From the Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802
Received for publication, January 29, 2001, and in revised form, April 16, 2001
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ABSTRACT |
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The bacteriophage T4 59 protein (gp59) plays a
vital role in recombination and replication by promoting the assembly
of the gene 41 helicase (gp41) onto DNA, thus enabling replication as well as strand exchange in recombination. Loading of the helicase onto
gp32 (the T4 single strand binding protein)-coated single-stranded DNA
requires gp59 to remove gp32 and replace it with gp41. Cross-linking studies between gp32 and gp59 reveal an interaction between Cys-166 of
gp32 and Cys-42 of gp59. Since Cys-166 lies in the DNA binding core
domain of gp32, this interaction may affect the association of gp32
with DNA. In the presence of gp32 or DNA, gp59 is capable of forming a
multimer consisting of at least five gp59 subunits. Kinetics studies
suggest that gp59 and gp41 exist in a one-to-one ratio, predicting that
gp59 is capable of forming a hexamer (Raney, K. D., Carver,
T. E., and Benkovic, S. J. (1996) J. Biol.
Chem. 271, 14074-14081). The C-terminal A-domain of gp32 is
needed for gp59 oligomer formation. Cross-linking has established that
gp59 can interact with gp32-A (a truncated form of gp32 lacking the A-domain) but cannot form higher species. The results support a model
in which gp59 binds to gp32 on a replication fork, destabilizing the
gp32-single-stranded DNA interaction concomitant with the oligomerization of gp59 that results in a switching of gp41 for gp32 at
the replication fork.
Assembly of the gp41 replicative helicase at the bacteriophage T4
DNA replication fork requires the displacement of single-strand DNA-binding proteins (gp32)1
coating the lagging strand. The helicase assembly protein, gp59, is
required to effectively load the helicase under these conditions, thus
assuming a critical role in bacteriophage T4 DNA replication as well as
recombination (1-3). Mutations in gene 59 result in arrested DNA
synthesis and reduced phage burst size (1), indicating that the gene
product is essential for recombination-dependent DNA
replication (which occurs in the late stages of Escherichia coli T4 infection). Since gp32 is also involved in early,
origin-initiated DNA replication, it is likely that gp59 also
facilitates replication in the early stages of T4 infection.
Furthermore, gp59 mutants exhibit recombination deficiency, UV
sensitivity, and sensitivity to chemical mutagens, suggesting a role of
gp59 in recombination and recombination-mediated DNA repair (2, 3).
gp59 is a basic (pI = 10.18), 26-kDa protein (4, 5). Hydrodynamic
studies indicate that it exists as a monomer in solution (4, 5). It has
a high affinity for DNA and has been shown to bind duplex DNA,
single-stranded DNA, and forked DNA substrates (4, 6). Moreover, Mueser
et al. (7) demonstrate that gp59 binds with a higher
affinity to a DNA fork than ssDNA. The crystal structure of gp59
reveals that it is composed predominantly of gp59 acts via direct contacts with gp41 and has been shown to interact
with the helicase both on and off DNA (5, 8). gp59 increases the
ATP-hydrolyzing activity of the helicase by accelerating the rate at
which it is loaded onto DNA but may also lower the
Km of the enzyme for ATP (8). In addition to
interacting with gp41, gp59 has also been shown to bind tightly to gp32
in both the presence and absence of DNA (5, 9, 10). Morrical et
al. (11) demonstrate that the C-terminal, acidic domain (A-domain)
of gp32 mediates the interaction between the two proteins. A truncated
form in which the A-domain has been removed (gp32-A) results in the
loss of interaction between gp32 and gp59 (11). Furthermore, gp59
contains distinct sites for gp32 and gp41 binding (8). As such, the
gp59-mediated assembly of gp41 onto gp32-coated DNA most likely
involves a ternary complex between the three proteins.
Although gp59 is a monomer in solution, evidence suggests that it may
exist as an oligomer in a complex with gp41 and gp32. Raney et
al. (12) demonstrate that the enhancement of the
ATP-dependent DNA unwinding ability of gp41 is maximal when
gp59 is present in a 1:1 molar ratio of gp59 monomer to gp41 monomer.
Since the helicase is hexameric under these conditions, gp59 may also
form a hexamer. In E. coli, the DnaB helicase requires a
protein analogous to gp59, DnaC, to promote loading. Cryoelectron
micrograph image reconstitutions indicate that DnaC also forms a
hexameric ring associated with the DnaB ring (13). By analogy, we
predict a similar arrangement of gp59 and gp41.
The actual mechanism of helicase assembly by gp59 has yet to be
elucidated. gp59 has been shown to lower the affinity of gp32 for
ssDNA, which may be a key factor in the removal of gp32 for helicase
loading (9). A number of mechanisms have been proposed to explain this
action of gp59. There could be a conformational change in gp32 upon
binding to gp59 that directly destabilizes the association between gp32
and ssDNA. The interaction between gp59 and gp32 could also lead to a
loss in cooperative binding between gp32 subunits, which in turn would
result in a change in affinity. On the other hand, the observed changes
could be due to an ability of gp59 to alter the structure of the
gp32-coated ssDNA, which may then lead to loss of cooperativity of gp32
subunits or a direct change in affinity for ssDNA (9).
The interaction between gp32 and gp59 was studied via a number of
cross-linking methods to understand the mechanism of gp59-mediated helicase assembly onto gp32-coated DNA. To determine whether the changes in the affinity of gp32 for ssDNA in the presence of gp59 could
be due to a direct interaction between the DNA binding core domain of
gp32 and gp59, this domain was labeled with a cross-linker, and the
interaction between the two proteins was analyzed. Furthermore, the
oligomerization of gp59 in the presence of gp32 and DNA was also
assessed by cross-linking. We have demonstrated that 1) the core domain
of gp32 interacts with gp59, and the site of this interaction was
localized to Cys-166 of gp32 and Cys-42 of gp59, 2) gp59 is capable of
forming at least a pentamer in the presence of gp32, and 3) gp59 can
interact with gp32-A, but the association is weaker than the gp32-gp59
interaction, and no gp59 oligomer formation is observed. These results
support a model in which helicase assembly requires the initial binding
of a gp59 monomer to both the replication fork and gp32 on the lagging
strand. A direct interaction between the core domain of gp32 and gp59
could destabilize the gp32-ssDNA interaction, which when followed by gp59 oligomer formation and binding to the helicase, results in displacement of gp32 from the DNA and loading of gp41 in its place. The
interactions between the core domain and gp59 as well as the interactions between the A-domain and gp59 play an important role in
the removal of gp32 from ssDNA.
Cloning and Purification of gp59--
The 59 gene was isolated
from bacteriophage T4 genomic DNA (Sigma) by polymerase chain reaction
amplification using the following primers: A,
5'-GCGGAATTCCTAGATGATTAAACTCCGC; B,
5'-GCGGAATTCGCTCTTCCGCAATACTTGCAAGATTTCAC. The product was
digested with BfaI and SapI and ligated into a custom IMPACT (New England Biolabs) vector placed under the
control of a T7 promoter (14) digested with NdeI and
SapI. This vector places a self-cleaving intein and chitin
binding domain at the C terminus of gp59 and allows affinity
purification on a chitin column followed by cleavage of the intein with
dithiothreitol (DTT) to yield the wild-type gp59. BL21(DE3) cells
containing the construct were grown to an
A600 of 0.5 and induced for 18 h at
30 °C with 0.1 mM
isopropyl-1-thio- Labeling of gp32 with a Trifunctional Cross-linker and
Photocross-linking to gp59--
gp32 was purified as described
previously (15). The synthesis of the trifunctional cross-linker (Fig.
1A) was described by Alley et al. (16). gp32 (25 µM) was dialyzed into buffer B to remove free thiols, and
the trifunctional cross-linker was added in 0.1 volume DMF (250 µM final). This mixture was incubated at 4 °C for
12 h in the dark and subjected to a 1-ml Sephadex G-25 column to
remove excess label. Labeled gp32 (500 nM final) was
incubated with 500 nM gp59 (in buffer B) in the presence or absence of 5 nM circular M13mp18 ssDNA (Bayou Biolabs) in a
total reaction volume of 20 µl. Photocross-linking was initiated by exposure to a 302-nm lamp with 1800 mW/cm2 output at a
distance of 15 cm for 7 min. The reaction was quenched with 5 µl of
buffer C (150 mM Tris, pH 6.8, 4% sodium dodecyl sulfate,
0.1% bromphenol blue, 30% glycerol, 20 mM
N-ethylmaleimide, and 20% DMF) for 10 min at 25 °C. If
reduction of the cross-linker was desired, 2 µl of a 1 M
DTT solution were added to the samples.
The products were subjected to SDS-PAGE (8% gel) and the gel-blotted
onto a nitrocellulose membrane (Micron Separations). The membrane was
blocked for 45 min with 3% bovine serum albumin in 20 mM
Tris, pH 7.6, 140 mM NaCl, and 0.1% Tween 20 (TBST), probed for 60 min with a 1:5000 dilution of streptavidin-horseradish peroxidase conjugate (SA-HRP, Life Technologies) in 3% bovine serum
albumin in TBST, washed twice for 10 min each with TBST, and developed
with a luminol/hydrogen peroxide mixture (Pierce). Chemiluminescence
was detected with BioMax film (Eastman Kodak Co.).
Activity Assay for gp32--
The activity of labeled and
unlabeled gp32 was measured by its ability to inhibit the ATPase
activity of gp41 by preventing it from binding to gp32-coated ssDNA in
the absence of gp59. The rate of ATP hydrolysis at 37 °C was
measured as previously described (17). The reaction mixture consisted
of 60 µl of 5× complex buffer (100 mM Tris, pH 7.5, 750 mM potassium acetate, 50 mM magnesium acetate),
18 µl of 50 mM phosphoenolpyruvate, 12 µl of 5 mM NADH, 6 µl of a mixture of pyruvate kinase (740 units/ml) and lactate dehydrogenase (1030 units/ml) enzymes (Sigma),
and 58 µl of H2O. To this solution 3.6 µl of M13mp18
ssDNA (560 ng/µl) and 55 µl of gp32 (27 µM) were
added, and the solution was incubated at 37 °C for 5 min. The
background ATPase activity was measured optically at 340 nm after the
addition of 60 µl of 10 mM ATP (warmed to 37 °C).
ATPase activity was then monitored after the addition of 15 µl of
gp41 (8 µM). After 5 min, 10 µl of gp59 (12 µM) was added, and the rate of ATP hydrolysis was measured.
The ATPase activity of gp41 was measured in the absence of DNA and
gp32. The background rate of gp59 ATPase activity was similarly measured in the absence of gp41.
Mapping of the Labeled Cysteine in gp32--
To label all of the
cysteine residues in gp32, 15 µl of a 27 µM protein
solution was denatured by boiling for 5 min, and 20 molar eq of
N-[6-(7-amino-4-methylcoumarin-3-acetamido)hexyl]-3'-(2'-pyridyldithio)propionamide (AMCA-HPDP, Fig. 1F, Pierce) was added in 0.1 volume DMF.
The protein was labeled for 12 h at 37 °C, and excess label was
removed with a 1-ml Sephadex G-25 column. The native protein (15 µl
of a 27 µM solution) was labeled with 10 eq of AMCA-HPDP
for 12 h at 4 °C to label only the solvent-exposed cysteine
residue(s). Excess label was removed as described above.
Cyanogen bromide (CNBr, Aldrich) was prepared by dissolving a
medium-sized crystal in 1 ml of 88% formic acid (J. T. Baker Inc.). The CNBr solution (100 µl) was added to both the denatured and
native-labeled proteins. The cleavage was allowed to proceed for
14 h at 25 °C in the dark. The resultant mixture was dried in a
speed vac and re-dissolved in 10 µl of H2O, and 5 µl of buffer C was added. The peptides were separated by SDS-PAGE
using a 10-20% Tris-Tricine gel (Bio-Rad) and visualized with a
Fluor-S Multiimager (Bio-Rad).
Label Transfer between gp32 and gp59--
gp32 (25 µM) was dialyzed into buffer A and incubated with
N-[6-(biotinamido)hexyl]-3'-(2'-pyridyldithio)propionamide(biotin)-HPDP, Fig. 1E, Pierce) in 0.1 volume DMF (250 µM
final) for 12 h at 4 °C. Excess label was removed with a 1-ml
Sephadex G-25 column. Labeled gp32 and unlabeled gp59 were mixed to a
final concentration of 1 µM each in a volume of 20 µl
for times of 0, 5, 10, and 15 min. The label transfer was quenched by
the addition of 5 µl of buffer C. The samples were analyzed by a
Western blot procedure as described in the previous section.
To map the site of label transfer between gp32 and gp59, 25 µM gp32 was labeled with AMCA-HPDP under the conditions
described above, and excess label was removed by Sephadex G-25. To this solution (200 µl) was added 200 µl of 25 µM gp59 in
buffer A. The reaction was quenched after 60 min by the addition of
N-ethylmaleimide in 0.1 volume of DMF (5 mM
final). A 10-µl aliquot of the label transfer reaction as well as 10 µl of the labeled gp32 was subjected to SDS-PAGE (8% gel) to assess
label transfer. The gel was placed on a transilluminator to visualize
the AMCA probe.
The product of the label transfer reaction was digested with 50 µg of
trypsin (L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-treated, from bovine pancreas, U. S. Biochemical Corp.) in 100 mM Tris, pH 8.5 at 37 °C for 12 h. This mixture was
separated by HPLC as described previously (16). The labeled peptide was selectively isolated by collecting eluent that absorbed at 345 nm (the
absorbance maximum of AMCA). The peptide was analyzed by reflector mode
MALDI mass spectrometry using an Thiol-Thiol Cross-linking--
Dibromobimane (DBB, Fig. 1B,
Molecular Probes) and 1,6-bis-maleimidohexane (BMH, Fig. 1C,
Pierce) were each dissolved in DMF to make 1.5 mM stock
solutions. gp32 was dialyzed into buffer A and labeled with 30 equivalents of either BMH or DBB for 60 min. Excess cross-linker was
removed by subjecting the mixture to a 1 ml of Sephadex G-25 column.
Labeled gp32 was mixed with gp59 to a final concentration of 5 µM each in a volume of 20 µl either in the absence or
presence of DNA. M13mp18 ssDNA was used in the reaction mixtures
containing DNA at a concentration of 5 nM. The reaction was
allowed to proceed for 10 min before quenching with DTT (10 mM final). The samples were denatured in 5 µl of buffer B
and subjected to SDS-PAGE. The gel was stained with Gel Code Blue (Pierce).
A similar procedure was conducted to verify that the putative
cross-link observed was an actual cross-link between gp32 and gp59.
1,4-Di-[3'-(2'-pyridyldithio)propionamido]butane (DPDPB, Fig.
1D, Pierce), a cleavable thiol-reactive cross-linker was used instead of DBB or BMH. The cross-link was visualized by negatively staining the gel with a copper stain (Bio-Rad). The cross-link band was
excised and placed in a 1% SDS solution containing 50 mM
DTT for 12 h at 25 °C. The eluted proteins were separated by SDS-PAGE and stained with Gel Code Blue.
To visualize the gp59 oligomers, gp32 and gp59 were mixed to a final
concentration of 5 µM each in a volume of 20 µl.
Cross-linking was initiated by the addition of 1 µl of 1.5 mM DBB or BMH. After 10 min the reaction was quenched with
10 mM DTT. Cross-linking studies using gp32 lacking the
A-domain (gp32-A, obtained as a generous gift from Dr. David Giedroc)
were performed under the same conditions, except that the reaction was
quenched after 30 min.
Labeling of gp32 with a Trifunctional Cross-linker and
Cross-linking to gp59--
The crystal structure of gp32 indicates
that only one of its four cysteine residues, Cys-166, is free and
exposed to solvent (18). The other three cysteine residues are involved
in ligation of a zinc ion (18). A 5,5'-dithio-bis(2-nitrobenzoic acid)
(DTNB) assay (19) confirmed the presence of a single free cysteine residue (data not shown). To ensure that we labeled this cysteine (Cys-166) and not one of the three cysteines involved in zinc ligation
in the cross-linking experiments described below, we mapped the site of
thiol modification. gp32 was labeled with AMCA-HPDP either under
denaturing or native conditions. The labeled proteins were then
digested with CNBr and subjected to SDS-PAGE, and the fluorescently
labeled peptides were identified with a Fluor-S Multiimager. If any of
the cysteine residues involved in zinc ligation had been labeled, the
expected CNBr fragment would be ~17 kDa, whereas modification at
Cys-166 would have produced a fragment of 7 kDa. Fragmentation of the
labeled, denatured protein, in which all cysteine residues should be
labeled, yielded two fluorescently labeled peptides, 17 and 7 kDa, when
separated by SDS-PAGE (Fig. 2A). Labeling of the
native protein followed by CNBr digestion and separation by SDS-PAGE
showed only one fluorescently labeled peptide with a molecular mass of
7 kDa, indicating that only Cys-166 was labeled (Fig.
2A).
We specifically labeled this cysteine residue with the trifunctional
cross-linker shown in Fig. 1A
(16). To ensure that the labeled protein was still active, an ATPase
activity of gp41 assay in the presence of gp32 was conducted. When
mixed with gp32-coated ssDNA, gp41 does not hydrolyze ATP due to its
inability to bind DNA. However, when gp59 is added, gp41 is loaded onto
DNA and subsequently hydrolyzes ATP. This is demonstrated by the gp41 ATPase activity summarized in Table I,
indicating that both the labeled and unlabeled proteins can bind to
ssDNA as well as interact with gp59. gp59 appears to increase the
ATPase activity of gp41 about 4-fold.
Our trifunctional cross-linker contains a thiol-reactive group, a
photoactivatable, aryl azide, a biotin affinity handle, and a cleavable
disulfide. This reagent allows detection of a cross-link by virtue of
the biotin group. The cross-linked products can be subjected to
SDS-PAGE, transferred to a nitrocellulose membrane, and detected with
streptavidin linked to horseradish peroxidase (SA-HRP) in a Western
blot procedure (16). Under nonreducing conditions, the cross-linked
product is visualized by a shift in mobility on the gel corresponding
to the sum of the molecular weight of the labeled protein plus that of
the cross-linked target protein. Under reducing conditions, however,
the biotin label will be transferred to the target protein after the
cleavage of the disulfide bond in the cross-linker. In this case, the
cross-link will no longer be visible by Western blot, but a band
corresponding to the molecular weight of the target protein will be visible.
The labeled gp32 was incubated with gp59 in the presence and absence of
ssDNA and subjected to ultraviolet light to initiate cross-linking
between gp32 and gp59. No gp32-gp32 cross-linking was seen either on or
off DNA (Fig. 2B). There was
some intramolecular gp32 cross-linking, as observed by the transfer of
biotin to gp32 under reducing conditions (Fig. 2B,
lane 9). When gp32 was mixed with gp59, a band corresponding
to the molecular weight of gp32 plus that of gp59 was visualized. This
indicates that Cys-166 in the core domain of gp32 is in close proximity
to gp59. However, a band corresponding to labeled gp59 was also
observed under these conditions. We did not expect to see any transfer
of the label to the target protein under nonreducing conditions. The
labeling of gp59 was not due to the presence of excess label, since
lane 3 (in which gp32 was denatured with SDS followed by
addition of gp59) shows no labeled gp59. The labeling of gp59 was most
likely due to a disulfide exchange. Since gp32 was linked to the
trifunctional cross-linker via a mixed disulfide, an interacting
protein containing a cysteine residue in close proximity could undergo
a disulfide exchange with the cross-linker, specifically labeling this
cysteine residue of the interacting protein with the probe.
Label Transfer between gp32 and gp59--
To determine if the
observations were due to a specific label transfer, we labeled gp32
with biotin-HPDP (Fig. 1E), a biotin probe that reacts with
a cysteine residue to form a mixed disulfide, and incubated the
conjugate with gp59. The two proteins were incubated over time periods
ranging from 0 to 15 min and then subjected to a Western blot using
SA-HRP as a probe. At the 0 min mark, all of the label was localized to
gp32 (Fig. 3, lane 1). Over time, the label was transferred onto gp59 so that by 15 min most of the
label was removed from gp32. This result indicated that one of the two
cysteine residues in gp59 is close to Cys-166 of gp32. This cysteine
residue may be more basic than Cys-166 of gp32, so the overall
equilibrium favors a complete transfer of label.
Thiol-Thiol Cross-linking between gp32 and gp59--
To confirm
the close proximity between the cysteine residues of the two proteins,
we subjected them to thiol-thiol cross-linking (Fig.
4A). BMH, a homobifunctional
thiol-thiol cross-linker (see Fig. 1C), and DBB, a shorter
reagent with similar reactivity (see Fig. 1B), were
employed. gp32 was labeled with each cross-linker individually and
mixed with gp59 in the presence and absence of M13 ssDNA. The products
of the cross-linking reaction were separated by SDS-PAGE and stained
with Gel Code Blue. No gp32-gp32 cross-links were detected with either
cross-linker.
Incubation of gp32 with gp59 resulted in a species that migrated with a
mobility on SDS-PAGE corresponding to the sum of the molecular weights
of gp32 plus gp59 regardless of which cross-linker was used (Fig.
4A). This indicates that Cys-166 of gp32 and one of the
cysteine residues of gp59 must be very close, since DBB has a very
short spacer region (~6 Å) between the reactive ends. The
cross-links were seen in the presence and absence of DNA.
We then confirmed that the putative cross-link band was indeed a
cross-link between gp32 and gp59. Cross-linking between gp32 and gp59
using DPDPB (Fig. 1D) results in the formation of a mixed disulfide as a means of covalently linking the two proteins. As such,
this cross-link can be cleaved under reducing conditions. The products
of the cross-linking reaction were subjected to SDS-PAGE and visualized
with a negative copper stain. The putative gp32-gp59 cross-link band
was excised, and the proteins extracted from the gel under reducing
conditions and then run on a second gel (Fig. 4B).
Coomassie staining of this gel showed two bands, one corresponding to
the molecular weight of gp32 and the other corresponding to the
molecular weight of gp59, verifying the identity of the cross-link.
Mapping the Interaction between gp32 and gp59--
gp59 contains
two solvent-accessible thiols, Cys-42 and Cys-215 (7). We utilized a
label-transfer experiment to determine which cysteine was interacting
with gp32. Since gp32 labeled with a biotin probe via disulfide results
in almost complete label transfer to gp59, we decided to use a similar
technique to map the site of interaction. gp32 was labeled with the
fluorescent probe AMCA-HPDP (Fig. 1F) and incubated with
gp59. Most of the fluorophore was transferred to gp59 after 30 min as
determined by separation of the proteins by SDS-PAGE followed by
visualization of the fluorescent bands on a transilluminator (data not
shown). The proteins were digested with trypsin and subjected to
reverse phase HPLC. The labeled peptide was selectively collected by
monitoring the absorbance of the fluorophore. The peptide was analyzed
by reflector mode MALDI mass spectrometry to determine the site of label transfer (Fig. 5). A monoisotopic
mass of 1289.58 ([M+H]+) was observed, which corresponds
to a gp59 fragment containing amino acids 39-44, in which Cys-42 was
labeled with the fluorophore. The theoretical molecular weight of the
protonated fragment plus the fluorescent probe is 1289.55.
Oligomerization of gp59 in the Presence of gp32--
It has been
previously suggested that gp59 exists as a higher oligomeric species in
a complex between gp41 and gp32 (12). Raney et al. (12)
demonstrate that the maximal stimulation of DNA unwinding occurred when
gp59 was in a 1:1 ratio with gp41 monomers. Furthermore, the analogous
E. coli protein, DnaC, exists as a hexameric ring when
associated with the DnaB helicase (13). gp59 is a monomer in solution
but may associate to form higher species upon binding to gp32 and DNA.
We subjected gp59 to thiol-thiol cross-linking in the presence and
absence of gp32 (Fig. 6). The proteins
were cross-linked with BMH and separated by SDS-PAGE. In the absence of
gp32, there were no higher gp59 oligomers except for a small amount of
gp59-gp59 dimer (Fig. 6, lane 5). This BMH cross-link is
believed to be initiated as a consequence of nonspecific disulfide
bridge formation between gp59 monomers under nonreducing conditions.
These disulfide-linked dimers may place the free cysteine residues on
each subunit in close proximity, allowing cross-link formation. No
species higher than the dimer is visualized by SDS-PAGE.
In the presence of gp32 both on and off DNA, higher multimers of gp59
were observed (Fig. 6, lanes 7 and 8). The
largest observable species migrated with a molecular weight consistent
with pentamer, although higher oligomer formation may be possible. In
the absence of gp32 but in the presence of ssDNA, gp59 also formed
multimers (Fig. 6, lane 6), indicating that gp59 may bind
ssDNA as a cluster. The most intense cross-link bands were observed
when gp59 was in the presence of both gp32 and ssDNA.
We also subjected gp59 to cross-linking in the presence of gp32-A. An
interaction is observed between gp59 and this truncated form of gp32,
indicating that the two proteins can still interact without the
A-domain (Fig. 7, lanes 5 and
6). The interaction does not appear to be as strong as the
gp32-gp59 association, since the cross-link band was not very intense
and required longer cross-linking times for visualization. No
self-association of gp59 beyond dimer is observed. Cross-linking of
gp59 on DNA in the absence of gp32-A resulted in the formation of
higher oligomeric species (Fig. 7, lane 7). The addition of
gp32-A to gp59 and DNA did not yield more oligomeric species than gp59
alone on DNA, indicating that gp32-A did not promote formation of the
gp59 oligomer.
gp59 is needed to displace gp32 to load the gp41 helicase onto the
replication fork. Formation of a ternary complex between gp59, gp32,
and gp41 may be an important step in loading of the gp41 helicase. In
the absence of gp32, gp59 has been shown to bind preferentially to a
replication fork over ssDNA or duplex DNA alone (6). gp59 and gp32 have
also been shown to co-occupy the same piece of ssDNA by contacting each
other as well as the DNA (9). Taken together, this suggests that an
initial step in the loading of the helicase involves the simultaneous
binding of gp59 to the replication fork and gp32. After formation of
this complex, gp41 binding and subsequent loading would follow. We have
characterized the interactions between gp59 and gp32 by cross-linking and have demonstrated that 1) the DNA-binding core domain of gp32 interacts with gp59, and this site of this interaction has been mapped
to Cys-166 of gp32 and Cys-42 of gp59; 2) gp59 is capable of forming at
least a pentamer in the presence of gp32 and DNA, and 3) gp59 can
interact with gp32-A, although weakly, and no oligomer formation is observed.
Interaction between gp32 and gp59--
Our cross-linking and
mapping experiments reveal an interaction between Cys-42 of gp59 and
Cys-166 of gp32. Based on our results and the crystal structures of
gp59 and gp32, we have proposed a model of gp32-gp59 association on DNA
(Fig. 8). The gp59 protein has been shown
to be a two-domain helical protein with an N-terminal domain that has
significant structural similarity to the DNA binding domain of
eukaryotic high mobility group family proteins (7). Mueser et
al. (7) describe a model of gp59 bound to fork DNA that showed the
N-terminal domain binding duplex DNA based on its similarity to high
mobility group proteins. In this model, the lagging strand passes
through a narrow groove that lies between the N- and C-terminal
domains, whereas the leading strand binds to the bottom surface of the
C-terminal domain. This model combined with our results positions gp59
on fork DNA such that Cys-42 is in close proximity to the lagging
strand and places gp32 in an orientation juxtaposing gp59 and gp32, as
demonstrated in Fig. 6. The A-domain of gp32 is predicted to interact
with the C-terminal domain of gp59. Our model of the gp32-gp59
interaction requires Cys-166 of gp32 to face the replication fork.
However, the crystal structure of gp32 bound to a poly(dT)
oligonucleotide shows the orientation to be the opposite of our model,
with the cysteine residue facing away from the replication fork if the
protein bound to the lagging strand with this polarity (18). There was
ambiguity, however, in assigning the polarity of the oligonucleotide in
the DNA binding cleft of gp32 (18). Our results indicate that gp32 binds to ssDNA in an orientation opposite that previously described, placing Cys-166 in a position facing the DNA replication fork to allow
binding to gp59.
Previous studies show that the C-terminal A-domain of gp32 is required
for an interaction between gp32 and gp59 (11). Removal of this domain
by proteolysis resulted in loss of interaction between the two proteins
and a failure of gp59 to load the helicase onto gp32-coated ssDNA (11).
However, our results indicate that the core domain of gp32, which
harbors Cys-166, must augment this interaction. Lefebvre et
al. (9) demonstrate that gp59 destabilizes the interaction between
gp32 and ssDNA through either a direct interaction with gp32, an
alteration in the structure of gp32 coated ssDNA, or both (9). Since
the core domain of gp32 is the site of ssDNA binding, our results
suggest that a direct interaction between gp59 and the core domain of
gp32 may be responsible for this destabilization. In work conducted by
Mosig et al. (20), it was shown that position 163 in gp32 is
important in interactions between the single strand binding protein and
a DNA replication protein, Tin. The Tin protein is believed to interact
with gp32 in this region of the core domain and inhibit normal function by disrupting either protein-protein or protein-DNA interactions (20),
suggesting that binding of gp59 to the same site could also alter gp32
function or DNA binding.
The affinity of gp32-A for ssDNA is decreased in the presence of gp59,
indicating that the interaction between the core domain and gp59 may be
independent of the C-terminal sequence, and this interaction is
sufficient to destabilize gp32-ssDNA binding (9). However, gp59 cannot
effectively load the helicase onto ssDNA coated with gp32-A (11),
suggesting that contacts between the A-domain and gp59 are required in
addition to those between the core domain and gp59.
Oligomeric State of gp59 in the Presence of gp32--
Although
gp59 exists predominantly as a monomer in solution, it may form a
higher oligomer to load the gp41 helicase. Studies demonstrate that the
maximal stimulation of the unwinding rate of gp41 by gp59 occurs when
the two proteins are in a one-to-one ratio, suggesting that gp59 is
hexameric under these conditions (12). The DnaC protein in E. coli is functionally similar to gp59 and forms a hexameric ring
that associates with the DnaB hexamer (13). Moreover, gp59 exhibits
cooperativity in binding of ssDNA (4) and forms condensed clusters with
gp32 on ssDNA (9), suggesting that oligomer formation may be an
important requirement for the action of gp59. Our
thiol-thiol-cross-linking studies showed that gp59 oligomerized in the
presence of gp32 as well as DNA. The largest species seen migrated with
a molecular weight consistent with a gp59 pentamer. Based on the
predicted 6:6 stoichiometry of gp59:gp41, we expect that gp59 is
capable of forming hexameric species. Interaction with the gp32-coated DNA or ssDNA most likely induces a conformational change in gp59 that
allows it to self-associate.
Cross-linking experiments between gp32-A and gp59 established that gp59
can still interact with gp32, even when the A-domain is absent. The
association was weak because the cross-link was difficult to detect and
required longer cross-linking times for visualization. gp59 did not
oligomerize under these conditions, indicating that the A-domain is
necessary to induce this association. Most likely, the A-domain is
responsible for most of the contact between gp32 and gp59, and binding
to this region causes a conformational change that allows oligomer
formation and may contribute to the destabilization of gp32-ssDNA binding.
The multimeric gp59 species observed with the thiol-thiol cross-linkers
indicate that both of the cysteine residues in gp59 must be in close
proximity to the gp59-gp59 subunit interface. Since gp32 interacts with
Cys-42 of gp59, it may also be binding at the gp59-gp59 subunit
interface. The stoichiometry of gp32 to gp59 was demonstrated to be one
to one,2 but the oligomeric
state of the complex may be much larger than a heterodimer. At least
two to three gp32 proteins (each with a binding site size of 8 nucleotides) must be released for loading of the helicase (which has a
binding site size of 12-20 nucleotides) (21). Formation of gp59-gp32
heterooligomers larger than dimers may not only facilitate the binding
and loading of the helicase but may also allow the binding and removal
of multiple gp32 proteins from the lagging strand. Replacement by the
helicase and possible departure of gp59 from the replication fork would
represent final events in the loading process that have strong
parallels to the successional steps observed in loading of the gp45
clamp protein by the gp44/62 clamp loader (22, 23).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices and contains
two domains, a C- and an N-terminal domain. Models generated from the
crystal structure of gp59 and its structural similarity to DNA-binding
proteins of the high mobility group family propose that duplex DNA
binds to the N-terminal domain, whereas ssDNA binds primarily to the
C-terminal domain (7).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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-D-galactopyranoside. The cells were
centrifuged at 4,200 × g for 30 min and resuspended in
20 mM Tris, pH 8.0, 500 mM NaCl, 10% glycerol,
0.1 mM EDTA (buffer A). The cells were subsequently lysed
by sonication, clarified by centrifuging at 20,000 × g
for 30 min, and applied to a 10-ml chitin column (New England
Biolabs). The column was washed extensively with 100 column
volumes of buffer A and incubated for 24 h in 100 mM
DTT dissolved in buffer A. The protein was eluted and dialyzed into 20 mM Tris, pH 7.5, 100 mM NaCl, and 10% glycerol
(buffer B).
-cyano-4-hydroxycinnamic acid matrix.
RESULTS
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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Fig. 1.
Structures of cross-linkers and probes
used. A, the trifunctional cross-linker containing a
thiol-reactive 2-thiopyridine mixed disulfide, a photoactivatable aryl
azide, and the affinity probe biotin. B, the short,
homobifunctional, thiol-reactive cross-linker DBB. C, BMH, a
homobifunctional, thiol-reactive cross-linker. D, DPDPB, a
cleavable, thiol-thiol cross-linker. E, biotin-HPDP, a
biotin probe containing a thiol-reactive 2-thiopyridine mixed
disulfide. F, the fluorescent, thiol-reactive probe
AMCA-HPDP.
ATPase activity of gp41 in the presence of labeled and unlabeled
gp32-coated DNA
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Fig. 2.
Photocross-linking reaction between gp32
labeled at Cys-166 with a trifunctional cross-linker and gp59.
A, labeling of the gp32 under denaturing conditions
(lane 1) with AMCA-HPDP followed by CNBr digest, separation
by SDS-PAGE, and identification of the labeled peptide by monitoring
fluorescence showed two fragments, 17 and 7 kDa, corresponding to
complete labeling of all cysteine residues. Labeling of the protein
under native conditions (lane 2) resulted in only one
fluorescent band after CNBr cleavage, indicating that only Cys-166 was
labeled. B, gp32 was labeled with the trifunctional
cross-linker at Cys-166, cross-linked to gp59, subjected to SDS-PAGE,
and blotted onto nitrocellulose, and biotinylated proteins were
detected with SA-HRP. Lanes 1-7 contain the following:
1, gp32 denatured with SDS followed by the addition of gp59
without exposure to light; 2, gp32 not exposed to light;
3, gp32 exposed to light; 4, gp32 mixed with gp59
without exposure to light; 5, gp32 mixed with gp59 with
exposure to light; 6, gp32, DNA, and gp59 not exposed to
light; and 7) gp32, DNA, and gp59 exposed to light. Lanes
8-13 are identical to lanes 2-7 except that DTT was
added after photocross-linking to reduce the disulfide bond in the
cross-linker. The bands corresponding to gp32, gp59, and the gp32-gp59
cross-link (designated gp32·gp59) are shown at the left.
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Fig. 3.
Label transfer between gp32 and gp59.
gp32 was labeled with the thiol-reactive probe biotin-HPDP and mixed
with gp59. The transfer of the label from gp32 to gp59 was measured by
Western blot using SA-HRP as a probe. Lane 1 contains gp32
mixed with gp59 and immediately quenched by the addition of SDS.
Lanes 2-4 contain gp32 incubated with gp59 for 5, 10, and
15 min, respectively.
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Fig. 4.
Thiol-thiol cross-linking between gp32 and
gp59. gp32 was cross-linked to gp59 using thiol-reactive,
homobifunctional cross-linkers. The two proteins were cross-linked with
DBB and BMH (panel A). Lanes 1-7 contain gp32
without cross-linker added (1), gp32 in the presence of
cross-linker (2), gp59 (3), gp32 labeled with DBB
and mixed with gp59 (4), gp32 labeled with DBB and mixed
with gp59 in the presence of DNA (5), gp32 labeled with BMH
and gp59 added (6), and gp32 labeled with BMH plus DNA and
gp59 (7). The proteins were also cross-linked with the
cleavable thiol-thiol cross-linker DPDPB (data not shown). The band
corresponding to the cross-link was excised, and proteins were
extracted, reduced with DTT, and separated by SDS-PAGE (panel
B).
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Fig. 5.
Mapping the site of interaction between gp32
and gp59. The site of label transfer on gp59 was mapped by
digesting the protein with trypsin and analyzing the labeled peptide by
MALDI mass spectrometry. A monoisotopic mass of 1290.58 ([M+H]+) is observed, which is consistent with the
tryptic fragment containing Cys-42 labeled with the fluorophore
(theoretical [M+H]+ = 1290.55).
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Fig. 6.
Oligomerization of gp59. gp59 was
subjected to BMH in the presence of DNA and gp32. The proteins were
separated by SDS-PAGE. Lanes 1-3 contain gp32
(1), gp32 and BMH (2), and gp32, DNA, and BMH
(3). Lanes 4-6 contain gp59 (4), gp59
and BMH (5), and gp59, DNA, and BMH (6). gp32,
gp59, and BMH were mixed in the absence (lane 7) or presence
(lane 8) of DNA.
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Fig. 7.
Interaction between gp32-A and gp59.
gp32-A and gp59 were mixed in the presence of BMH, and the products of
the reaction were separated by SDS-PAGE. The contents of the gel are
gp32-A (lane 1), gp32-A, DNA, and BMH (lane 2),
gp59 (lane 3), gp59 + BMH (lane 4), gp32-A, gp59,
and BMH (lane 5), gp32-A, gp59, DNA, and BMH (lane
6), gp59, DNA, and BMH (lane 7).
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Fig. 8.
Model of the gp32-gp59 complex.
gp59 is predicted to associate with the replication fork such that the
N-terminal domain binds to the duplex and the lagging strand traverses
a groove between the N- and C-terminal domains (7). This
orientation of gp59 on DNA places Cys-42 in close contact with Cys-166
of gp32, which is bound to ssDNA. The A-domain of gp32 (shown in
blue) most likely interacts with the C-terminal domain of
gp59.
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ACKNOWLEDGEMENT |
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We acknowledge Dr. A. Daniel Jones for assistance with the MALDI mass spectrometry analysis and Dr. M. Uljana Mayer for critical reading of this manuscript and invaluable discussions.
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FOOTNOTES |
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* 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.
To whom correspondence should be addressed: Dept. of Chemistry,
414 Wartik Laboratory, University Park, PA 16802. Tel.: 814-865-2882; Fax: 1-814-865-2973; E-mail: sjb1@psu.edu.
Published, JBC Papers in Press, April 17, 2001, DOI 10.1074/jbc.M100783200
2 S. W. Morrical, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: gp32-A, the truncated form of gp32 lacking the A-domain; ssDNA, single-stranded DNA; DTT, dithiothreitol; TBST, Tris-buffered saline with Tween 20; SA-HRP, streptavidin-horseradish peroxidase conjugate; AMCA-HPDP, N-[6-(7-amino-4-methylcoumarin-3-acetamido)hexyl]-3'-(2'-pyridyldithio)propionamide; CNBr, cyanogen bromide; biotin-HPDP, N-[6-(biotinamido)hexyl]-3'-(2'-pyridyldithio)propionamide; DBB, dibromobimane; BMH, 1,6-bismaleimidohexane; DPDPB, 1,4-di-[3'-(2'-pyridyldithio)propionamido]butane; DMF, N,N-dimethylformamide; PAGE, polyacrylamide gel electrophoresis; Tris-Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; HPLC, high performance liquid chromatography; MALDI, matrix-assisted laser desorption ionization.
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