked gp43 by size exclusion
chromatography, and the protein was digested with trypsin. The
cross-linked fragment was isolated by reverse phase HPLC by monitoring
the absorbance of the fluorophore, and the identity of the cross-linked
fragment was elucidated by MALDI mass spectrometry as described
previously (19) (Fig. 2B). A mass of 3450.86 was observed,
consistent with the expected mass of 3446.78 for a tryptic fragment
containing Cys-507 cross-linked to a second tryptic fragment containing
Cys-507. Because other possible cysteine-cysteine crosslinks have
masses within this m/z envelope, additional fragments are being
examined (Fig. 2B).

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Fig. 2.
Mapping the site of cross-linking between
gp43·gp43. A, the thiol-thiol cross-linker AMCA-TMEA
contains a fluorescent group that allows detection of cross-linked
peptides after the proteins have been cross-linked and digested with
trypsin. B, MALDI mass spectrometry was used to identify the
cross-linked peptides. A mass of 3450.86 was observed, consistent with
the expected mass of 3446.78 for a tryptic fragment containing Cys-507
cross-linked to a second tryptic fragment containing Cys-507. Other
cross linked peptides may exist within the broad mass peak.
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Interaction between the Primosome and gp43 by
Cross-linking--
Our general methodology for photocross-linking
involves specifically labeling a protein of interest with a
cross-linker at a unique site (typically a cysteine residue), followed
by the addition of the interacting protein for cross-linking. The
primosome proteins, gp41, gp59, and gp61, were specifically labeled
with the trifunctional cross-linker shown in Fig.
3A at Cys-316, Cys-42, and
Cys-144. The trifunctional cross-linker contains a biotin affinity
handle, a photoactivable arylazide, and a cleavable disulfide bond. The
structure of the cross-linker is such that after the labeled protein is
cross-linked to an interacting protein, cleavage of the disulfide bond
results in transfer of the biotin group to the interacting protein. In
these experiments, gp43 was mixed with all of the primosome proteins
(gp41, gp59, and gp61) in the presence of a forked DNA substrate
(either the 34/62/73-mer or 34/62/36-mer), where one of the primosome
proteins was labeled with the trifunctional cross-linker. The samples
were exposed to ultraviolet light to initiate cross-linking, separated
by SDS-PAGE in the presence or absence of DTT, and then subjected to a
Western blot using SA-HRP as a probe.

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Fig. 3.
Cross-linking strategy for detecting
interactions between gp43 and the primosome. A, this
trifunctional, thiol-reactive cross-linker was used to identify
interactions between the primosome and holoenzyme. The cross-linker
also contains a biotin group, a photoactivable arylazide, and a
cleavable disulfide bond. B, cross-linking and FRET
experiments were performed on the forked DNA substrates (34/62/73-mer
or 34/62/36-mer). The lagging strand contained a region of 18 nucleotides that were complimentary to the leading strand, and a 55- or
18-base region that was single-stranded.
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When labeled gp59 was mixed with gp43 and exposed to UV light, a higher
molecular weight band was seen on the Western blot, consistent with a
cross-link between gp43 and gp59 (Fig.
4A, lane 2). The
addition of DTT results in cleavage of the disulfide bond in the
cross-linker and the resultant transfer of the biotin label to gp43
(Fig. 4A, lane 4), thus verifying the
interaction between gp59 and gp43. There was no difference in
cross-linking between assembly of the complex on the 34/62/73 fork
versus the 34/62/36 fork (data not shown). In contrast,
cross-links between gp41 and gp43 or between gp61 and gp43 were not
observed consistently, and when they were observed, only trace amounts
of cross-linking could be seen (data not shown). gp41c (gp41 with a
cysteine added to the C terminus) was also labeled and subjected to
cross-linking with gp43 but did not show any interaction.

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Fig. 4.
gp43·gp59 interaction detected by
cross-linking. A, gp59 was labeled with the
cross-linker shown in Fig. 3A and mixed with gp43. The
proteins were separated by SDS-PAGE after cross-linking, and a Western
blot using SA-HRP was performed. The contents of the blot are as
follows. Lane 1, labeled gp59, gp61, gp41, gp43,
and DNA (32/64/73-mer) without exposure to UV light; lane
2, same components as lane 1 but
exposed to UV light; lanes 3 and 4,
same as lanes 1 and 2, but DTT was
added to reduce the disulfide bond in the cross-linker. B,
gp43 was labeled with the amine-reactive cross-linker Sulfo-SBED and
mixed with the primosome proteins in the presence of the fork DNA
substrate. Cross-links and label transfer were detected as previously
described using a Western blot with SA-HRP. The proteins were mixed in
the absence (lane 1) or presence (lane
2) of UV light. Lanes 3 and
4 were again the same as lanes 1 and
2, but DTT was added. C, a rolling circle
reaction was conducted in which gp59 was labeled with the cross-linker
shown in Fig. 3A and mixed with the replisome proteins in
the presence of minicircle DNA, dNTPs, and rNTPs. After
cross-linking, the products were separated by SDS-PAGE and detected by
a Western blot using SA-HRP. The lanes on the Western blot correspond
to the following. Lane 1, labeled gp59 mixed with
the replisome proteins, dNTPs, rNTPs, and DNA in the absence of UV
light; lane 2, same as lane
1 but exposed to UV light; lanes 3 and
4, same as lanes 1 and 2,
but DTT was added. Note in A, B, and C
(lane 4), there is intraprotein cross-linking of the probes
within the protein that serves as the initial site of attachment.
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Since the labels attached to gp41 and gp61 may not have been in close
proximity to gp43, gp43 was labeled with the trifunctional cross-linker
Sulfo-SBED and mixed with the primosome proteins. Sulfo-SBED is similar
to the trifunctional cross-linker used above, with the exception that
it is amine-reactive instead of thiol-reactive. Due to the large number
of lysine residues on gp43, numerous labels can be placed throughout
the protein, maximizing the probability of capturing an interacting
protein. In the presence of the primosome proteins and fork DNA, no
cross-linking was observed between gp43 and gp41 or gp61. However,
cross-linking between gp43 and gp59 was again observed, since the
biotin label was transferred from gp43 to gp59 upon reduction of the
cross-linker (Fig. 4B, lane 4).
The interaction between gp59 and gp43 was investigated further on an
active rolling minicircle. The replisome was assembled on DNA in the
presence of the minicircle using gp59 labeled with the thiol-reactive
trifunctional cross-linker. After the initiation of replication,
cross-linking was induced by exposure to UV light. The products of the
reaction were subjected to SDS-PAGE and Western blot as described
above. Cross-link bands are observed, and when DTT is added, the biotin
label is transferred to proteins corresponding to the molecular weights
of gp61 and gp43, indicating that interactions between gp59, the
primase, and the polymerase occur during active replication (Fig.
4C, lane 4).
These collective results suggest that the primary protein-protein
interaction between gp43 and the primosome occurs via gp59, whereas
gp41 and gp61 do not interact with gp43, or the interaction is too
transient to capture with this technique. Additionally, the anticipated
contact between the primase and gp59 protein is verified.
Interaction between gp59 and gp43 by FRET--
Interactions
between the primosome and holoenzyme proteins were also explored by
FRET. gp43(OGN) was mixed with either gp59(CPM), gp61(TMR), or
gp41(TMR) in the presence of the other two unlabeled primosome proteins
and the 32/64/73-mer fork substrate. No FRET was observed between
gp43(OG) and gp41(TMR) or gp61(TMR), consistent with the cross-linking
results above that failed to demonstrate an interaction between gp43
and gp41 or gp61. However, FRET was observed between gp59(CPM) and
gp43(OGN), as shown in Fig.
5A. Fluorescence emission was
lost from gp59(CPM) (donor quenching) and gained by gp43(OGN) (acceptor
sensitization). A FRET signal shows an R6
distance dependence on fluorophore separation (see "Experimental Procedures") with an RO value of 48 Å (50% of
the maximum, obtainable signal) for the CPM-OG pair. As such, detection
of FRET-induced spectral changes requires intimate protein-protein contacts. A distance of 44 ± 5 Å was calculated between the FRET pair on gp43 and gp59, underscoring the requirement of intimate protein-protein contacts for FRET detection.

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Fig. 5.
Steady-state FRET experiments detect
replisome interactions. A, an interaction between
gp59(CPM) and gp43(OGN) as found by equilibrium FRET in the presence of
32/64/73 mer. The blue trace corresponds to
gp59(CPM) fluorescence; the pink trace reflects
CPM quenching and OG sensitization due to FRET within a
gp59(CPM)·gp43(OGN)·gp41·gp61·DNA complex. B, the
FRET signal between gp59(CPM) and gp32(OG) is lost upon the addition of
gp43. The blue trace represents CPM quenching and
OG sensitization due to FRET (in an assembled complex of gp59(CPM),
gp32(OG), gp41, gp61, and DNA); the pink trace
corresponds to loss of FRET as gp43 is added.
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Kinetics of gp43 Binding to gp59--
A presteady-state analysis
of the binding to gp43 to the primosome was conducted. gp43(OGN) was
mixed with gp59(CPM) associated with gp41, gp61, and DNA in a stopped
flow fluorimeter, and the change in FRET was monitored over a 2-s time
course (Fig. 6A). The trace
obtained fit best to a double exponential, where the initial binding
step of gp43(OGN) to gp59(CPM) was fluorescently silent. The binding
step was simulated using an approximate
KD with KinTekSim to obtain a
forward rate constant of 300 ± 100 µM
1 s
1 and a reverse rate
constant of 30 ± 10 s
1. The proposed kinetic scheme
for binding of gp43 to the primosome is given in Fig. 6B.
The first observable fluorescent change is a first order process
(changing the concentration of gp43(OGN) relative to gp59(CPM) causes
no change in rate) that corresponds to a conformational change or
orientation change between the two proteins such that the two probes
move away from each other. This is followed by another first order step
that is rate-limiting for the complex assembly.

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Fig. 6.
Presteady-state FRET detects the kinetic
interaction between gp43 and gp59. A, the kinetics of
gp43 binding to gp59 were monitored by time-dependent
gp59(CPM) quenching in the presence of gp43(OGN). B, the
data were fit using KinTekSim to the model shown to obtain rate
constants for the process.
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Displacement of gp32 upon gp43 Binding--
We demonstrated that
the gp32 single-stranded binding protein remains part of the primosome
(bound proximal to gp59) even after gp59 has assembled gp61 and gp41
onto DNA.2 This raises the
question as to whether gp43 binding would displace gp32. A FRET
experiment was conducted such that gp59(CPM) was mixed with gp32(OG) in
the presence of gp41, gp61, and DNA. FRET (quenching of donor emission
and enhancement of acceptor emission) is observed between gp59(CPM) and
gp32(OG) under these conditions (Fig. 5B). However, when
gp43 is added, the FRET signal is lost, indicating that gp32 has been
displaced from gp59. It is not yet clear whether gp43 competes with
gp32 for a binding site on gp59 or whether it induces a conformational
change in the helicase-loading protein, causing a decreased affinity
for gp32.
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DISCUSSION |
Formation of protein complexes is a common feature in biological
processes. In DNA replication in particular, formation of protein
complexes drives DNA synthesis. In the past, the bacteriophage T4 DNA
replication complex has been studied primarily as separate units, where
holoenzyme and primosome were treated as distinct complexes. Moreover,
the holoenzyme on leading strand DNA and that on the lagging strand
have also been considered separate entities. However, since these parts
function together as a whole to synthesize DNA, they must communicate
with each other, through noncovalent physical interaction.
Previously, Salinas and Benkovic (10) demonstrated coordinated leading
and lagging strand synthesis and suggested that coupling of the
polymerases may occur by formation of a gp43 dimer. However, more
recent studies failed to find any stable interaction between the gp43
protein subunits (16, 17). Second, coordinated replication requires
molecular interactions between the holoenzyme and primosome. Efficient
T4 DNA replication proceeds through the unwinding of double-stranded
DNA by the helicase component of the primosome. On the leading strand,
the holoenzyme may travel juxtaposed to the primosome, incorporating
nucleotides into the growing daughter strand. The obvious question then
arises as to whether the holoenzyme and primosome are coupled through
noncovalent interactions. It is difficult to imagine that these two
units function separately, especially in light of studies conducted on
similar systems, which demonstrated this type of contact. For example,
the helicase/primase protein in bacteriophage T7 forms a complex with
the T7 polymerase, as does the replicative helicase and DNA polymerase
III in E. coli (11, 12).
We investigated complex formation between components of the
bacteriophage T4 replisome and showed that 1) gp43 dimerizes in the
presence of DNA and is dimeric in an actively replicating rolling
circle system, 2) a physical interaction between the gp59 in the
primosome and polymerase in the holoenzyme exists, and 3) the
polymerase displaces gp32 from gp59 as part of the primosome upon binding.
Dimerization of gp43--
Salinas and Benkovic (10) proposed that
coupling may be at least in part mediated by an interaction between the
two polymerases. Using a two-hybrid system, they showed that a region
containing amino acids 401-600 was capable of dimerization (10).
Moreover, Alberts and co-workers (30) showed that a gp43 affinity
column retained gp43 when exposed to a clear lysate of T4-infected
E. coli that contained radiolabeled T4 early proteins.
However, more recently, studies aimed at detecting interactions between
gp43 subunits have failed to demonstrate a dimerization. Kadyrov and Drake (17) investigated the quaternary nature of gp43 by size exclusion
chromatography and did not observe any higher order species. Moreover,
Delagoutte and von Hippel (16) studied interactions between gp41 and
gp43 as well as gp43 and gp43 by analytical ultracentrifugation and
also failed to see any complex formation between either of the species.
However, both of these studies were conducted in the absence of DNA.
Since DNA could induce conformational changes in gp43 that stabilized
dimerization, we used a cross-linking approach to study oligomerization
of the protein in the presence of a DNA substrate.
gp43 was found to dimerize in the presence of a partially
double-stranded DNA (34/62-mer), indicating that interaction of the
protein with DNA could induce changes that allowed dimerization. No
dimerization was observed in the absence of DNA. A second experiment was conducted to study association of gp43 subunits during active DNA
replication. gp43 dimerization was seen on a replicating rolling minicircle, indicating that the interaction occurs during the course of
replication and is physiologically relevant.
The site of cross-linking between gp43 subunits was mapped using an
approach described previously (19). The region of interaction, Cys-507
of one subunit to Cys-507 of a second subunit, lies in an antiparallel
coiled-coiled extension of the finger domain. This structure has
previously been suggested to play a role in the interaction of two
polymerases and was shown to be capable of dimerization in a two-hybrid
system (10, 31, 32). A model of the gp43·gp43 interaction in shown in
Fig. 7A. The gp43
three-dimensional structure was created from a homology model of the
bacteriophage RB69 polymerase by submission to SWISS-MODEL (33-35).
The two proteins are identical or chemically similar at 74% of all
amino acid sites. In this model, the protrusion of the extension of the
finger domain is in close proximity to the same structure on a second
subunit, such that Cys-507 of one subunit and Cys-507 of a second
subunit are juxtaposed. It is presumed that this structure forms only in the presence of DNA, although the DNA was omitted in this model to
simplify the diagram. The linked polymerases are shown such that they
are antiparallel. However, the relative orientation of the two subunits
is unknown and is currently being investigated further. Nevertheless,
this physical coupling of the two holoenzyme complexes could ensure the
rapid and efficient transfer of the lagging strand polymerase to the
next primer after completion of an Okazaki fragment.

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Fig. 7.
Representation of macromolecular interactions
in the bacteriophage T4 replisome. A, a model of
gp43·gp43 dimerization is shown, such that the extensions of the
finger domain come in contact. The regions, containing Cys-507, were
determined to be in close proximity. It is assumed that this structure
forms on DNA, although the DNA is omitted to simplify the diagram.
B, the current model of the replisome complex shows a
primosome composed of three hexameric rings (gp41, gp61, and gp59),
where gp59 contacts the polymerase on the leading strand. The leading
strand polymerase subsequently dimerizes with the lagging strand
polymerase.
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Interaction between the Primosome and Holoenzyme--
The
interaction between primosome proteins and the holoenzyme complex was
investigated using cross-linking and fluorescent techniques. This
allowed the study of protein interactions either on DNA or, as in the
case of cross-linking, during active DNA replication. FRET experiments
showed an interaction between gp59 and gp43 in the presence of gp41,
gp61, and DNA. The distance between the fluorescent probes (Cys-42 of
gp59 and the N terminus of gp43) was calculated to be 44 Å, reflective
of the close physical contact. The kinetics for establishing this
contact follow a three-step process involving binding followed by two
first-order steps. The rate-limiting step of ~1 s
1 is
typical of steps that characterize holoenzyme and primosome assembly
(25, 36, 37). No FRET was observed between gp43 and gp41 or gp61.
The interaction between gp59 and gp43 also was detected on a static
fork using a specific trifunctional cross-linker. The experiment was
conducted using two types of forks, one containing a 55-nucleotide
lagging strand (32/64/73-mer) and the other with an 18-nucleotide
(32/64/36-mer) lagging strand. The substrate containing the shorter
lagging strand region is not long enough to support binding of the
polymerase when gp41 and gp61 are present. Since cross-linking is seen
between gp59 and gp43 on both substrates, it is the leading strand
holoenzyme most likely that interacts with gp59. Interaction between
the lagging strand holoenzyme and gp59 may still be possible but will
require further study.
When the cross-linking experiment was repeated in an active replication
system, cross-linking between gp59 and gp43 was again observed,
demonstrating that this interaction occurs during active DNA synthesis.
Cross-links were observed between gp59 and gp61 as well under these
conditions. No interactions between gp61 and gp59 were observed on a
static fork, indicating that conformational changes may occur during
the course of replication that place the label on gp59 closer to the
cross-linking site on gp61. One cannot, however, rule out crosslinking
within interactive replisomes. Cross-linking between gp59 and gp32 was
not observed, nor between gp59 and gp41. In the latter case, the label
on gp59 (at Cys-42) is not in close proximity to gp41 (19). However, it
was previously demonstrated that gp59 and gp32 formed a complex where
Cys-42 of gp59 is in close proximity to gp32 (21). The cross-linking experiment on the rolling circle was engineered such that initiation of
cross-linking did not occur until after a few minutes of DNA synthesis,
thus allowing time for assembly of the replisome. Most likely, the
gp32-gp59 interaction occurs as the primosome is assembled but
disappears once replication has started.
Previous kinetic evidence suggested an interaction between the
primosome proteins, gp41 and gp43. Dong et al. (15)
showed that a minimal complex of gp41, gp43, and DNA can perform strand displacement synthesis at close to physiologic rates in the presence of
polyethylene glycol (15). However, no interaction between gp41 and gp43
(or gp61 and gp43) was detected by either FRET experiments or
cross-linking. Moreover, analytical ultracentrifugation experiments conducted in the von Hippel laboratory also failed to detect any interaction between gp43 and gp41 (16). It is possible that the
interaction between gp41 and gp43 is unstable but is affected by the
addition of polyethylene glycol. More likely, the contact between the
primosome and holoenzyme is mainly mediated through gp59. If any area
of contact between gp43 and gp41 exists, it may be small, resulting in
only transient interactions between the two proteins in the absence of gp59.
FRET studies between gp32(OG) and gp59(CPM) in the presence of gp43,
gp41, gp61, and DNA support the departure of gp32 from gp59 after
primosome assembly and their lack of cross-linking during active
replication. We have demonstrated that gp32, gp59, gp61, and gp41
formed a complex on DNA.2 However, the addition of gp43
results in loss of the FRET signal between gp59(CPM) and gp32(OG),
indicating a loss of close contact between these two proteins.
Presumably, displacement of gp32 is needed for polymerase binding. gp59
has been shown to contact both gp61 and gp41, perhaps having only a
limited area exposed for other protein binding. It is not clear,
however, whether gp32 remains part of the primosome. Previous studies
showed that gp32 and gp61 can interact, implicating this complex in
transfer of the primer synthesized by the primase to the lagging strand
gp43 (22).
The bacteriophage T4 replisome is comprised of eight proteins that form
distinct units within the replication complex. These units, the
primosome, leading and lagging strand holoenzyme, and single-stranded
binding proteins, combine to form the replisome that is responsible for
efficient DNA replication. A summary model of the contacts demonstrated
to date between the elements of the primosome and holoenzyme is shown
in Fig 7B. In this diagram, the primosome is coupled to the
holoenzyme on the leading strand via interactions between gp59 and
gp43. The lagging strand holoenzyme is linked to the leading strand
holoenzyme via contacts in the extension of the finger domain. These
physical linkages allow molecular communication between the primosome
and holoenzyme as well as leading and lagging strand holoenzymes and
may serve to coordinate both leading and lagging strand synthesis as
well as DNA unwinding and polymerase synthesis.