(Received for publication, May 5, 1995)
From the
The bacteriophage T7 gene 4 protein, like a number of helicases,
is believed to function as a hexamer. The amino acid sequence of the T7
gene 4 protein from residue 475 to 491 is conserved in the homologous
proteins of the related phages T3 and SP6. In addition, part of this
region is conserved in DNA helicases such as Escherichia coli DnaB protein and phage T4 gp41. Mutations within this region of
the T7 gene 4 protein can reduce the ability of the protein to form
hexamers. The His
Ala and Asp
Gly mutant proteins show decreases in nucleotide hydrolysis,
single-stranded DNA binding, double-stranded DNA unwinding, and primer
synthesis in proportion to their ability to form hexamers. The mutation
Arg
Ala has little effect on oligomerization, but
nucleotide hydrolysis by this mutant protein is inhibited by
single-stranded DNA, and it has a higher affinity for dTTP, suggesting
that this protein is defective in the protein-protein interactions
required for efficient nucleotide hydrolysis and translocation on
single-stranded DNA. Gene 4 protein can form hexamers in the absence of
a nucleotide, but dTTP increases hexamer formation, as does dTDP, to a
lesser extent, demonstrating that the protein self-association affinity
is influenced by the nucleotide bound. Together, the data demonstrate
that this region of the gene 4 protein is important for the
protein-protein contacts necessary for both hexamer formation and the
interactions between the subunits of the hexamer required for
coordinated nucleotide hydrolysis, translocation on single-stranded
DNA, and unwinding of double-stranded DNA. The fact that the gene 4
proteins form dimers, but not monomers, even while hexamer formation is
severely diminished by some of the mutations, suggests that the
proteins associate in a manner with two separate and distinct
protein-protein interfaces.
Gene 4 of bacteriophage T7 encodes two proteins that provide
helicase and primase activities required for
replication(1, 2, 3, 4, 5, 6) .
Along with the T7 DNA polymerase, a 1:1 complex of T7 gene 5 protein,
and Escherichia coli thioredoxin, these proteins catalyze the
reactions required for leading and lagging strand synthesis during
phage DNA replication(7, 8) . The product of gene 2.5,
a ssDNA()-binding protein, is required for DNA replication
although its specific role is not fully understood(9) .
An internal translation initiation sequence in the gene 4 transcript results in expression of the encoded protein as two colinear forms: the 63-kDa gene 4A protein and the 56-kDa gene 4B protein(1) . The 63-kDa gene 4 protein exhibits helicase activity and, by virtue of an amino-terminal zinc binding motif, catalyzes the template-directed synthesis of tetraribonucleotides that are used as primers by the T7 DNA polymerase(10, 11) . The 56-kDa protein lacks the 63 amino-terminal residues that form the zinc binding motif and consequently catalyzes only helicase activity(12) . Since the 63-kDa protein (primase) can provide both primase and helicase activities, it is both necessary and sufficient for productive infection by T7 phage(13, 14) .
While the gene 4 primase is sufficient for T7 DNA replication, there is considerable evidence that the helicase and primase proteins interact cooperatively. For example, the 56-kDa helicase stimulates template-dependent tetraribonucleotide synthesis by the 63-kDa primase and enhances DNA replication in vivo(14, 15) . Further evidence of this interaction was derived from studies (16) with a gene 4 protein containing a defective nucleotide binding site (NBS). The NBS mutant protein inhibits nucleotide hydrolysis by wild-type gene 4 proteins through direct protein-protein interactions, demonstrating the importance of cooperation between the gene 4 proteins in order to translocate on ssDNA and to catalyze the unwinding of double-stranded DNA. Also, the low level of primer synthesis catalyzed by the NBS mutant primase is increased by wild-type helicase, suggesting that the wild-type protein forms a complex with the mutant primase enabling it to translocate along the template DNA to a primase recognition site(17) .
The precise mechanism of strand separation by a DNA helicase is not known. However, all helicases examined thus far function as multimeric proteins and use the energy of nucleotide hydrolysis to unwind dsDNA (18, 19, 20) . Two forms of helicase multimers have been identified: dimer and hexamer. The E. coli Rep protein is the best characterized example of a dimeric helicase(21) . The reported group of helicases that form hexamers currently includes proteins such as the T7 gene 4 protein(22) , E. coli proteins DnaB(23) , Rho(24) , and RuvB(25) , SV40 large T antigen(26) , and the bacteriophage T4 gp41(18) .
The T7 gene 4 protein is one of a group of bacterial and bacteriophage helicases known as the ``DnaB helicase family'' that share multiple regions of amino acid sequence similarity(27) . Notably, this group includes E. coli DnaB, bacteriophage T4 gp41, and the gene 4 protein of phage T3. Four regions of sequence similarity were identified in the DnaB helicase family; the first two motifs are known to be associated with nucleotide binding, while the roles of the third and fourth motifs are not yet known. We compared the amino acid sequence of the T7 gene 4 protein with its homologs in the closely related phages T3 and SP6. Other than the NBS, the only continuous region of conserved amino acid sequence among these three proteins occurs toward their carboxyl terminus and corresponds to T7 gene 4 residues 475-491 (refer to Table 1). This region of the T7 gene 4 protein overlaps the fourth DnaB helicase family motif, corresponding to T7 gene 4 protein residues 481-500. Interestingly, the phage SP6 gene 4 protein has sequence similarity to the fourth motif of the DnaB family of helicases only within the common overlapping region (corresponding to T7 gene 4 protein residues 481-491).
Since the function of this highly conserved carboxyl-terminal region is not known, we have investigated its role in T7 gene 4 protein through site-directed mutagenesis. We show that mutations within this region affect both the formation of gene 4 protein hexamers and cooperative protein-protein interactions within the hexamer that are required for nucleotide hydrolysis and translocation on ssDNA.
Figure 1:
SDS-PAGE analysis of the purification
of the T7 gene 4A mutant protein R487A. Protein samples were separated
on a 10% SDS-polyacrylamide gel by electrophoresis at 20 V/cm for
approximately 50 min and stained with Coomassie Brilliant Blue. Lanes: 1, fraction I, cleared lysate from induced culture of E. coli HMS 174 (DE3)/pGP4-G64R487A; 2,
fraction II, resuspended polyethylene glycol precipitate; 3,
fraction III, phosphocellulose chromatography pool; 4,
purified gene 4A R487A protein, fraction IV, agarose-hexane-ATP column
pool; 5, purified R487A protein overloaded to demonstrate
purity. Lanes 1-4 contain approximately 1.2 µg of
total protein/lane, and lane 5 contains approximately 5 µg
of protein. The positions of the molecular mass standards (kDa) are
indicated to the left of the
figure.
DNA binding experiments were performed using
the same gel and electrophoresis buffer system described for the native
PAGE. The 10-µl binding reactions contained 40 mM
Tris-HCl, pH 7.0, 50 mM NaCl, 10 mM MgCl,
10 mM DTT, 0.1 µM oligonucleotide, 1 mM
,
-methylene dTTP, and gene 4A protein at various
concentrations. The samples were incubated at room temperature for 5
min, then loaded onto the gel for electrophoresis. The 35-base
oligonucleotide used for ssDNA binding experiments had the sequence:
5`-CAGATGCGCGCCTCCTGGCTTATCGGTGTACTTGG-3` and was end-labeled in a
standard reaction with [
-
P]ATP and T4
polynucleotide kinase; unincorporated label was removed using a
spin-column (Microspin S300, Pharmacia Biotech Inc.). After
electrophoresis, the gels were fixed, stained with Coomassie Brilliant
Blue, and dried. The amount of DNA that co-migrated with the gene 4
protein was determined by PhosphorImager (Molecular Dynamics)
analysis.
UV-mediated cross-linking of gene 4 proteins to
radiolabeled (dT) was performed as described previously
for UV-cross-linking of gene 4 proteins to dTTP(16) . The
10-µl reactions contained 0.2 µM gene 4A protein, 0.01
µM radiolabeled (dT)
, 40 mM Tris-HCl, pH 7.0, 10 mM DTT, 100 mM NaCl, 50
µg/ml bovine serum albumin, and 10% glycerol. The (dT)
was 5`-end labeled with [
-
P]ATP and
T4 polynucleotide kinase. When included in the reactions, MgCl
was at 10 mM, and nucleotides, dTTP or
,
-methylene dTTP were at 2 mM. The reactions were
incubated for 10 min at 30 °C, then placed on ice and exposed to
the UV source (1.0 milliwatts/cm
) for 15 min. The reaction
mixtures were then subjected to SDS-PAGE, and the amount of labeled DNA
bound to the gene 4 protein was determined by phosphorimage analysis.
The helicase assay measures the ability of the gene 4 protein to separate the partially complementary oligonucleotides of the helicase substrate. The helicase reactions (50 µl) contained dTTPase buffer, 2 mM dTTP, 44 nM helicase substrate, and 20 nM gene 4A protein. The reaction mixtures were assembled on ice, and the reaction was started by the addition of gene 4 protein and incubation at 30 °C. At timed intervals, 7.5-µl samples were removed from the reaction and added to 7.5 µl of stop buffer containing 40 mM EDTA and 25% glycerol. The reaction samples were analyzed by nondenaturing 20% PAGE and phosphorimage analysis. The decrease with time in the amount of radiolabeled 25-mer migrating as duplex DNA with the 75-mer was measured, and the percent oligonucleotide displaced was calculated as described previously(35) .
The effect these mutations
have on the ability of gene 4A protein to support T7 bacteriophage
reproduction in vivo was examined by complementation analysis.
Gene 4-deleted T7 phage (T7 4-1) will not lyse E. coli unless a functional copy of gene 4 is provided in trans.
Previous studies have shown that the 63-kDa form of gene 4 protein is
sufficient for T7 phage replication (13, 14) . E.
coli DH5
-carrying plasmids encoding the wild-type and mutant
gene 4A proteins were infected with wild-type T7 or T7
4-1 phage,
and the number and size of the plaques produced were determined. When
infected with T7
4-1, the strains carrying the mutations H475A and
D485G produced 20.8- and 3.3-fold fewer plaques, respectively, than
wild-type gene 4A (Table 2). Also, both of these mutations
resulted in plaques that were on average smaller in diameter (pinpoint
to 2.5 mm) than those produced by wild-type gene 4 (2.5 to 5 mm) under
the same conditions. No plaques were produced by phage T7
4-1 when
plated on cells containing the plasmid with gene 4A mutation R487A.
Figure 2:
Native PAGE analysis of the gene 4A
wild-type and mutant proteins. The purified gene 4A proteins were
examined by electrophoresis under nondenaturing conditions using
4-15% polyacrylamide gels that were then silver-stained for
analysis. The nucleotides present in the preincubation and
electrophoresis buffers for each experiment are indicated at the top of each panel: A, no nucleotide; B,
dTDP; C, ,
-methylene dTTP; and D, dTTP. The
lanes in each panel contained approximately 20 pmol (1.26 µg) of
the indicated gene 4A protein: W, wild-type; H,
H475A; D, D485G; and R, R487A. The positions of the
hexamer bands (open arrows) and the dimer bands (closed
arrow) are indicated in each panel. The positions of the native
PAGE protein standards and their molecular masses (kDa) are indicated
for the gel in D.
The
effect of various nucleotides on the association state of the gene 4A
proteins can be observed by including the nucleotide in the reaction
and electrophoresis buffers. In the presence of dTTP, the preferred
nucleotide substrate for T7 gene 4 protein, each of the gene 4A
proteins form predominantly hexamers (Fig. 2D and Table 3). However, a significant portion of the H475A and D485G
proteins still migrate as dimers (Fig. 2D, closed
arrowhead). Also, with ,
-methylene dTTP, the wild-type
and mutant R487A proteins form hexamers almost exclusively (Fig. 2, C and D). In contrast, in the
presence of dTDP, none of the proteins formed hexamers as readily as
they did with dTTP (Fig. 2B). The H475A and D485G
proteins are principally dimers in the presence of dTDP and
,
-methylene dTTP, with few distinct high molecular weight
bands visible, indicating either that they do not form discrete
complexes or that the complexes are unstable under the conditions used
for electrophoresis (Fig. 2, B and C). In
addition to further demonstrating the effect of the mutations in this
region on oligomer formation, these results indicate that the gene 4
protein-protein affinity (association affinity) will vary depending on
whether a nucleoside di- or triphosphate is bound.
Figure 3: Visualization of wild-type and mutant T7 gene 4A proteins. T7 gene proteins were prepared for EM by negative staining of unfixed samples with 1% uranyl acetate and mounting on thin carbon foils. A, a field of wild-type gene 4A protein complexes, largely hexamers, in the presence of 0.6 mM dTTP. The open arrowhead indicates a hexamer, and the closed arrowheads indicate smaller oligomers. B, images of individual hexamers formed by the wild-type and mutant gene 4A proteins in the presence of 0.6 mM dTTP. The identity of the gene 4A protein in each panel of images is indicated on the right. The white scale bars equal 50 nm (A) and 10 nm (B).
Figure 4: Effect of increasing substrate concentration on nucleotide hydrolysis by the wild-type and mutant T7 gene 4A proteins in the presence and absence of ssDNA. The reactions were performed as described under ``Experimental Procedures.'' All reactions contained gene 4A protein at 200 nM, and the nucleotide concentrations were as indicated. A, the nucleotide hydrolysis reactions contained 50 µM M13mp6 ssDNA (nucleotide equivalents). B, reactions were performed as in A but in the absence of ssDNA. The curves are labeled: WT, wild-type; H/A, H475A; D/G, D485G; and R/A, R487A. Each curve represents the average of two experiments, and each experiment was performed in duplicate.
The velocity of the dTTP hydrolysis reaction catalyzed by the R487A protein decreases in the presence of ssDNA, the opposite of the reaction observed with wild-type protein. This apparent inhibition of activity by ssDNA was confirmed in experiments where nucleotide hydrolysis was measured before and after the addition of ssDNA to reactions with the R487A protein. The rate of hydrolysis by the R487A protein decreased immediately after the addition of ssDNA to the reaction mixtures (data not shown). In addition, this mutant protein has an almost 5-fold higher affinity for dTTP than the wild-type protein and forms hexamers as well as the wild-type protein. The results show that the ability of the mutant protein to hydrolyze NTP is intact, but the R487A mutation interferes with the mechanism by which the gene 4 proteins within the hexamer interact in the presence of ssDNA to coordinately hydrolyze nucleotides.
Figure 5:
ssDNA binding by the wild-type and mutant
T7 gene 4A proteins. Reactions were performed as described under
``Experimental Procedures.'' Increasing concentrations of the
gene 4A proteins were incubated with a constant amount of radiolabeled
oligonucleotide, and the reactions mixtures were separated by
nondenaturing PAGE. The amounts of bound and unbound oligonucleotide
were quantified by phosphorimage analysis of the respective bands. Each
reaction contained the indicated concentrations of gene 4A protein, 1
mM ,
-methylene dTTP, and 0.1 µM radiolabeled 35-mer. The curves are labeled: WT,
wild-type; H/A, H475A; D/G, D485G;
and R/A, R487A.
The fact that the R487A protein bound ssDNA so poorly as
measured by the gel-shift assay was puzzling. This mutant protein forms
hexamers as well as the wild-type protein and must interact with DNA
since its dTTPase activity is inhibited by ssDNA. One possible
explanation for the poor binding is that ,
-methylene dTTP
imparts a conformation to this mutant protein that differs somewhat
from that imparted by dTTP. However, this gel-shift assay cannot be
used with dTTP, because the gene 4 protein translocates off the ssDNA
and is then separated from the DNA during electrophoresis.
Consequently, to examine DNA binding by the R487A protein with greater
sensitivity and determine if there are differences attributable to the
nucleotide present, we employed a UV-mediated cross-linking assay (Fig. 6). With this assay we demonstrated that the R487A protein
bound radiolabeled (dT)
approximately 75% as well as the
wild-type gene 4 protein (indicated by the arrowheads in Fig. 6). Cross-linking occurred only when both nucleotide and
magnesium are present in the reaction mixtures, thus demonstrating a
specific interaction between the gene 4 protein and the
oligonucleotide. The labeled oligonucleotide primarily reacted with a
single monomer of gene 4A protein (closed arrow, Fig. 6); however, a fraction of the oligonucleotide was
cross-linked to multiple gene 4 protein monomers (open arrow, Fig. 6). It is unclear if this result was due to multiple
proteins cross-linked to a single oligonucleotide or cross-linked
proteins bound to a single oligonucleotide. The nucleotide present in
the assay has a slight influence on ssDNA binding; both wild-type and
R487A proteins were cross-linked to the labeled oligonucleotide only
86% as well with dTTP as with
,
-methylene dTTP. This result
confirms that the R487A protein interacts with ssDNA, but does not
reveal the relative strength of the interaction. It may be that this
mutant has a tenuous hold on ssDNA that is not sufficiently strong to
maintain contact during the gel-shift assay, but is strong enough to
inhibit nucleotide hydrolysis.
Figure 6:
UV-mediated cross-linking of gene 4A
wild-type and mutant R487A proteins to ssDNA. The reaction mixtures
contained 0.2 µM gene 4A protein, 0.01 µM radiolabeled (dT), 40 mM Tris-HCl, pH 7.0,
10 mM DTT, 100 mM NaCl, 50 µg/ml bovine serum
albumin, and 10% glycerol, plus 10 mM MgCl
and 2
mM nucleotide as indicated at the top of the figure.
The reaction mixtures were preincubated for 10 min at 30 °C, placed
on ice, and exposed to the UV source for 15 min. The samples were
examined by SDS-PAGE and PhosphorImager analysis. The closed arrow indicated gene 4A monomer (63-kDa) cross-linked to the labeled
oligonucleotide, and the open arrow indicates a dimer
(120-kDa); higher molecular weight species are also visible. Unbound
radiolabeled (dT)
is visible at the bottom of the
figure.
Figure 7:
Helicase activity of the wild-type and
mutant gene 4A proteins. The helicase substrate consists of a 75-base
oligonucleotide with 17 bases of a 25-base oligonucleotide annealed to
its 3`-end as diagramed in the inset. The 25-mer was
5`-P-end-labeled so that its position in the gel can be
detected and quantified. The reactions mixtures contained 20 nM gene 4A protein, 44 nM helicase substrate, and 2 mM dTTP in a 50-µl volume. Samples were removed at the indicated
time, and the reaction was stopped by the addition of buffer with
excess EDTA. The reactions were quantified by PhosphorImager analysis
of the nondenaturing PAGE. The change in the amount of labeled 25-mer
separated from the 75-mer was calculated and expressed as a percent of
the total helicase substrate. The curves are labeled: WT,
wild-type; H/A, H475A; D/G, D485G;
and R/A, R487A.
Figure 8:
The effect of increasing concentrations of
T7 gene 4A wild-type and mutant proteins on RNA- primed DNA synthesis
by T7 DNA polymerase. The concentrations of gene 4A proteins are
indicated. In addition, the 30-µl reactions contained 40 nM T7 DNA polymerase, 40 µM M13 ssDNA, 0.3
µM NTP, 0.3 µM d(G,A,C)TP, and 1 mM [-
P]dTTP in dTTPase reaction buffer
plus 50 mM potassium glutamate. The incorporation of label
into DNA was determined using DEAE-filters (Whatman DE81) and
scintillation counting. The curves are labeled as in Fig. 7.
The hexameric nature of the T7 gene 4 protein was previously demonstrated by EM, gel filtration analysis, and chemical cross-linking experiments(22, 38) . Studies with a T7 gene 4 protein having mutations in its NBS support this physical evidence(16) . In these latter studies, we exploited the ability of the NBS mutant gene 4 protein to inhibit ssDNA-dependent nucleotide hydrolysis by the wild-type protein to investigate the stoichiometry of the gene 4 protein complex on ssDNA. Both this inhibition reaction and the stoichiometry of ssDNA binding indicated that the gene 4 protein was active as a hexamer. The ability of the NBS mutant protein to inhibit completely the activity of the wild-type gene 4 protein also demonstrated the importance of cooperative nucleotide hydrolysis within the hexamer.
In the present study, we have identified mutations in the gene 4 protein that affect its ability to form hexamers and to interfere with coordinated interactions between the subunits of the hexamer. The mutations lie within a conserved domain in the carboxyl-terminal region of the protein. This domain spans amino acid residues 475 to 500 and shares sequence similarities with the gene 4 proteins of phages T3 and SP6, the DnaB proteins of E. coli and Salmonella typhimurium, the phage T4 gp41 helicase and others (refer to Table 1of this report and Ilyina et al.(27) ). Moreover, the core of this region, T7 gene 4 protein residues 481 to 491, is highly conserved in each protein of the DnaB helicase family, many of which form hexamers. Our finding that this conserved domain is responsible for oligomerization and protein-protein interactions required for nucleotide hydrolysis on ssDNA suggests that this related group of NTP-dependent hexameric helicases may share a common mechanism for translocation on ssDNA and unwinding dsDNA.
Each of the mutations within this domain affected
the ability of the gene 4A protein to complement a T7 phage lacking
gene 4. The H475A and D485G mutations decreased T7 4-1 plaque size
and number, and the R487A mutation prevented growth of this phage. EM
analysis revealed that each of the mutant gene 4 proteins form
hexagonal rings, and, morphologically, these hexamers were
indistinguishable from those formed by the wild-type protein. Further
analysis of the mutant proteins revealed that the amino acid
substitutions cause two distinct yet related changes in the properties
of the gene 4 protein. The H475A and D485G mutations decrease the
ability of the proteins to form hexamers, and the R487A mutation
affects the ability of the gene 4 protein hexamer to use the energy of
nucleotide hydrolysis for translocation on ssDNA.
The H475A and D485G proteins have significantly lower ssDNA-dependent dTTPase activity than does the wild-type protein. Since nucleotide hydrolysis catalyzed by the gene 4 protein is stimulated by ssDNA, and since only gene 4 protein hexamers bind ssDNA (data not shown, refer to (41) ), it appears that the reduced dTTPase activity of these mutants is due to less efficient hexamer formation. Consistent with this is the finding that the loss of nucleotide hydrolysis activity resulting from these mutations is proportional to their decreased ability to form hexamers as measured by native PAGE (refer to Table 3). The results of the ssDNA binding and helicase assays also support the conclusion that the primary defect caused by the H475A and D485G mutations is a lower protein-protein binding affinity and not a defect in nucleotide binding or hydrolysis. Based on these findings, we conclude that the activities of the gene 4 protein, ssDNA-dependent nucleotide hydrolysis, ssDNA binding, translocation on ssDNA, and DNA strand separation are all dependent on hexamer formation.
The R487A mutation affects the activity of the gene 4 protein in a different manner. This mutant protein forms hexamers as well as wild-type protein and hydrolyzes nucleotides better than wild-type in the absence of ssDNA. However, nucleotide hydrolysis by this protein is inhibited, instead of stimulated by ssDNA. In addition, the R487A protein does not bind ssDNA tightly or exhibit any ability to unwind dsDNA. Together, these findings strongly suggest that the R487A mutation affects the ability of the monomers within a hexamer to interact properly during nucleotide hydrolysis and translocation on ssDNA.
The 63-kDa gene 4 protein, by virtue of its unique amino-terminal domain, also catalyzes template-dependent synthesis of tetraribonucleotides(10) . Each mutant protein is able to synthesize primers for T7 DNA polymerase, indicating that the mutations in this conserved carboxyl-terminal region of the protein do not directly affect its ability to function as a primase. Similar results were observed in our analysis of a NBS mutant gene 4A protein(17) . This latter mutant protein could not hydrolyze dTTP, and therefore could not translocate on ssDNA, but it could synthesize template-directed primers, presumably through random interaction with DNA.
The D485G mutation found in our original clone of gene 4 protein can be attributed to the lethality of the wild-type protein to E. coli. In fact, in the process of recloning wild-type gene 4, we found that a high frequency of clones contained mutations, at least one of which was defective in nucleotide hydrolysis (data not shown). The primary defect caused by this mutation resulted in our initial inability to demonstrate the oligomeric nature of the gene 4 protein by gel filtration(12, 15) . In this report, we have shown that the D485G protein retains all of the catalytic properties of the wild-type protein, but in each assay the specific activity of this mutant protein is lower than that of the wild-type protein. Nonetheless, the overall observations made with this cloned mutant protein are consistent with those made with gene 4 protein purified from phage-infected cells. This is not a surprising result since the mutant protein does support T7 replication and growth(14, 37) . Therefore, we do not believe that any of our earlier results obtained with this protein will differ significantly from those of the wild-type protein. Rather, we believe that certain reactions requiring tight protein-protein interactions, such as the coupling of DNA polymerase and helicase/primase activities at the T7 DNA replication fork (35) will be augmented.
A recent EM analysis of the gene 4 protein revealed that ssDNA passes through the center of the gene 4 protein hexamer(38) . It was also demonstrated that the E. coli RuvB branch migration protein, which has helicase activity, forms double hexameric rings around DNA (25) . The ring structure of these protein complexes raises the intriguing question of how these hexagonal rings load onto the DNA. Since the gene 4 protein tightly binds circular ssDNA (40) we can rule out mechanisms requiring loading via free ends. Therefore, in order to bind ssDNA, the gene 4 hexamer must either assemble around the DNA or the preformed hexameric ring must open to load onto the ssDNA. As observed with other hexameric helicases, such as DnaB and T4 gp41(18, 42) , nucleoside triphosphate binding facilitates hexamer formation by the gene 4 proteins(22) . In this study, we also observed an increase in hexamer formation by the gene 4 protein upon binding dTTP (Fig. 2D and Table 3and Table 4). The product of the hydrolysis reaction, dTDP, does not induce hexamer formation to the same extent as does dTTP (Fig. 2B and Table 4). This result suggests that NTP binding leads to a conformational change in the gene 4 protein that increases the protein-protein binding affinity and that conformational changes during the hydrolysis of the NTP to NDP decreases the association affinity. When bound to ssDNA, these conformational changes are rapid and result in translocation. In the absence of bound ssDNA, the hexamer will have more time to partially dissociate due to the relatively slow rate of the hydrolysis reaction. The decreased protein self-association affinity that occurs as dTTP is hydrolyzed to dTDP may therefore be a necessary step for the hexameric ring formed by the gene 4 protein to open and bind ssDNA.
The ssDNA binding experiments demonstrate that
nucleotide binding and hexamer formation are not sufficient for tight
DNA binding, nor is nucleotide hydrolysis required for the gene 4
protein to bind ssDNA, since the nonhydrolyzable analog
,
-methylene dTTP promotes strong binding(40) . The
R487A protein forms hexamers as well as wild-type protein and binds
ssDNA almost as well in the cross-linking assay. It may be that the
hexamers formed by the R487A protein interact with DNA, but cannot
undergo the conformational changes required for the hexamer to
``grip'' the DNA and translocate. Consequently, it slips off
the end of the oligonucleotide during electrophoresis, and, thus,
binding cannot be detected in the gel-shift assay.
We find it of
interest that the two closely spaced mutations D485G and R487A have
such differing effects on the oligomerization and enzymatic activities
of the gene 4 protein. The residues Asp and Arg
are at the center of the core region of homology with the DnaB
family of helicases and so suggest a common mechanism of action for
hexameric helicases. Thus, we speculate that interactions mediated by
this domain between the subunits in a hexamer may be responsible for
the presence of three high and three low affinity NBS observed in
studies of the DnaB hexamer(43) . This hypothesis is supported
by the close proximity of the residues directly involved in the
protein-protein interactions. It is very likely that the protein domain
responsible for communication of conformational changes between the
hexamer subunits during nucleotide hydrolysis and the domain
responsible for protein-protein interactions would be integrated.
It should be noted that the gene 4 proteins H475A and D485G, while defective in hexamer formation, do not migrate as monomers in native PAGE analysis. Although it is possible that the fastest migrating protein band visible on the silver-stained native gels is actually a monomer with aberrant migration, this seems unlikely since a ``Ferguson'' analysis (44) of both the protein standards and the estimated molecular weight of each of the visible gene 4 protein bands generates a linear plot. Additionally, small zone gel filtration analysis of gene 4 protein at various concentrations in the presence and absence of nucleotide detected only two species of oligomer, estimated to be dimer and hexamer(22) . If the proteins interact in a ``head-to-tail'' configuration, it is difficult to imagine how mutations that influence hexamer formation would not affect dimerization. This head-to-tail configuration predicts a single interface, the disruption of which would lead to the appearance of monomers (Fig. 9). On the other hand, if dimer formation is represented as a ``head-to-head'' interaction of the monomers, and the dimers then interact through ``tail-to-tail'' contacts to form hexamers, we would predict two types of protein-protein interface (Fig. 9). Mutations at one interface would affect hexamer formation, but would have no influence on dimer formation. Precisely such a model was proposed by Dong et al.(18) for nucleotide-induced hexamer formation by the phage T4 gp41 helicase. They observed the formation of dimers at low protein concentrations and hexamers at higher concentrations and proposed the existence of two interfaces with different protein association strengths. The gene 4 protein-protein interactions appear to be much stronger than those of T4 gp41 as hexamers of gene 4 protein were detected in the absence of nucleotide by native PAGE at protein concentrations of 2 µM and by electron microscopy at very low protein concentrations (175 nM), whereas T4 gp41 does not form hexamers at low protein concentrations unless a nucleotide is present(18) .
Figure 9: Model for association of gene 4 proteins in a hexamer. A, head-to-tail association of monomers showing six protein-protein interfaces of a single type. B, head-to-head and tail-to-tail association of three dimer pairs of gene 4 protein with two types of interface.