(Received for publication, October 17, 1994; and in revised form, November 28, 1994)
From the
Genes 41 and 59 of bacteriophage T4 are involved in DNA recombination as well as in DNA replication. The 41 protein has a DNA helicase activity. The 59 protein has been recently purified and found to have a specific affinity for both 32 protein (single-stranded DNA-binding protein) and 41 protein (Yonesaki 1994, J. Biol. Chem. 269, 1284-1289). We examined the effects of 59 protein on ssDNA-dependent ATPase activity and DNA helicase activity of 41 protein in the presence or absence of 32 protein. The ATPase activity of 41 protein was strongly inhibited by 32 protein over a wide range of amounts from subsaturation to oversaturation of ssDNA. The 32 protein was also inhibitory toward DNA helicase activity. Addition of 59 protein effectively eliminated these inhibitory effects of 32 protein. Moreover, 59 protein facilitated 41 protein to overcome the barrier to initiate the unwinding reaction with a duplex flanking a single-stranded DNA gap. Intriguingly, 32 protein at an amount optimal for saturation of ssDNA stimulated the overcoming of the barrier when 59 protein was present. For the best circumvention of this initiation barrier, only eight monomers of 59 protein/one DNA substrate molecule containing 2900 nucleotides of ssDNA were required. These results strongly suggest that 59 protein modulates 41 protein activities by forming a complex with 41 protein and that 41 protein can produce recombinogenic ssDNA with the aid of 32 and 59 proteins.
The protein encoded by gene 41 (41 protein) of bacteriophage T4
has a DNA helicase activity. This protein facilitates the unwinding of
double-stranded DNA ahead of the advancing DNA polymerase and
accelerates the movement of the replication fork (Nossal and Alberts,
1983). Recently, gene 41 has been further disclosed to participate in
DNA recombination (Yonesaki, 1994a). T4 DNA recombination that is
promoted by uvsX protein, a T4 homolog of the Escherichia coli recA protein, requires ssDNA ()with a free end as a
substrate (Yonesaki and Minagawa, 1985, 1989; Formosa and Alberts,
1986). An exonuclease encoded or controlled by 46 and 47 recombination
genes can create ssDNA by expansion of a ssDNA nick into a gap on dsDNA
(Hosoda and Prashad, 1972). However, the ssDNA in this gap would not be
available for strand transfer by uvsX protein because it has no free
end. Alternatively, the gapped DNA would offer a good substrate for 41
protein, which could bind to ssDNA in the gap, hydrolyze ATP or GTP,
and slide in the 5` to 3` direction to progressively unwind the duplex
when the protein travels beyond the gap (Liu and Alberts, 1981;
Richardson and Nossal, 1989). The additional ssDNA thus created has a
free 3` end. Therefore, 41 protein is suggested to function in
production of recombinogenic ssDNA.
The 32 protein, a ssDNA-binding protein, has a high affinity for ssDNA and binds to ssDNA with a strong cooperativity (Alberts and Frey, 1971). The synthesis of this protein is self-regulated according to the concentration of intracellular ssDNA (von Hippel et al., 1983). Therefore, inside a T4-infected cell, ssDNA is considered to be covered by 32 protein. Since 32 protein-covered ssDNA is inaccessible to 41 protein or 32 protein inhibits the binding of 41 protein to ssDNA (Barry and Alberts, 1994), another protein should be required for promotion of the function of 41 protein.
The 59 protein exists as a monomer and dispersively binds to ssDNA. The protein has a specific affinity for both 32 and 41 proteins and is capable of rapid binding to ssDNA covered with 32 protein (Yonesaki, 1994b). Barry and Alberts(1994) have found that 59 protein rescues the ssDNA-dependent GTP hydrolysis activity of 41 protein from inhibition by 32 protein, suggesting that 59 protein promotes the binding of 41 protein to ssDNA when this is inhibited by 32 protein. In fact, addition of 59 protein to the in vitro DNA replication system reconstituted by seven purified proteins (32, 41, 44/62, 43, 45, and 61 proteins) greatly stimulates DNA synthesis, presumably by helping quick loading of 41 protein onto 32 protein-covered ssDNA (Barry and Alberts, 1994). Besides acting in DNA replication, gene 59 is also involved in DNA recombination. Like a recombination-deficient mutation of gene 41 (Yonesaki, 1994a), a mutation of gene 59 diminishes the frequency of genetic recombination (Shah, 1976), increases UV sensitivity (Wakem and Ebisuzaki, 1981), and impairs the formation of ssDNA longer than one unit length of T4 genome, which is produced by DNA recombination (Wu and Yeh, 1974). These facts suggest that the 59 protein is necessary for the normal functioning of 41 protein in DNA recombination.
Here, we characterize the functional interactions of 32, 41, and 59 proteins in terms of the ssDNA-dependent ATPase activity and DNA helicase activity of 41 protein. Our results strongly suggest that 59 protein modulates activities of 41 protein by forming a binary complex of these proteins. Our results also reveal that 59 protein has an essential role in the helicase activity with a single strand-gapped DNA as a substrate.
Figure 1: A set of substrates for helicase assay. A, base sequences of synthetic 50- and 80-mer. B, schematic draws of two substrates prepared by annealing of 50- or 80-mer to pBluescript II KS+ ssDNA. Arrows indicate the direction of movement of 41 protein along ssDNA.
Figure 6:
Effects of preincubation. Two 30-µl
reaction mixtures were prepared; one contained 60 fmol of substrate A
and 27 pmol of 32 protein without ATP (32/DNA mixture) and the other
contained 8.4 pmol of 41 protein and 4 mM ATP (41/ATP
mixture). These mixtures were incubated at 37 °C for 2 min, and 5.3
pmol of 59 protein was then added to the 32/DNA mixture () or to
the 41/ATP mixture (
). Following a 3-min incubation with 59
protein, the two mixtures were combined at time 0. Alternatively, after
32/DNA and 41/ATP mixtures had been incubated for 2 min, they were
combined and incubated for 3 min. Then 59 protein was added to the
mixture at time 0 (
). A 10-µl aliquot was withdrawn at each
time point indicated on the abscissa to measure released 50
mer ssDNA as described under ``Materials and
Methods.''
To measure ATPase activity, we
added [-
P]ATP (ARC, 7000 Ci/mmol) to the
reaction mixture. After the reaction, radioactivity of the inorganic
phosphate released was measured as described previously (Yonesaki and
Minagawa, 1985). To measure DNA helicase activity, we monitored the
release of short ssDNA fragments as follows. After the reaction, 0.1%
SDS and 500 µg/ml proteinase K were added, and incubation was
continued for a further 5 min. The DNAs were electropho-resed through a
12% polyacrylamide gel (160
130
1 mm) in a buffer of 89
mM Tris, 89 mM boric acid, and 2 mM EDTA (pH
8.0), at a constant current of 20 mA for 2 h, and the product DNAs were
detected by exposing the gel to an x-ray film or an image plate of a
Fujix Bas 2000 image analyzer. The radioactivity found in a DNA band
was quantified with an image processor installed in the Fujix Bas 2000
image analyzer.
Figure 2:
Effects of 32 and 59 proteins on ATPase
activity of 41 protein. The measurement of ATP hydrolysis by 41 protein
was described under ``Materials and Methods.'' Fifty fmol of
pBluescript II KS- ssDNA, 7.0 pmol of 41 protein, and various amounts
of 32 protein as indicated on the abscissa were included in a
10-µl reaction mixture with or without 4.6 pmol of 59 protein.
, with 59 protein;
, without 59
protein.
Even with a lower than saturating amount, 32 protein was also highly inhibitory. For example, 4.5 pmol of 32 protein, which corresponds to 20% of the saturating amount, inhibited 80% of the ssDNA-dependent ATP hydrolysis activity. Since 80% of the ssDNAwas 32 protein-free at this subsaturating level, the observed inhibition cannot be explained by the inhibition of 41 protein binding to ssDNA (see ``Discussion''). The 59 protein had no detectable ATP hydrolysis activity. However, when 59 protein was added together with 32 protein, the inhibitory effect of the latter over the whole range of amounts tested was completely eliminated.
The two substrates were simultaneously added to a reaction mixture, and the fragments released from large circular ssDNA by unwinding of the duplex were analyzed by 12% polyacrylamide gel electrophoresis. Fig. 3shows the efficiencies of fragment release with various combinations of 32, 41, and 59 proteins. A duplex in substrate B was much more efficiently released by 41 protein than that in substrate A. This result is consistent with the result of Richardson and Nossal(1989) and strongly suggests that a duplex flanking an ssDNA gap represents a barrier for 41 protein to initiate unwinding.
Figure 3: DNA helicase activity with a set of substrates. Each reaction mixture (10 µl) contained 10 fmol each of substrates A and B. Proteins included in various combinations as indicated on the figure were 4.5 pmol of 32 protein, 1.2 pmol of 41 protein, and 1.7 pmol of 59 protein. The reaction conditions and detection of DNA products are described under ``Materials and Methods.''
Under the present conditions, neither 32 nor 59 protein had detectable helicase activity. Instead, 59 protein stimulated the helicase activity of 41 protein; the release of fragments from substrate A was undetectable with 41 protein alone, but it was discernible when both 41 and 59 proteins were included. Although the stimulatory effect of 59 protein with substrate B was not clear in this autoradiogram, experiments in which these substrates were separately assessed revealed that 59 protein also stimulated the helicase activity (Fig. 4B). On the other hand, 32 protein showed an inhibitory effect on the helicase activity with both substrates A and B ( Fig. 3and Fig. 4).
Figure 4:
Differential effects of 32 protein on DNA
helicase activity in the presence of 59 protein. A, a
10-µl reaction mixture contained 10 fmol of substrate A, 1.2 pmol
of 41 protein, and various amounts of 32 protein as indicated on the abscissa, with () or without (
) 110 fmol of 59
protein. B, a 10-µl reaction mixture contained 10 fmol of
substrate B, 0.3 pmol of 41 protein, and various amounts of 32 protein
as indicated on the abscissa, with (
) or without (
)
110 fmol of 59 protein. DNA fragment released by unwinding was measured
as described under ``Materials and
Methods.''
An intriguing effect of 32
protein was found by the observation that 32 protein further stimulated
the helicase activity with substrate A in the copresence of 59 protein (Fig. 3). The stimulation was significant over a limited range
of amounts of 32 protein (Fig. 4). As described under the
Introduction, 32 protein is considered to cover ssDNA inside a
T4-infected cell. This consideration led us to examine the quantitative
requirement for the 32 protein stimulation. When substrate A was
increased from 10 to 30 fmol in the presence of the same amounts of 41
and 59 proteins as indicated in Fig. 4, the amount of 32 protein
required for maximal activity was shifted about 3-fold, or from 3.0 to
4.5 pmol for 10 fmol DNA (Fig. 4) to 13.5 pmol for 30 fmol DNA
(data not shown). Since 450 monomers of 32 protein were necessary to
saturate one monomer of pBluescript ssDNA, these results suggest that
the DNA helicase activity is maximally stimulated when all ssDNA is
just covered by 32 protein. Fig. 4also suggests that excess 32
protein is inhibitory to the reaction even with 59 protein. This
inhibition could be explained by the possibility that free 32 protein
binds to 59 protein and sequesters 59 protein molecules available for
interaction with 32 proteinssDNA complex (see below), inasmuch as
these proteins have an affinity for each other (Yonesaki, 1994b). In
this context, the lack of apparent inhibition of ssDNA-dependent ATPase
activity by 32 protein in the presence of 59 protein, as shown in Fig. 2, would be attributable to the abundant presence of 59
protein.
In the reaction including substrate B, 32 protein over a wide range of amounts was always inhibitory to the reaction regardless whether or not 59 protein was present. However, the stimulatory effect of 59 protein was still remarkable, and the inhibition of the reaction in the presence of 59 protein was much weaker than that in its absence (Fig. 4). The contrasting effects of 32 protein on substrates A and B in the presence of 59 protein should be a reflection of the difference in DNA structure.
Figure 5:
Effects of 32 and 59 proteins on DNA
helicase activity with a gapped DNA as a substrate. A 10-µl
reaction mixture contained 10 fmol of substrate A and various amounts
of 41 protein as indicated on the abscissa, with or without
4.5 pmol of 32 protein and 55 fmol of 59 protein. DNA fragment released
by unwinding was measured as described under ``Materials and
Methods.'' Proteins included were as follows: , 41 protein
alone;
, 32 and 41 protein;
, 41 and 59 proteins;
,
32, 41, and 59 proteins.
Figure 7:
Stoichiometry of 32 protein-covered ssDNA
and 59 protein. A 10-µl reaction mixture contained 0.8 pmol of 41
protein and one of various amounts of 59 protein as indicated on the abscissa. Substrate A and 32 protein included were
respectively 10 fmol and 4.5 pmol () or 30 fmol and 13.5 pmol
(
). DNA fragment released by unwinding was measured as described
under ``Materials and Methods.'' The percentage of fragment
released in the absence of 59 protein was subtracted for the correction
of each series of experiments.
In the present study, we examined the effects of 59 protein on ssDNA-dependent ATPase activity and DNA helicase activity of 41 protein in the presence or absence of 32 protein. The results show that 59 protein has profound effects on these activities, particularly in the presence of 32 protein, and thereby that it may act as an enhancer of 41 protein. Since 59 protein has a specific affinity for both 32 and 41 proteins and can rapidly bind to ssDNA covered by 32 protein (Yonesaki, 1994), 59 protein would have a central role in the interactions of 32, 41, and 59 proteins.
The ssDNA-dependent ATP hydrolysis activity was completely suppressed by 32 protein at concentrations sufficient to saturate ssDNA, and 59 protein completely saved the activity (Fig. 2). This result is consistent with the observation of Barry and Alberts(1994) with ssDNA-dependent GTPase activity and follows their suggestion that 59 protein mediates the rapid loading of 41 protein onto 32 protein-covered ssDNA. However, their results were inconclusive about whether 59 protein is also necessary for the translocation of 41 protein on 32 protein-covered ssDNA once the 41 protein has been bound. As shown in Fig. 2, even a partial coverage of ssDNA by 32 protein exerted strong inhibition of ssDNA-dependent ATPase activity; one-fifth of the saturation amount of 32 protein on circular pBluescript II ssDNA inhibited 80% of the activity. Since up to 80% of the ssDNA region is free from 32 protein under these conditions, this 80% inhibition cannot come from the inhibition of 41 protein binding to ssDNA. Rather, clustered 32 protein molecules bound to ssDNA could conceivably slow down or block the movement of 41 protein along the ssDNA strand, and such interference might either halt the ATP hydrolysis by 41 protein or cause 41 protein molecules to fall off the DNA. The overcoming of such interference caused by the 32 protein would require a continuous action of 59 protein until the 41 protein passed over the region covered by a cluster of 32 proteins. This would be achieved by formation of a binary complex of 41 and 59 proteins and migration of this complex along the ssDNA strand.
In Barry and Alberts(1994), the effects of 59 protein on 41 protein helicase activity was measured indirectly, by measuring the rate of replication fork movement. In the present experiments, we have measured the DNA helicase activity directly, using helicase substrates, and the results have shown several important aspects of the functional interactions of three proteins. A set of substrate DNAs are helpful to distinguish the steps of the helicase reaction (Fig. 1). The 41 protein alone can efficiently cause progression of the unwinding of a duplex in which initiation of unwinding has already been initiated. Addition of 32 protein to the reaction is quite inhibitory. Similar to its effect on the ssDNA-dependent ATP hydrolysis reaction, 59 protein has a stimulatory effect on unwinding and largely suppresses the inhibition by 32 protein (Fig. 4). These effects also support the hypothesis that 59 protein promotes the binding of 41 protein when this is inhibited by 32 protein (Barry and Alberts, 1994).
By contrast, the initiation of unwinding by 41 protein alone is so weak that a duplex flanking a ssDNA gap must be a barrier for 41 protein to initiate the reaction. Addition of 59 protein, but not 32 protein, is stimulatory to the reaction. However, addition of 32 protein in the presence of 59 protein can further drive the reaction forward ( Fig. 3and Fig. 4). Thus, the initiation barrier seems to be best circumvented by the synergistic action of 32 and 59 proteins, suggesting a novel role for each of these proteins. Since a stoichiometric amount of 32 protein that just saturates ssDNA is required to sustain the maximal stimulation in the reaction, we propose that the 32 protein in the vicinity of the border of the duplex helps 41 protein to initiate unwinding, or that the binding of 32 protein to complementary ssDNA produced by unwinding keeps the strands from reannealing. The complex formation of 59 protein with 41 protein, as suggested from the results of the experiment on the ssDNA-dependent ATP hydrolysis reaction, may help 41 protein activity to overcome the initiation barrier. For the maximal activity of the 41 protein, eight monomers of 59 protein were calculated to be required per one 2900-nucleotide long ssDNA molecule covered with 32 protein (Fig. 7). The extremely low stoichiometry of the protein to ssDNA nucleotides supports the idea that 41 and 59 proteins comigrate after they form a complex on ssDNA.
Preincubation of 41 protein with 59 protein in the absence of DNA and 32 protein was found to inactivate the helicase activity (Fig. 6). We also found that insoluble complexes were formed when 41 and 59 proteins at high concentrations (1.2 µM each) were incubated together (data not shown). These facts strongly suggest that 59 protein stably binds to 41 protein and may support the ability of 41 and 59 proteins to form a binary complex on ssDNA.
Our present study reveals that 59 protein has an essential role in production of ssDNA when 41 protein reacts with a gapped DNA as a substrate. This feature is favorable to the suggestion that 41 protein functions in in vivo production of recombinogenic ssDNA and may account for involvement of both 41 and 59 genes in DNA recombination (Wu and Yeh, 1974; Shah, 1976; Wakem and Ebisuzaki, 1981; Yonesaki, 1994a). Based on the present results, Fig. 8schematically depicts the process for production of recombinogenic ssDNA. It will be important to examine whether ssDNA produced by cooperation of 32, 41, and 59 proteins is indeed utilized by a protein machine for single strand transfer, which is composed of uvsX, uvsY, and 32 proteins (Yonesaki and Minagawa, 1989).
Figure 8:
Production of recombinogenic ssDNA by a
synergistic action of 32, 41, and 59 proteins. A ssDNA in a gap
presumably generated by an exonuclease activity controlled by gene 46
and 47 is covered by 32 protein. The affinity between 32 and 59
proteins helps 59 protein to invade the 32 proteinssDNA complex.
Then, 41 protein forms a complex with 59 protein on the ssDNA, and the
complex of these two proteins migrates in a 5`
3` direction,
producing a ssDNA strand with a 3`-free
end.