(Received for publication, June 1, 1995; and in revised form, July 20, 1995)
From the
The bacteriophage T7 gene 4 protein is a multifunctional enzyme
that has DNA helicase, primase, and deoxyribonucleotide
5`-triphosphatase activities. Prior studies have shown that in the
presence of dTTP or dTDP the gene 4 protein assembles into a
functionally active hexamer prior to binding to single-stranded DNA. In
this study, we have examined the effects of different nucleotide
cofactors on the conformation of the gene 4 protein in the presence and
absence of DNA. Gel retardation analysis, partial protease digestion,
and DNA footprinting all suggest that the gene 4 protein undergoes a
conformational change when dTTP is hydrolyzed to dTTP and that in the
presence of dTDP the complex with DNA is more open or extended. We have
also found that the dissociation constant of the gene 4
proteinDNA complex in the presence of dTDP was 10-fold lower than
that determined in the presence of dTTP, further suggesting that these
cofactors exerts different allosteric effects on the DNA-binding site
of the gene 4 protein.
The bacteriophage T7 gene 4 protein is crucial in the replication of the phage genome. In this process, it functions as a DNA helicase (Kolodner et al., 1978; Kolodner and Richardson, 1978; Matson et al., 1983) and primase (Strätling and Knippers, 1973; Hinkle and Richardson, 1975; Romano and Richardson, 1979a, 1979b). In addition, the gene 4 protein can hydrolyze deoxyribonucleotide triphosphates (dNTPs) to diphosphates, with dTTP being the preferred substrate (Matson and Richardson, 1983). The energy released from hydrolysis of dTTP is used to fuel the other biological activities of this enzyme, such as its unidirectional (from 5` to 3`) translocation along single-stranded DNA (Tabor and Richardson, 1981) and its DNA unwinding activity.
Recently, it has been shown that the gene 4 protein is
capable of forming a hexamer (Hingorani and Patel, 1993) that assembles
in solution in the presence of either dTTP or dTDP, and in this form
binds to single-stranded DNA. Following stabilization by cross-linking
with gluteraldehyde, this hexameric complex can be isolated and was
shown to retain both its DNA helicase and dTTPase activities implying that the functionally active form of the gene 4 protein
is the hexamer. The hexameric structure formed by the gene 4 protein is
capable of making simultaneous interactions with both strands of DNA, a
complex in which two or three subunits are contacting the same strand
of DNA. (
)
Hydrolysis of dTTP by the gene 4 protein is required for both translocation along single-stranded DNA and unwinding of double-stranded DNA. However, little is known about how the gene 4 protein couples its dTTPase activity to the movement necessary for both of these biological processes. It is likely that a conformational change driven by the energy released from hydrolysis of dTTP may play a pivotal role producing the requisite movement that mediates the translocation and actual strand separation by the gene 4 protein. Indeed, nucleotide-induced conformational changes have been observed for several DNA helicases. These include Escherichia coli dna B protein (Nakyama et al., 1984) and E. coli Rep and Helicase II proteins (Chao and Lohman, 1990; Wong and Lohman, 1992). For the gene 4 protein, additional conformational changes might be induced by binding to single-stranded DNA since the dTTPase activity of the gene 4 protein is DNA-dependent (Matson and Richardson, 1983). It is possible that a structural alteration in the gene 4 protein triggered by the interaction with single-stranded DNA is necessary to initiate the hydrolysis of dTTP.
For these reasons it seemed that a knowledge of the allosteric effects on the structure of the gene 4 protein that are induced by nucleotide cofactors and the interaction with single-stranded DNA might help us understand how this enzyme couples the energy released by dTTP hydrolysis into molecular movement along the DNA strand. In the present study, we have examined the structural alternations in the gene 4 protein using gel retardation analysis, partial proteolysis, and nuclease protection assays. The results from this study clearly show that the gene 4 protein undergoes conformational changes upon hydrolyzing dTTP to dTDP and upon binding to single-stranded DNA.
log[Pt]=log(f1-f)+log K
K was the dissociation constant of the
DNA-binding reaction, [Pt] was the total concentration of the
protein present in the reaction, and f was the ratio of the
amount of the bound oligonucleotides over the total amount of the
oligonucleotides present in the reaction. The logarithm of
[Pt] was plotted against the logarithm of (f/(1-f), and the intercept at the vertical axis
represented the logarithm of K
.
Figure 1:
The proteinDNA complexes formed
by the gene 4 protein and oligonucleotides in the presence of dTDP and
dTTP. The DNA binding reactions and the separation of the reaction
products were carried out as described under ``Experimental
Procedures.'' An oligonucleotide molecule 60 bases long (60-mer)
was used in this experiment. The compositions of these reactions are as
indicated and the positions of the free 60-mer, and the
protein
DNA complexes formed by the gene 4 protein and the 60-mer
are indicated. The positions of the origins of the lanes are marked by
the arrow.
More interesting is the difference in
mobility of the faster moving complexes formed in the presence of dTTP versus dTDP. The proteinDNA complex formed by the gene 4
protein and the 60-mer in the presence of dTDP displayed a clearly
slower electrophoretic mobility than the complex formed at low (80
nM) concentrations of gene 4 protein in the presence of dTTP (Fig. 1). As described above, these complexes have identical
molecular weights in an SDS gel, suggesting that the gene 4
protein
DNA complex may undergo a conformational change when the
nucleotide cofactor is switch from dTTP to dTDP, a process that occurs
during hydrolysis of dTTP. This observation also suggests that the gene
4 protein
DNA complex may adopt a more extended conformation in
the presence of dTDP since this structure migrates more slowly in the
gel than the complex formed in the presence of dTTP.
In the presence of
dTTP, the gene 4 protein could bind very weakly to an oligonucleotide
as short as 12 bases in length (Fig. 2B). However,
there appeared to be a sharp decrease in the formation of the
proteinDNA complex when the oligonucleotide probe was shorter
than 13 bases long. As the length of the oligonucleotide probe used in
the binding reaction increased, there was not only a general increase
in the amount of the protein
DNA complex observed, but also the
formation of a second complex with a lower electrophoretic mobility. In
addition, the ratio of the second complex over the first increased
steadily as the length of the oligonucleotide probe grew. This
observation further substantiated the hypothesis that the
slower-migrating complex was the result of contiguous binding of two
gene 4 protein hexamers to a single oligonucleotide molecule.
Figure 2:
The minimum length of oligonucleotides
required to form proteinDNA complexes with the gene 4 protein. A
group of oligonucleotide molecules ranging from 11 to 54 bases in size
was prepared through chemical cleavage of a 54-mer and were then used
in the gel retardation assays as described under ``Experimental
Procedures.'' The oligonucleotide molecule used in each reaction
and the compositions of these reactions are indicated. A, dTTP
was present as the nucleotide cofactor. B, dTDP was used as
the nucleotide cofactor.
When
dTDP was present in the DNA-binding reaction as the nucleotide
cofactor, the gene 4 protein could bind to a 21-mer efficiently; but
when a 20-mer was used there was a dramatic decrease in the formation
of the proteinDNA complex, and below this chain length no complex
was observed (Fig. 2A). This suggested that in the
presence of dTDP, an oligonucleotide of at least 21 bases long was
required to provide sufficient interaction with the DNA-binding site of
the gene 4 protein to obtain a stable protein
DNA complex. These
observations also suggest that when the nucleotide cofactor associated
with the gene 4 protein is switched from dTTP to dTDP, the allosteric
effect induced by this transition caused structural perturbations that
lead to an alternation in the DNA-binding site and presumably results
in a more extended conformation since only longer oligonucleotides are
bound.
A fixed
amount of the appropriate oligonucleotide probe was titrated with
increasing concentrations of the gene 4 protein and the
proteinDNA complexes formed were monitored by gel mobility-shift
assay (Fig. 3A). In the presence of dTTP, the binding
reaction reached a plateau when about 90% of the oligonucleotide probes
were bound. However, when dTDP was present, the binding reaction reach
a plateau after about 40% of the probes were associated with the gene 4
protein (Fig. 3A). These data were analyzed by a Hill
plot (Fig. 3B) and the dissociation constants (K
) calculated as described under
``Methods''. In the presence of dTTP, the K
was 0.13 µM while dTDP gave a K
of 1.3 µM. These results suggest the protein-DNA
complex formed in the presence of dTTP was approximately 10 times more
stable than the complex formed in the presence of dTDP. In fact, the
difference in the K
values reported here might
well be underestimated because of the shorter length of the
oligonucleotide molecule used in the presence of dTTP than in the
presence of dTDP. In any case, these results are additional evidence
that the transition from a dTTP-associating state to a dTDP-associating
state has a major impact on the DNA-binding site of the gene 4 protein.
Figure 3:
Determination of the Kof the gene 4 protein
DNA complex.
The DNA binding reactions of the gene 4 protein, the separation and
quantitation of the reaction products, and the analysis of experimental
data were carried out as described under ``Experimental
Procedures.'' The DNA binding reactions contained 6.7 nM of either a 25-mer (in the presence of dTTP) or a 60-mer (in the
presence of dTDP); and 83.3, 125, 167, 250, 333, 500, 667, 833, 1000,
and 1250 nM of the gene 4 protein, respectively. A,
the percentages of all the oligonucleotide molecules bound by the gene
4 protein are plotted against the concentration of the gene 4 protein.
Each data point represented the mean of at least three different
experiments with the standard error indicated. B, Hill plot of
the data obtained from the experiments.
Digestion of the gene 4 protein with trypsin produced a maximum of 14 discrete proteolytic fragments ranging from 21.2 to 53.8 kDa (Fig. 4). Because the amino acid sequence of the gene 4 protein contains 65 potential trypsin cleavage sites, it is possible that other proteolytic fragments also existed but were too short to detect in our electrophoretic system. Alternatively, many of the tryptic cleavage sites may reside at positions not accessible to the protease. A summary of the presence of these fragments under various reaction conditions is shown in Table 1.
Figure 4: Limited proteolysis of the gene 4 protein. Proteolysis of the gene 4 protein with trypsin was carried out as described under ``Experimental Procedures.'' Lanes A and J in all panels are molecular weight markers; lanes I are trypsin. The compositions of each reaction are as indicated. The concentrations of trypsin present in lanes B through H are 0, 0.1, 0.2, 0.5, 1.0, 2.0, and 5.0 ng/µl, respectively. The positions of the undigested gene 4 protein molecules in all the panels are indicated by open arrows, and the positions of trypsin are indicated by closed arrows. The molecular weight of the markers are indicated on the left of each panel, the positions and the molecular weight of the proteolytic fragments are indicated on the right.
When neither single-stranded M13 DNA or nucleotide cofactors were present, limited tryptic digestion revealed six proteolytic fragments (Fig. 4A and Table 1). Addition of single-stranded DNA alone caused no detectable changes in the proteolytic pattern (Fig. 4B and Table 1). This is consistent with the previous observations that nucleotide cofactors are absolutely required by the gene 4 protein in order to bind to single-stranded DNA (Matson and Richardson, 1985), and thus in their absence there is no interaction between the gene 4 protein and DNA and, therefore, no effect on the conformation of the gene 4 protein.
The addition of dTDP alone resulted in a few subtle differences in the digestion pattern (Fig. 4C). Under these condition, the 32.3- and the 21.2-kDa proteolytic fragments were not detected (Table 1). Most of the other fragments were fainter than their counterparts in Fig. 4, A and B, the exception being the 48.2-kDa fragment. In addition, the gene 4 protein was much more resistant to tryptic digestion in the presence of dTDP than when the reaction contained no nucleotides as shown by the level of gene 4 protein that remained when dTDP was present (cf.Fig. 4C with A and B). When the gene 4 protein was digested in the presence of both dTDP and single-stranded M13 DNA (Fig. 4D), the proteolytic pattern was drastically different from all those described above. At intermediate trypsin concentrations a total of 14 fragments were detected (Fig. 4D and Table 1), some of which were not observed in the prior experiments. In addition, the gene 4 protein was more susceptible to the attack of the protease than when only dTDP was present. Taken together, these results suggest that the association with dTDP and the subsequent binding to single-stranded DNA triggered a major perturbation in the structure of the gene 4 protein and also that under these conditions the gene 4 protein was more susceptible to digestion, possibly because of a more open conformation.
When the gene 4 protein was preincubated in a reaction mixture containing dTTP but without single-stranded DNA, a total of five fragments were detectable (Fig. 4E and Table 1). Subtle differences could be observed when this was compared with the experiment where only dTDP was present (Fig. 4C). First, the 35.5-kDa fragment was more resistant to tryptic digestion in the presence of dTTP. Second, we could observe the disappearance of the 26.4-kDa fragment and the appearance of a fragment 38.1 kDa in size. Third, the gene 4 protein appeared to be generally more resistant to the digestion of trypsin, as indicated by the relatively larger amount of the uncleaved gene 4 protein molecules left at the end of the protease digestion. Taken together, these results indicated that the gene 4 protein adopted a different conformation when it was bound to dTTP than when bound to dTDP.
Finally, limited proteolysis of the
gene 4 protein was carried out in the presence of both dTTP and
single-stranded M13 DNA (Fig. 4F). Under these
conditions, a total of nine proteolytic fragments were generated.
Compared with the profile where only dTTP was present (Fig. 4E), the proteolytic fragments, in general,
started appearing at much lower concentrations of trypsin and with a
much stronger intensity, suggesting that upon binding to
single-stranded DNA, the gene 4 protein underwent structural
alterations so that the trypsin cleavage sites involved were more
accessible. When this profile was contrasted with that where both dTDP
and single-stranded M13 DNA were present (compare Fig. 4F with Fig. 3D), the difference was even more
drastic. In the presence of dTTP and single-stranded DNA (Fig. 3F), not only were the 53.8-, 45.9-, 44.4-,
34.1-, and the 22.6-kDa fragments missing, but the others like the
32.2-, 25.1-, and the 23.8-kDa fragments were also much lower in their
intensities and/or started appearing only at higher trypsin
concentrations than in the presence of dTDP and DNA (Fig. 4D). In addition, in the presence of dTTP and
DNA, the 35.5-kDa fragment appeared to be much more resistant to
tryptic digestion than when dTDP was present, suggesting that the
three-dimensional structure of this part of the gene 4 protein was
different from the structure when dTDP was present. Finally, the
dTTP-gene 4 proteinDNA complex was more resistant to tryptic
digestion than the dTDP-gene 4 protein complex, as judged by the
difference in the amount of the intact gene 4 protein molecules left
after proteolysis.
The gene 4 protein hydrolyzes dTTP to dTDP in the
presence of single-stranded DNA; therefore, after the gene 4 protein is
incubated with dTTP and single-stranded M13 DNA, the nucleotide present
would actually become a mixture of both dTTP and dTDP. Thus, it is
possible that the protease digestion profile observed in the presence
of dTTP resulted from the joint effects of dTTP and dTDP which were
present in the ratio of 3:1 at the end of the protease digestion (data
not shown). In order to exclude this possibility, we used a
nonhydrolyzable analog of dTTP, ,
-methylene dTTP, which also
induces strong binding of the gene 4 protein to single-stranded
DNA.
Limited tryptic digestion of this DNA complex gave
results identical to that which were observed in the presence of dTTP
(data not shown).
Overall, these results suggested that nucleotide cofactors triggered structural alterations in the gene 4 protein. The subsequent binding of the gene 4 protein to single-stranded DNA caused additional conformational changes that appeared to make it more open or accessible than in the absence of DNA. More importantly, the gene 4 protein adopted different conformations in the presence of dTTP versus dTDP and appeared to be more open when bound to dTDP.
The gene 4 protein was preincubated with the appropriate nucleotide cofactors and poly(dT) molecules with an average length of 2000 bases, and then micrococcal nuclease was added. When dTDP was present, the ladder produced by the oligonucleotide fragments protected by the gene 4 protein centered at about 11-12 bases in length at low nuclease concentrations (Fig. 5A, lane F). As the amount of micrococcal nuclease used in the digestion increased, the fragments were further shortened to a lower limit of around five to six bases. It was possible that the 11-12-base long fragments reflected the physical dimension of each individual gene 4 protein molecule within the hexameric complex whereas the five to six-base fragments might represent the size of the DNA-binding site of the gene 4 protein.
Figure 5: DNA footprinting analysis of the gene 4 protein[chempDNA complex. The binding of the gene 4 protein to single-stranded homopolymers (poly(dT) or poly(dA)) and the subsequent nuclease digestion were carried out as described under ``Experimental Procedures.'' The concentrations of micrococcal nuclease used in these reactions was 0.2 (lanes A and C), 0.5 (lanes B and D), 1.0 (lanes F and J), 2.0 (lanes G and K), 4.0 (lanes H and L), and 8.0 (lanes I and M) units/µl, respectively, for both panels A and B. Lanes E and N are size markers generated by combining the A, G, C, and T Sanger's dideoxy sequencing reaction mixtures. The primer-template complex used in the sequencing reaction contains a 5` end-labeled 11-mer annealed to a 48-mer. A, poly(dT); B, poly(dA).
The micrococcal nuclease digestion profile in the presence of dTTP
was then determined. At low concentrations of micrococcal nuclease, the
fragments protected by the gene 4 protein appeared to segregate into
two groups; the ladder generated by the slower migrating group centered
at about 21-22 bases while that produced by the faster migrating
group centered at about seven to eight bases (Fig. 5A, lane J). As the concentration of the nuclease used in the
digestion increased, the overall pattern was similar, except that the
average length of the upper ladder shrank to about 18-19 bases
and that of the lower ladder declined to about four to five. One
interpretation of these results (see ``Discussion'') is that
the slower moving material represents protection by two adjacent gene 4
protein monomers while the faster bands again represent that within the
DNA-binding site. This interpretation is consistent with our
observation that two of the monomers simultaneously interact most
strongly with the DNA.
Taken together, these results indicate a marked difference in the profile of protection from nuclease digestion by the gene 4 protein in the presence of dTDP and dTTP. One interpretation of the data is consistent with the model that the gene 4 protein has a more open or extended conformation in the presence of dTDP.
DNA helicases are a class of enzyme indispensable for the process of DNA replication. These proteins precede the other members of the replication complexes to unwind the DNA duplex so that the genetic information carried in DNA is available to the DNA polymerase. How DNA helicases carry out this unwinding process has long been an intriguing and elusive question (Lohman, 1993). All known DNA helicases require the binding and subsequent hydrolysis of either ribo- or deoxyribonucleoside 5`-triphosphates. It has been proposed that conformational changes in DNA helicases driven by the free energy released from hydrolysis of NTPs or dNTPs are essential for their DNA unwinding activity and for their translocation along single-stranded DNA (Hill and Tsuchiya, 1981, Kolodner and Richardson, 1977; Brown and Romano, 1989). Recently, nucleotide- and DNA-induced conformational changes have been observed in E. coli DnaB helicase (Nakayama et al., 1984), Helicase II, and Rep helicase (Chao and Lohman, 1990).
The T7 gene 4 protein, a helicase necessary for phage T7 to
replicate its genome, has been shown to form a hexamer (Hingorani and
Patel, 1993; Patel and Hingorani, 1993) that is apparently capable of
interacting with both strands of DNA at a replication fork. In this study, we present several experiments each of which
suggests that the gene 4 protein adopts a different conformation when
bound to DNA in the presence of either dTTP or dTDP and that the
complex formed in the presence of dTDP is more open or extended. First,
we have shown that the gene 4 protein
DNA complex formed in the
presence of dTDP migrates slightly more slowly in a gel retardation
experiment than the complex formed in the presence of dTTP. This is
consistent with the generally observed result that complexes that
occupy more space as a result of being more extended migrate more
slowly in this type of analysis. Second, we found that the gene 4
protein can bind to much smaller oligonucleotides in the presence of
dTTP than it can in the presence of dTDP. Third, the dissociation
constant for the gene 4 protein complex is 10-fold lower in the
presence of dTDP suggesting a major difference in the conformation
occurs when dTTP is hydrolyzed. Fourth, limited proteolysis indicates
that the gene 4 protein adopts a more accessible conformation when it
binds to single-stranded DNA and that the complex formed in the
presence of dTDP is more accessible than that formed in the presence of
dTTP. Finally, DNA footprinting suggests that in the presence of dTDP a
larger DNA fragment is protected from hydrolysis.
The finding that
the gene 4 protein requires a minimum length oligonucleotide ligand in
order to form a stable proteinDNA complex provides some important
clues for the nature of the protein-DNA interaction involved. This
requirement most likely reflects a need to establish a threshold level
of contact between this enzyme and single-stranded DNA. Further
information regarding the DNA-binding site size of the gene 4 protein
protomers can be gleaned from the DNA footprinting experiments. We
found that the binding of the gene 4 protein to single-stranded DNA
protects short stretches of DNA from micrococcal nuclease digestion and
that the sizes of these fragments depend on the nucleotide cofactors
involved. The observation that the length of the DNA fragments
protected by the gene 4 protein is shorter in the presence of dTDP than
in the presence of dTTP is initially surprising because this appears to
be contradictory to what has been suggested by all our previous data.
However, this inconsistency may be explained if two gene 4 protomers
are required for binding and if the nuclease can cleave between
protomers more easily when dTDP is present. Thus, in the presence of
dTTP, the DNA fragments protected by the gene 4 protein appear to
segregate into two size groups: one centered around seven nucleotides
and the other around 21 nucleotides in length. From the physical
dimensions of the gene 4 protein monomer, it is possible that the
21-nucleotide-protected region represents the binding of two protomers
and the seven-nucleotide region represents the less frequent nuclease
attack between two adjacent protomers.
In the presence of dTDP, the pattern of nuclease protection showed a dramatic difference from that observed in the presence of dTTP. Only one group of protected fragments is observed with the size of these fragments centering at about 11-12 bases in length at a low concentration of micrococcal nuclease and five to six bases long as the concentration of the nuclease increases. We propose that the 11 to 12-base fragments represent the overall physical dimension of each gene 4 protomer in the presence of dTDP and that as the amount of micrococcal nuclease increases, those nucleotide residues only partially protected by the gene 4 protomer are excised leaving a shorter stretch that is protected by the actual DNA-binding site in the gene 4 protein. The failure to detect a second ladder in the presence of dTDP may be explained by either a different conformation of the protein that allows easier access to the interprotein regions or by the weaker binding in the presence of dTDP that allows release of one of the two bound protomers.
The results presented in this paper have important implications with
regard to the mechanism of molecular movement by gene 4 protein. We
find that dTDP and dTTP exert different allosteric effects on the gene
4 protein suggesting that the gene 4 protein alternates between these
two different conformations during hydrolysis of dTTP. The differences
in K for these two complexes suggest that when
dTDP is bound, the protein
DNA complex is at a higher energy
state, and therefore the displacement of dTDP by dTTP in the
nucleotide-binding site of the gene 4 protein is favored and would
result in release of free energy. This, in turn, implies that the
energy input used to drive the transition from a dTDP-induced
conformation to a dTTP-induced one could potentially be provided by the
free energy released during this process, while the energy required to
effect the structural alternation in the opposite direction is supplied
through hydrolysis of dTTP. It is likely that the hydrolysis of dTTP to
dTDP and the subsequent displacement of dTDP by a dTTP molecule cycles
the gene 4 protein through these different conformational changes and
somehow results in unidirectional translocation and the helicase
activity.
We have also shown that when the gene 4 protein binds to single-stranded DNA in the presence of dTDP, it adopts a conformation that is more open or extended and interacts with a longer stretch of DNA than the conformation formed in the presence of dTTP. During hydrolysis this conformation change occurs within the context of a hexameric complex and therefore would introduce structural constraints into the complex itself. It is thought that the gene 4 protein translocates along single-stranded DNA in the 5` to 3` direction, raising the possibility that any conformational changes that bring about these movements are also directional. Thus, it is tantalizing to hypothesize that the sequential vectored extension of the structure of these individual protomers causes the protein complex to move in a single orientation in a rolling type process (Lohman, 1993).
Recently an EM study of the gene 4 protein hexamer bound to
single-stranded DNA suggested that the DNA passes through the center of
the ringed hexameric structure (Egelman et al., 1995).
Although our data do not exclude this possibility, an alternative
model, in which the DNA contacts two or three monomers on the outside
of the hexameric ring (Debyser et al., 1994) would allow a
``rolling'' mechanism for unwinding (Lohman, 1993), and is
supported by several experiments. First, we find that micrococcal
nuclease is able to digest all but five to six nucleotides, a fact that
can be accommodated in the ``outside binding'' model if this
is the size of the DNA occluded in the DNA-binding site. From the
dimensions of the gene 4 protein hexamer as visualized in the EM, the
alternative ``inside binding'' model predicts that each
hexamer would protect approximately 25-30 nucleotides (Egelman et al. 1995). Second, it is well established that the gene 4
protein forms hexamers in the presence of nucleotide cofactor in the
absence of DNA (Patel and Hingorani). In order for this
complex to then bind to circular DNA by the inside binding model, one
would need to postulate that the hexamer disassembles prior to DNA
binding. Third, we
and others (Egelman et al.,
1995) have shown that protein-protein cross-links do not form a complex
with single-stranded linear DNA that is stable to
denaturation, although this complex apparently is stable if the DNA is
circular (Egelman et al., 1995). It is possible that the
single-stranded DNA slips out through the inside of the hexameric ring
after denaturation; however this seems unlikely for DNA that is 7000
bases long and bound to multiple gene 4 protein complexes. Finally, UV
cross-linking of the gene 4 hexamer to DNA showed very strong and
simultaneous binding between two of the subunits and one DNA
strand.
The inside binding model not only predicts
difficulty in forming a UV cross-link because of the inaccessibility of
the DNA to the UV light, it also predicts a more symmetrical
interaction between the six subunits and the DNA. Which of these two
possible models is correct awaits further study.