From the Department of Biological Chemistry and Molecular Pharmacology, Harvard University Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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The gene 2.5 single-stranded DNA (ssDNA) binding protein of bacteriophage T7 is essential for T7 DNA replication and recombination. Earlier studies have shown that the COOH-terminal 21 amino acids of the gene 2.5 protein are essential for specific protein-protein interaction with T7 DNA polymerase and T7 DNA helicase/primase. A truncated gene 2.5 protein, in which the acidic COOH-terminal 21 amino acid residues are deleted no longer supports T7 growth, forms dimers, or interacts with either T7 DNA polymerase or T7 helicase/primase in vitro. The single-stranded DNA-binding protein encoded by Escherichia coli (SSB protein) and phage T4 (gene 32 protein) also have acidic COOH-terminal domains, but neither protein can substitute for T7 gene 2.5 protein in vivo. To determine if the specificity for the protein-protein interaction involving gene 2.5 protein resides in its COOH terminus, we replaced the COOH-terminal region of the gene 2.5 protein with the COOH-terminal region from either E. coli SSB protein or T4 gene 32 protein. Both of the two chimeric proteins can substitute for T7 gene 2.5 protein to support the growth of phage T7. The two chimeric proteins, like gene 2.5 protein, form dimers and interact with T7 DNA polymerase and helicase/primase to stimulate their activities. In contrast, chimeric proteins in which the COOH terminus of T7 gene 2.5 protein replaced the COOH terminus of E. coli SSB protein or T4 gene 32 protein cannot support the growth of phage T7. We conclude that an acidic COOH terminus of the gene 2.5 protein is essential for protein-protein interaction, but it alone cannot account for the specificity of the interaction.
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INTRODUCTION |
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The gene 2.5 protein encoded by bacteriophage T7 is a single-stranded DNA (ssDNA)1 binding protein similar to the Escherichia coli SSB protein and the T4 gene 32 protein (1, 2). The gene 2.5 protein, like its counterparts in the E. coli and T4 phage systems, is essential for DNA replication and plays key roles in recombination and DNA repair (3-6). Biochemical studies have shown that gene 2.5 protein modulates several essential reactions of DNA replication and recombination; it interacts physically with T7 DNA polymerase to stimulate its activity (7) and with the T7 helicase/primase to increase the efficiency of RNA primer synthesis (8).
The ability of ssDNA-binding proteins to bind tightly to ssDNA is undoubtedly essential for their roles in replication, but their interactions with other replication proteins underlie their ability to coordinate reactions at a replication fork. Not surprisingly, the physical interactions of the ssDNA-binding proteins with other proteins show considerable specificity. In the case of T7 replication, neither E. coli SSB protein nor T4 gene 32 protein can substitute for gene 2.5 protein to support the growth of T7 phage (3).2 The inability of T4 gene 32 protein to replace T7 gene 2.5 protein in vivo can be explained biochemically. Whereas T7 gene 2.5 protein physically interacts with T7 DNA polymerase to stimulate its activity on ssDNA templates, T4 gene 32 protein has only a minor effect (7). Likewise, T7 gene 4 helicase/primase is unable to load onto ssDNA coated with T4 gene 32 protein, a reaction that occurs readily with T7 gene 2.5 protein-coated DNA (4). On the other hand, the inability of E. coli SSB protein to substitute for gene 2.5 protein is more difficult to explain. For example, E. coli SSB protein stimulates the activity of T7 DNA polymerase equally as well as does the gene 2.5 protein (7), and it interacts with T7 gene 4 protein to allow its entry onto ssDNA (4, 8). The only observable difference between the two proteins resides in their effects on primase activity. Whereas gene 2.5 protein increases the frequency of initiation of lagging strand synthesis by greater than 10-fold, E. coli SSB protein has no such effect (8). The possibility remains, of course, that there are other, as yet unrecognized, interactions involving the gene 2.5 protein, interactions that are specific for the gene 2.5 protein.
It is well documented that T4 gene 32 protein and E. coli SSB protein interact with their cognate proteins. For example, Formosa et al. (9) used affinity chromatography to demonstrate that T4 gene 32 protein physically interacts with at least 10 T4-encoded proteins including T4 DNA polymerase, dda helicase, and UvsX, UvsY proteins, which are involved in T4 DNA replication, recombination, and repair. By DNA synthesis assay and density gradient centrifugation, it was shown that E. coli SSB protein interacts with E. coli DNA polymerase II (10), exonuclease I (11), and a component of the primosome complex, protein n (12). Some preliminary results also suggest that E. coli SSB protein interacts with Rep and uvrD proteins (13, 14). The specificity of the protein-protein interactions involving T4 gene 32 protein and E. coli SSB protein has also been documented. E. coli SSB protein stimulates the polymerase activity of both E. coli DNA polymerases II and III, but not T4 DNA polymerase (10, 15-17), and T4 gene 32 protein specifically stimulates T4 DNA polymerase activity (18).
Numerous studies on the ssDNA-binding proteins have provided insight
into the nature of the DNA binding domains and the domains involved in
the specific interactions with their cognate replication proteins
(19-21). Two regions of the T4 gene 32 protein, amino acid residues
9-21 and 253-275, are particularly susceptible to proteolytic
cleavage (22-27). Cleavage at both sites leaves a core protein that
retains its ability to bind to ssDNA. Loss of the NH2-terminal 9-21 residues abolishes cooperative binding
of the protein to ssDNA. Deletion of the COOH-terminal region, which is
relevant to our current studies, leaves a truncated T4 gene 32 protein
that no longer interacts with either the T4 DNA polymerase or the gene
61 primase (24, 28-30). Williams et al. (30) have shown
that the acidic COOH-terminal domain (23, 31, 42, or 62 amino acids) of
E. coli SSB protein can be removed by limited proteolysis
and that the resulting COOH-terminal truncated protein, like the
truncated T4 gene 32 protein, retains its ability to bind to ssDNA.
However, the question as to their ability to participate in
protein-protein interactions remains unanswered. The similar arrangement of domains in E. coli SSB protein and T4 gene 32 protein suggests that the acidic COOH-terminal domains of both proteins are functionally homologous. The COOH terminus of the T7 gene 2.5 protein is also involved in protein-protein interactions. A truncated
gene 2.5 protein, gene 2.5-21C protein, which lacks the
COOH-terminal 21 amino acids, cannot support the growth of phage T7,
and the purified mutant protein does not form dimers and does not
interact with T7 DNA polymerase or T7 helicase/primase (31). The
amino-terminal region of gene 2.5 protein contains a
tyrosine-rich putative ssDNA binding motif shared by other
ssDNA-binding proteins (32, 33).
Inasmuch as the COOH terminus of E. coli SSB protein, T4 gene 32 protein, and T7 gene 2.5 protein confers the ability of each protein to interact with its cognate replication proteins, the question arises as to the presence of a distinguishing structure or motif in this region. The COOH-terminal 25-amino acid sequence of each of the three proteins is shown in Fig. 1. The only distinctive feature of the COOH termini of all three proteins is the relatively high content of acidic residues. Of the carboxyl-terminal 21 residues, 6 in T4 gene 32 protein, 5 in E. coli SSB protein, and 15 in T7 gene 2.5 protein are acidic. No homology exists in this region among the three proteins.
To determine if the acidic COOH-terminal domain of the E. coli SSB protein, the T4 gene 32 protein, and the T7 gene 2.5 protein is solely responsible for the specificity of protein-protein interactions observed in vivo and in vitro, we have constructed chimeric ssDNA-binding proteins and examined their ability to support phage growth and to interact with other proteins in vitro. In this paper, we show that the COOH-terminal domain of either the E. coli or T4 ssDNA-binding protein can substitute for the COOH-terminal domain of the T7 gene 2.5 protein. However, neither E. coli SSB protein nor T4 gene 32 protein in which the COOH-terminal domain has been replaced by the comparable domain of T7 gene 2.5 protein can substitute for T7 gene 2.5 protein in vivo.
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EXPERIMENTAL PROCEDURES |
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E. coli Strains and Bacteriophages--
E. coli
strain HB101 (F mcrB mrr
hsdS20(rB
,
mB
)recA13 supE44
ara14 galK2 lacY1 proA2
rpsL20(Smr) xyl5
l
leu
mtl1) was used as
the host for plasmids pGP2.5-1, pGEM-gp2.5-ssb, and
pGEM-gp2.5-32. Phage T7
2.5 constructed by Kim and Richardson (3)
was prepared by amplifying this phage in E. coli W3110 (thyA
supo) harboring the
plasmid pGP2.5-1, which expresses the wild-type gene 2.5 protein.
Growth of phage T7 or T7
2.5 on HB101 or W3110 in liquid or solid
media was carried out as described (34). Burst sizes were determined as
described previously (35, 36). Bacteriophage T4D was kindly provided by
Dr. Ken Kreuzer (Duke University). The plasmids expressing wild-type
gene 32 protein were provided by Dr. David P. Giedroc (Texas A & M
University) and Dr. Yousif Shamoo (Yale University).
Plasmids, DNA, and Proteins--
Plasmid pGP2.5-1, which
contains wild-type T7 gene 2.5 under the control of the tetracycline
gene promoter, was constructed by Kim and Richardson (3). E. coli HB101 strain harboring the plasmid pGP2.5-1 was used for the
growth of phage T72.5; in this phage, the entire gene 2.5 is deleted
from the T7 genome. Plasmid vector pGEM-4Z harboring the promoter for
T7 RNA polymerase was purchased from Promega and was used to construct
the expression plasmids for the chimeric ssDNA-binding proteins
gp2.5-ssb and gp2.5-32. The partial DNA fragments of T7
gene 2.5, T4 gene 32, and E. coli gene ssb were
obtained by polymerase chain reaction from the genomic DNA of phage T4,
T7, and E. coli strain W3110, respectively. All
oligonucleotides were purchased from Integrated DNA Technologies,
Inc.
Construction of Plasmids Containing Genes for Chimeric
ssDNA-binding Proteins--
If not otherwise indicated, DNA
manipulations were performed according to the protocol described (39)
or according to the instruction of the supplier. T7
gp2.5-ssb and T7 gp2.5-32 are chimeric proteins consisting
of the NH2-terminal 209 amino acids from the
NH2-terminal region of T7 gene 2.5 protein and the
COOH-terminal 25 amino acids from the COOH-terminal region of E. coli SSB protein or the COOH-terminal 22 amino acids from the
COOH-terminal region of the T4 gene 32 protein, respectively. To
construct the genes encoding the two chimeric proteins, we first
inserted the polymerase chain reaction-amplified DNA fragments encoding
the NH2-terminal 209 amino acids of T7 gene 2.5 protein and
the ribosome binding site into the plasmid pGEM-4Z at PstI
and XbaI sites. Two oligonucleotide primers, one
containing a PstI site
(5'-GCGCGCCTGCAGTCTGAGAAACCAAACGAAACC-3') and the other a
XbaI site
(5'-GGCGGATCTAGATGGTTTGCTCGCTTTGGCAGA-3'), were used to
amplify the T7 DNA sequence 9119-9787. The polymerase chain
reaction fragment was purified by agarose gel electrophoresis, digested with restriction enzymes PstI and XbaI,
and inserted into plasmid pGEM-4Z at PstI and
XbaI sites to create plasmid pGEM-gp2.5-22C. To construct
the plasmid encoding T7 gp2.5-ssb, two oligonucleotides,
5'-CTAGACCGCAGCAGTCCGCTCCGGCAGCGCCGTCTAACGAGCCGCCGATGGACTTTGATGATGACATTCCGTTCTAAG-3' and
5'-AATTCTTAGAACGGAATGTCATCATCAAAGTCCATCGGCGGCTCGTTAGACGGCGCTGCCGGAGCGGACTGCTGCGGT-3', which are designed to encode COOH-terminal 23 amino acids of E. coli SSB protein, were annealed and inserted into pGEM-2.5-
22C at XbaI and EcoRI sites to yield plasmid
pGEM-gp2.5-ssb. In a similar method, the plasmid encoding T7
gp2.5-32 was constructed by annealing the two oligonucleotides,
5'-CTAGAAGCTCAAGCTCTGGTAGTTCATCTAGTGCTGATGACACGGACCTGGATGACCTTTTGAATGACCTTTAAG-3' and
5'AATTCTTAAAGGTCATTCAAAAGGTCATCCAGGTCCGTGTCATCAGCACTAGATGAACTACCAGAGCTTGAGCTT-3', which encode the COOH-terminal 22 amino acids of the T7 gene 32 protein, and inserting the resulting duplex into pGEM-gp2.5-
22C at
XbaI and EcoRI sites to yield plasmid
pGEM-gp2.5-32. An XbaI recognition sequence, 5'-TCTAGA-3',
is present in both the chimeric genes and encodes amino acids Ser-Arg.
The positions of Ser-Arg at chimeric protein gp2.5-ssb are
identical to that found at the identical position in the COOH terminus
of E. coli SSB protein. Therefore, the chimeric protein
gp2.5-ssb has the NH2-terminal 209 amino acids
from T7 gene 2.5 protein and the COOH-terminal 25 amino acids from
E. coli SSB protein. The gp2.5-32 chimeric protein has the
NH2-terminal 209 amino acids from T7 gp2.5 and the
COOH-terminal 22 amino acids from T4 gene 32 protein. The Ser-Arg
encoded by the XbaI recognition sequence is located at the
junction.
Overproduction and Purification of Chimeric Proteins gp2.5-ssb
and gp2.5-32--
E. coli HB101 cells carrying plasmid
pGEM-gp2.5-ssb or pGEM-gp2.5-32 were grown at 30 °C in
500 ml of medium consisting of 2% tryptone, 1% yeast extract, 0.5%
NaCl, 0.2% casamino acids, 40 mM
K3PO4, pH 7.4, and 50 µg/ml ampicillin. At an
A590 of 1.0, bacteriophage CE6 carrying the
gene encoding T7 RNA polymerase was added to the culture at a
multiplicity of infection of 8. Incubation was continued for an
additional 3 h, and the cells were harvested and stored at
80 °C. The chimeric proteins were purified by the procedure used
for purification of wild-type gene 2.5 protein (4).
Oligoribonucleotide Synthesis by 63-kDa T7 Gene 4 Primase--
Oligonucleotide synthesis by the T7 DNA primase (63-kDa
gene 4 protein) was determined by measuring the amount of radioactively labeled oligonucleotide after electrophoretic separation (37). The
reaction (10 µl) contained 40 mM Tris-Cl (pH 7.5), 10 mM MgCl2, 5 mM dithiothreitol
(DTT), 50 mM potassium glutamate, 0.1 mM dTTP, 0.5 mM concentration each of ATP, CTP, UTP, and GTP, 10 ng
of M13mp18 ssDNA, 400 ng of ssDNA-binding protein. After incubation at
37 °C for 5 min to allow ssDNA-binding protein to bind to ssDNA M13mp18, 5 µCi of [-32P]CTP and 100 ng of gene 4 protein were added to the reaction. After incubation at 37 °C for an
additional 40 min, the reaction was stopped by the addition of 50 mM EDTA. Products of the reaction were analyzed by 25%, 1 M urea-PAGE as described previously (37).
DNA Synthesis Catalyzed by T7 DNA Polymerase-- The T7 DNA polymerase assay was a modification of that described by Tabor and Richardson (40). The reaction mixture (40 µl) contained 250 ng of M13mp18 ssDNA primed by a 51-nucleotide oligonucleotide, 50 mM Tris-Cl, pH 7.5, 10 mM MgCl2, 5 mM DTT, 50 mM NaCl, 0.3 mM dNTPs, 0.4 µCi of [3H]dGTP, 10 ng of T7 DNA polymerase, and the indicated amount of ssDNA-binding protein. The reaction was incubated at 30 °C for 3 min and stopped by the addition of EDTA to 50 mM, and then the reaction was transferred to Whatman DE81 filter. The DE81 filters were kept at room temperature to dry for 30 min and then washed with 0.3 M ammonium formate (pH 8.0) four times and once with 95% ethanol. The filters were dried thoroughly, and the radioactivity retained on the filters was determined by scintillation counting.
Affinity Chromatography--
T7 gp2.5-21C,
gp2.5-ssb, and gp2.5-32 were covalently coupled to Affi-Gel
10 following the manufacturer's instruction. The efficiency of
coupling to the resin was 87% for T7 gp2.5-
21C, 90% for
gp2-5-ssb, and 91% for gp2.5-32. T7 DNA polymerase (0.2 mg) was mixed with 0.1 ml (drained volume) of the Affi-Gel 10 covalently linked to each of the three ssDNA-binding proteins and
incubated for 15 min at 4 °C with gentle mixing. The mixture was
transferred to a pipette tip column, and the column was washed with 1 ml of 20 mM Tris-Cl (pH 7.5), 0.1 mM EDTA, 0.1 mM DTT, and 10% glycerol (buffer A) to remove any unbound
T7 DNA polymerase. A step gradient (1 ml) of buffer A containing 50, 100, 150, 200, or 250 mM NaCl was used to elute T7 DNA
polymerase bound to the affinity column. All fractions were analyzed by
absorbance at 280 nm, and the presence of T7 DNA polymerase in each
fraction was confirmed by SDS-PAGE.
Molecular Weight Determination of Chimeric Proteins by Gel
Filtration--
The native molecular weights of the chimeric proteins,
gp2.5-ssb and gp2.5-32, were determined by gel filtration
on a Superose 12 column (0.79 cm2 × 47.5 cm). The buffer
for all experiments was 50 mM
K3PO4, pH 7.0, 150 mM NaCl, 0.1 mM EDTA, 0.1 mM DTT, and 10% glycerol. Gel filtration was carried out at 4 °C with a flow rate of 0.1 ml/min. 100 µg of gp2.5-ssb or gp2.5-32 at concentrations of 0.20 and 0.28 mg/ml, respectively, were applied to the column, and fractions (0.25 ml for each fraction) were collected. The presence of protein in
each fraction was detected by 10% PAGE. A standard curve of Kav versus
log10Mr was determined by
chromatographing separately standard proteins (low molecular weight gel
filtration calibration kit from Pharmacia). Blue dextran and xylene
were used to determine the void volume (vo) and the
total volume (vt), respectively.
Kav, the fractional retention, was calculated
according to the formula Kav = (ve vo)/(vt
vo), where ve is the peak
elution volume for each protein.
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RESULTS |
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Construction of Chimeric ssDNA-binding Proteins--
A genetically
altered T7 gene 2.5 protein, gene 2.5-21C protein, lacking the
COOH-terminal 21 amino acid residues, no longer interacts with T7 DNA
polymerase or helicase/primase and cannot support the growth of T7
phage lacking gene 2.5. These results suggest strongly that interaction
between the gene 2.5 protein and other T7 DNA replication proteins are
essential for T7 DNA replication and phage growth. Unresolved, however,
is whether the acidic COOH-terminal domain confers specificity on the
interaction with T7 DNA polymerase or the helicase/primase or merely
provides a proper conformation or electrostatic charge to the protein
while specificity of the protein-protein interactions resides elsewhere in the protein.
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Chimeric ssDNA-binding Proteins Support T7 Phage Growth--
To
determine if the COOH-terminal domain of E. coli SSB protein
or T4 gene 32 protein could substitute for the COOH-terminal domain of
T7 gene 2.5 protein to support phage growth, we transformed E. coli HB101 with the two plasmids encoding each of the two chimeric proteins. In the experiment presented in Table
I, we examined the ability of T7 phage
lacking gene 2.5, T72.5, to produce plaques on E. coli
cells harboring each of the plasmids encoding the chimeric proteins.
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Chimeric ssDNA-binding Proteins Form Dimers--
T7 gene 2.5 protein, T4 gene 32 protein, and E. coli SSB protein all
form multimers. Gene 2.5 protein is a dimer in solution (1); T4 gene 32 protein exists as a monomer or dimer at low concentration but can form
higher molecular weight species at higher concentrations (2). E. coli SSB protein, on the other hand, is a tetramer of four
identical subunits (41). Removal of the COOH-terminal domain diminishes
the stability of the tetramers (30). In the case of T7 gene 2.5 protein, the acidic COOH-terminal domain is not only essential for
interactions with other replication proteins, but it is also essential
for interactions with other gene 2.5 proteins to form a dimer; T7 gene
2.5-21C protein, lacking the 21 COOH-terminal residues, exists as a
monomer in solution although it is fully soluble and binds normally to
ssDNA (31).
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Physical Interaction between the Chimeric Proteins, gp2.5-32 and
gp2.5-ssb, and T7 DNA Polymerase--
T7 gene 2.5 protein physically
interacts (Kd of 1.1 µM) with T7 DNA
polymerase to form a 1:1 complex as measured by steady-state
fluorescence emission anisotropy (7). In contrast, T7 gene 2.5-21C
protein, lacking the COOH terminus, does not physically interact with
T7 DNA polymerase (31). If protein-protein interactions involving the
gene 2.5 protein are essential for T7 DNA replication, it seems likely
that the chimeric proteins, since they support T7 phage growth, should
physically interact with T7 DNA polymerase.
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Stimulation of T7 DNA Polymerase--
T7 gene 2.5 protein, like
E. coli SSB protein and T4 gene 32 protein (10, 18, 42),
stimulates the activity of its cognate DNA polymerase (7, 38, 43-45).
T7 gene 2.5-21C on the other hand has no affect on T7 DNA polymerase
activity, although it retains its ability to bind to ssDNA (30), a
result that is not surprising in view of its inability to interact
physically with T7 DNA polymerase as presented above. We have compared
the ability of wild-type gene 2.5 protein, T7 gp2.5-
21C, and the two
chimeric ssDNA-binding proteins to stimulate DNA synthesis catalyzed by
T7 DNA polymerase on primed M13 DNA as a primer-template (Fig.
5). Such a comparison is of interest,
since the four gene 2.5 proteins vary in their affinity for T7 DNA
polymerase in the relative order
gp2.5>gp2.5-ssb>gp2.5-32>gp2.5-
21C.
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Stimulation of Gene 4 Primase--
Gene 4 of phage T7 encodes a
full-length 63-kDa protein and a second, co-linear protein, the 56-kDa
gene 4 protein (37). The 56-kDa gene 4 protein, the T7 DNA helicase, is
expressed from an internal ribosome binding site and start codon
located 189 bases from the 5'-end of the gene 4 open reading frame
(46). The 63-kDa gene 4 protein, by virtue of the additional 63 amino-terminal residues, in addition to being a helicase, is also a
primase that catalyzes the template-directed synthesis of
oligoribonucleotides, which in turn function as primers for T7 DNA
polymerase (37, 47). The 63-kDa gene 4 protein can thus supply both
helicase and primase activities at the replication fork, and hence it
alone is sufficient to support T7 DNA replication and phage growth (48, 49). Gene 2.5 protein physically interacts with both molecular weight
forms of gene 4 protein (7) and stimulates the synthesis of
oligoribonucleotides by the 63-kDa primase (8). Again, the COOH-terminal domain of gene 2.5 protein has been shown to be essential
for the interaction of gene 2.5 protein with gene 4 protein (4, 31). T7
gp2.5-21C, lacking the COOH terminus, cannot bind to gene 4 protein
(31), and it inhibits the gene 4 protein-mediated strand transfer
reaction, presumably due to the inability of the gene 4 protein to bind
to gp2.5-
21C coated DNA (4).
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Stimulation of T7 DNA Polymerase and Primase by Gene 2.5 Proteins Is Not Affected by COOH-terminal Peptides of T7 Gene 2.5 Protein or T4 Gene 32 Protein-- Although the COOH-terminal domains of all three ssDNA-binding proteins used in this study are sufficient to allow gene 2.5 protein to interact with other proteins, it is not known if the acidic COOH-terminal domains themselves directly dock with T7 DNA polymerase or T7 DNA helicase/primase. To address this point, we have examined the effect of synthetic peptides having the same amino acid sequence as that of the 21 COOH-terminal residues of gene 2.5 protein or the 24 COOH-terminal residues of T4 gene 32 proteins on the ability of T7 gene 2.5 protein or the chimeric gp2.5-32 protein, respectively, to stimulate T7 DNA polymerase and T7 gene 4 primase (Fig. 7). The results show that the COOH-terminal peptides, even at relatively high molar ratios, do not impair the ability of the gene 2.5 protein or the gp2.5-32 protein to stimulate either reaction. The results suggest that the peptides do not bind to T7 DNA polymerase or to T7 DNA helicase/primase.
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DISCUSSION |
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ssDNA-binding proteins such as the T7 gene 2.5 protein, the T4 gene 32 protein, and E. coli SSB protein discussed in this paper are involved in DNA replication, recombination, and repair (2). All three proteins are essential for phage or bacterial growth (2, 3). In vitro studies with these proteins have revealed that they modulate a large number of reactions involving proteins that interact with DNA. Herein lies a major difficulty in assigning relative importance to these in vitro effects, since a complete genetic analysis of the multiple effects has not yet been compiled. A second complication in addressing the essential role of these proteins in vivo is that both the ability of the protein to bind to ssDNA and its ability to interact with other proteins of DNA metabolism must be considered. One approach to this problem is to examine each property separately.
We have shown previously that the acidic COOH-terminal domain of the T7
gene 2.5 protein is required for several of the known protein-protein
interactions involving this essential protein (31). Deletion of the
COOH-terminal 21 acidic residues of gene 2.5 protein yields a truncated
protein, T7 gene 2.5-21C, that retains its ability to bind to ssDNA
with the same affinity as does wild-type gene 2.5 protein. The
retention of DNA binding is not surprising, since the domain putatively
responsible for the binding, a domain found in a number of
ssDNA-binding proteins (32), is present in the amino-terminal region of
gene 2.5 protein. However, T7 gene 2.5-
21C protein no longer
interacts with itself to form dimers or with T7 DNA polymerase or the
T7 gene 4 helicase/primase to stimulate their activities (4, 31). Our
findings that dimer formation is not necessary for binding to ssDNA
demonstrated the essential nature of protein-protein interactions; gene
2.5-
21C protein cannot support the growth of T7 phage lacking gene
2.5. Likewise, the loss of a physical interaction of gene 2.5-
21C with T7 DNA polymerase and the helicase/primase and a loss of its
ability to stimulate their activities further emphasize the importance
of these protein-protein interactions.
In the present study, we have attempted to address the specific role of the COOH terminus of gene 2.5 protein in protein-protein interactions. The approach we have used is based on the presence of a similar COOH-terminal domains in T4 gene 32 protein and in E. coli SSB protein (2). A number of studies have shown a similar separation of domains in the T4 and E. coli proteins. In E. coli SSB protein, the ssDNA binding domain has been shown to reside, at least partly, in the amino-terminal region (29, 30, 53-56). More important to the current work are a number of studies that implicate the acidic COOH-terminal domains of the T4, T7, and E. coli proteins in specific protein-protein interactions. In the case of T4 gene 32 protein, limited proteolysis has been used to generate three active fragments (23, 28, 54). Cleavage between residues 9 and 21 removes the NH2-terminal region, producing gp32-B, while cleavage between residues 253 and 275 removes the acidic COOH-terminal A region, producing gp32-A. Cleavage at both sites results in gp32-(A + B). As expected from the above discussion, the B domain was implicated in cooperative ssDNA binding, but the acidic A domain was found to be responsible for interactions with other T4 replication proteins. Similar proteolytic studies have shown that the COOH-terminal domain of E. coli SSB protein is involved in stabilizing the tetrameric structure of the protein (30).
The COOH termini of all three of the prokaryotic ssDNA-binding proteins discussed above are highly acidic, and deletion of these COOH-terminal acidic residues eliminates their ability to interact with their cognate DNA replication proteins. This raises the question as to whether or not the specificity for these interactions resides exclusively within this domain. The lack of homology among the COOH-terminal domains of T4, T7, and E. coli ssDNA-binding proteins shown in Fig. 1 seems to imply their roles in specific protein-protein interaction in vitro and to account for the inability for one ssDNA-binding protein to substitute for another in vivo. In this study, we have used the direct approach of substituting the COOH-terminal domains of the T4 gene 32 protein and the E. coli SSB protein for the corresponding COOH-terminal domain of T7 gene 2.5 protein and likewise replacing the COOH-terminal domains of the former two proteins with the COOH-terminal domain of the T7 gene 2.5 protein. These chimeric proteins were then examined for their ability to support the growth of T7 phage and to interact with DNA replication proteins encoded by phage T7.
The chimeric proteins bearing the COOH-terminal domain of gene 2.5 protein were not able to support growth of T7, not a surprising result in view of the fact that the major portions of these three ssDNA-binding proteins are nonhomologous and are certain to have different tertiary structures. This result may further indicate that the NH2-terminal region may have functions, in addition to its binding to ssDNA, such as in specific protein-protein interaction. Although no further studies were carried out with these chimeric proteins, it would be of interest to see if they could physically interact with T7 DNA polymerase and the gene 4 protein to stimulate their activities and to determine if they are monomers, dimers, or tetramers in solution.
Our results show that the acidic COOH-terminal domain of either T4 or E. coli SSB protein can substitute for the COOH-terminal region of gene 2.5 protein to provide for T7 DNA replication and phage growth, albeit slightly less efficiently compared with wild-type gene 2.5 protein. Thus, the COOH-terminal domain of gene 2.5 protein is essential for mediating protein-protein interactions, but the specificity for a functional interaction must reside elsewhere on the protein. This in vivo interpretation was confirmed in vitro by the demonstration that purified chimeric proteins physically interact with T7 DNA polymerase and helicase/primase and stimulate both the polymerase and the primase. Furthermore, the chimeric proteins readily form dimers in solution as does the wild-type gene 2.5 protein, a property that is dependent on the presence of the COOH terminus (31). This latter observation also suggests that, although the COOH terminus is necessary for dimerization, the determinant for dimer formation might reside elsewhere in the gene 2.5 protein. E. coli SSB protein is a tetramer in solution, and its COOH terminus has also been implicated in tetramer formation (30).
What precisely is the role of the acidic COOH-terminal domain of ssDNA-binding proteins? The fact that this domain is essential for interacting with other proteins but not for specificity suggests that it interacts with other residues within the protein to induce a conformational change that is required for protein interaction and multimer formation. Such a role would provide a number of mechanisms for modulating its affinity for replication proteins and perhaps for ssDNA to which it also binds. In fact, the removal of COOH termini of E. coli SSB protein and T4 gene 32 protein increases the helix-destabilizing ability of these two proteins (2, 30). Our observation that the synthetic COOH-terminal peptides of the ssDNA-binding protein do not compete with the gene 2.5 protein for binding with either T7 DNA polymerase or primase support the interpretation that the COOH terminus does not directly dock with these proteins. It is interesting that gene 2.5 protein is a dimer, a structure that is dependent on the presence of the COOH terminus, yet interacts with T7 DNA polymerase and presumably gene 4 protein as a monomer (7). This result implies that either the COOH-terminal domains of the two gene 2.5 proteins in the dimer contact one another and are thus not available to bind with other proteins or else that conformational changes in the protein dictate the specificity of binding. In the latter case, the dimer must dissociate in order for the COOH terminus of the gene 2.5 protein to mediate another conformational change suitable for binding to T7 DNA polymerase.
Our earlier results on helicase-mediated strand transfer suggested that
the interaction of gene 2.5 protein with T7 gene 4 helicase was
essential for binding of the helicase to gene 2.5 protein-coated ssDNA
(4). The presence of T7 gene 2.5-21C protein on ssDNA entirely
prevented the gene 4 protein from entering the strand transfer
reaction. In this same reaction, T4 gene 32 protein also inhibits
whereas E. coli SSB protein does not. Likewise, T4 gene 32 protein only slightly stimulates T7 DNA polymerase, whereas E. coli SSB protein markedly stimulates the polymerase-catalyzed reaction (7). In the case of E. coli SSB protein, a physical interaction with the T7 replication proteins can be invoked, since E. coli SSB protein has been shown to physically interact
with T7 DNA polymerase by sedimentation analysis (11). It may well be
that T7 has evolved such that both its DNA polymerase and helicase can
interact with E. coli SSB protein, since upon infection of E. coli there is an abundance of SSB protein. If so, then
one must postulate other reactions involving gene 2.5 protein,
reactions that are unique for gene 2.5 protein. Such alternatives may
include the ability of gene 2.5 protein to facilitate homologous base pairing (4) and its role in establishing a functional replisome at the
T7 replication fork.3
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ACKNOWLEDGEMENTS |
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We thank Young Tae Kim for plasmid pGP2.5-1
and phage T72.5, Dr. Ken Kreuzer for providing T4D phage, and Dr.
Giedroc and Dr. Shamoo for supplying the plasmids containing wild-type
T4 gene 32. We also are very grateful to U. I. Richardson and T. Kusakabe for comments and constructive criticisms on the
manuscript.
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FOOTNOTES |
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* This work was supported by U.S. Public Health Services Grant AI-06045 and by American Cancer Society Grant NP-1Z.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 Biological
Chemistry and Molecular Pharmacology, Harvard University Medical School, 250 Longwood Ave., Boston, MA 02115. Tel.: 617-432-1864; Fax:
617-432-3362; E-mail: ccr{at}bcmp.med.harvard.edu.
1 The abbreviations used are: ssDNA, single-stranded DNA; SSB, ssDNA-binding protein; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis.
2 D. Kong and C. C. Richardson, unpublished results.
3 J. Lee and C. C. Richardson, unpublished results.
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REFERENCES |
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