From the
Department of Biological Chemistry and
Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115
and the
Lineberger Comprehensive Cancer Research
Center, University of North Carolina, Chapel Hill, North Carolina 27599
Received for publication, April 2, 2003 , and in revised form, May 9, 2003.
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ABSTRACT |
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INTRODUCTION |
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In addition to binding ssDNA and physically interacting with T7 DNA
polymerase, gene 2.5 protein also facilitates the annealing of complementary
strands of DNA (10,
11,
16). Homologous DNA annealing
is a vital activity during the process of DNA replication, recombination, and
repair (17). A number of
proteins have evolved to carry out this vital function, such as the RecA
protein (18,
19) and members of the single
strand annealing family that includes the E. coli RecT protein, the
Red protein from bacteriophage
, and the eukaryotic annealing
protein Rad52 (17,
2023).
Unlike the RecA protein, the gene 2.5 protein does not require ATP
(16), and it cannot mediate
strand transfer (11,
16). Gene 2.5 protein bears
some similarity to the RecT protein and its family members, proteins that also
mediate DNA annealing in an ATP-independent fashion
(17). Structurally, gene 2.5
protein differs from members of this family, which form multimeric ring
structures in the presence and absence of ssDNA
(2426).
Gene 2.5 protein, on the other hand, is a dimer in solution
(2), and its three-dimensional
structure resembles that of other ssDNA-binding proteins
(27). Similar to T4 gene 32
protein and E. coli SSB protein, gene 2.5 protein features an
oligonucleotide/oligosaccaride binding fold
(Fig. 1) (27). Although both T4 gene 32
protein and E. coli SSB protein have been shown to mediate DNA
annealing (28,
29), T7 gene 2.5 protein does
so much more efficiently
(16).
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The biochemical basis of the efficient DNA annealing activity of gene 2.5
protein is unknown. It seems likely that it involves interactions between two
gene 2.5 protein-coated ssDNA molecules. A previous study has shown that the
ability to bind ssDNA is critical for this reaction to occur
(30). It is also possible that
interactions at the dimer interface are involved in this process. Two gene 2.5
proteins with alterations in the dimer interface retained the ability to
mediate DNA annealing, in a manner similar to the WT protein, whereas a third
did so in a slightly longer time period
(31). We have recently
employed a genetic screen to identify functional domains in gene 2.5 protein
(31). One of the alterations
uncovered by the screen mapped to a loop connecting the prominent
-helix to the
-barrel portion of the structure
(Fig. 1). The exact residue,
Arg-82, resides in a disordered region of the structure. Here we describe the
purification and characterization of this protein and show that it is
defective in DNA annealing.
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EXPERIMENTAL PROCEDURES |
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Plasmids, Oligonucleotides, and ProteinsThe following
oligonucleotides were purchased from Oligos Etc.: T72.5NdeI
(5'-CGTAGGATCCATATGGCTAAGAAGATTTTCACCTC-3'), T72.5BamH1
(5'-CGTAGGATCCACTTAGAAGTCTCCGTC-3'), and Oligo 70
(5'-GACCATATCCTCCACCCTCCCCAATATTGACCATCAACCCTTCAC
CTCACTTCACTCCACTATACCACTC-3'). The oligonucleotide BCMP206
(5'-TAACGCCAGGGTTTTCCCAGTCACG-3') was synthesized by the
Biopolymer Laboratory, Harvard Medical School. M13 (mGP1-2) DNA and T7 DNA
polymerase lacking exonuclease activity
(30) were kindly provided by
Stanley Tabor (Harvard Medical School). Gene 2.5 protein-26C was
provided by Edel Hyland (Harvard Medical School). His-gene 2.5
protein-
26C was provided by James Stattel (Harvard Medical School). T7
DNA polymerase was provided by Donald Johnson and Joon-Soo Lee (Harvard
Medical School). Purification of WT gene 2.5 protein and His-gene 2.5 protein
was described previously (31).
E. coli SSB protein was purchased from U.S. Biochemical Corp. All
other proteins were purified as described below.
In Vivo DNA Synthesis AssayPhage DNA synthesis was
determined as described previously
(31). E. coli HMS262
cells transformed with pETGP2.5-R82C were grown in Davis minimal media
supplemented with ampicillin at 30 °C. Cells were infected with
T72.5 phage at a multiplicity of infection of 7. At 5-min intervals
postinfection, 200-µl samples were removed. [3H]thymidine
(50µCi/ml) was added, and after a 90-s incubation at 30 °C, 40 µl of
an ice-cold solution of 50 mM Tris-HCl (pH 7.5), 2 mM
EDTA, 2% SDS was added to the sample. The lysed cells were then spotted onto
DE81 filters, washed, and air-dried. [3H]Thymidine incorporation
into DNA was then measured by liquid scintillation counting.
Protein PurificationGene 2.5 protein-R82C was overproduced and purified using a procedure previously employed to purify WT gene 2.5 protein (31). A 1-liter culture of E. coli BL21(DE3) (Novagen) expressing gene 2.5 protein-R82C was grown, and gene 2.5 protein-R82C was purified by precipitation in polyethyleneimine (pH 7.5), followed by fractionation on an HQ column and a gel filtration column. The protein was 99% pure as determined by denaturing polyacrylamide gel electrophoresis followed by Coomassie Blue staining and was free of contaminating deoxyribonuclease activity (data not shown). Protein concentrations were calculated from UV spectrophotometer readings at 280 mM, using the calculated extinction coefficient at 280 nm of 2.59 x 104 M1 cm1 for gene 2.5 protein-R82C (32). His-tagged gene 2.5 protein-R82C was purified using a previously described method (31).
Determining DNA Binding Affinity by Electrophoretic Mobility Shift
AssayThe ssDNA binding activity of gene 2.5 protein was assessed
by a electrophoretic mobility shift assay
(31). Gene 2.5 proteins
(diluted in a buffer of 20 mM Tris (pH 7.5), 10 mM
-mercaptoethanol, and 500 µg/ml bovine serum albumin) were incubated
with 3 nM33P-end-labeled 70-mer oligonucleotide, 15
mM MgCl2, 5 mM dithiothreitol, 50
mM KCl, 10% glycerol, 0.01% bromophenyl blue. ssDNA was separated
from ssDNA-protein complex on 10% TBE Ready Gels (Bio-Rad) running in
0.5x Tris/glycine buffer (12.5 mM Tris base, 95 mM
glycine, 0.5 mM EDTA). Gels were dried and exposed to a Fujix
phosphor imager plate, and the amount of radioactivity was calculated using
ImageQuant software.
DNA Annealing AssayThe ability of WT gene 2.5 protein to mediate the annealing of homologous strands of DNA was assessed using an in vitro annealing assay developed by Tabor and Richardson (16). A 310-nucleotide internally labeled ssDNA fragment was generated as described previously (16, 31). DNA annealing was assayed in reactions containing 4 nM32P-labeled ssDNA fragment, 20 µM M13 mGP1-2 ssDNA, 20 mM Tris-Cl (pH 7.5), 1 mM dithiothreitol, 10 mM MgCl2, 50 mM NaCl, and various concentrations of gene 2.5 protein. Unless noted otherwise, reactions were incubated at 30 °C for 8 min. Time course experiments were carried out at 30 °C with 10 µM gene 2.5 protein, and the reaction was stopped by the addition of SDS to a final concentration of 0.5%. Reaction products were analyzed on a 0.8% agarose gel at 75 V for 1 h at room temperature, dried under vacuum, and exposed to a Fujix phosphor imager plate, and radioactivity was calculated using ImageQuant software. Plots of the data represent the background-corrected average of three experiments.
Electron MicroscopyWT and altered gene 2.5 proteins or E. coli SSB protein were diluted to 500 ng/µl in 20 mM Hepes/NaOH, (pH 7.5), 20% glycerol, mixed with single-stranded WT M13 DNA at 25 ng/µl in a buffer containing 10 mM Hepes/NaOH (pH 7.5), 50 mM NaCl final concentration. MgCl2 was added to the reaction buffer to 10 mM where indicated. Binding reactions with protein/DNA ratios (µg/µg) ranging from 40:1 for WT gene 2.5 protein to 10:1 for mutants and E. coli SSB protein were incubated for 15 min at room temperature in a 50-µl total reaction volume.
Following the binding reactions, the samples were fixed with an equal volume of 1.2% glutaraldehyde for 5 min at room temperature and then loaded onto a 2-ml column of Bio-Gel A-5m previously equilibrated in 10 mM Tris·HCl (pH 7.5), 0.5 mM EDTA. The same buffer was then used to elute the sample from the column and 250-µl fractions were collected. Aliquots of the protein-DNA containing fractions were mixed with a buffer containing spermidine (33) for 3 s and quickly applied to a mesh copper grid coated with a thin carbon film, glow-charged shortly before sample application. Following adsorption of the samples to the electron microscopy support for 12 min, the grids were subjected to a dehydration procedure in which the water content of the wash solutions was gently replaced by a serial increase in ethanol concentration to 100% and then air-dried. The samples were then rotary shadowcast with tungsten at 107 torr and examined in a Philips CM 12 TEM instrument at 40 kV. Micrographs, taken at x 46,000, were scanned using a Nikon LS-4500AF film scanner, and panels were arranged using Adobe Photoshop.
Gel Filtration AnalysisGel filtration analysis was performed as previously described (31). Fifty µg of gene 2.5 protein-R82C diluted in buffer S (final concentration 4 µM) were loaded on a Superdex 75 column (Amersham Biosciences). A standard curve of Kav versus log Mr was generated by applying low molecular weight protein standards (Amersham Biosciences) to the column under the same conditions.
Analysis of Protein-Protein Interaction by Surface Plasmon
ResonanceThe interaction between gene 2.5 protein and T7 DNA
polymerase was measured by SPR using the BIACORE 3000 system as described
previously (31). Briefly, 10
µl of 500 nM histidine-tagged gene 2.5 protein, gene 2.5
protein-R82C, and gene 2.5 protein-26C were immobilized onto separate
lanes of a nickel-charged sensor chip NTA (BIAcore). This amount of protein
correlated to
7,000 resonance units. Ten µl of 500 nM T7
DNA polymerase or bovine serum albumin were passed over the chip, and
dissociation of T7 DNA polymerase was monitored for 10 min while passing 100
µl of running buffer over the chip. Each analysis was performed in
triplicate and repeated on three separate days. The kinetics of the gene 2.5
protein-T7 DNA polymerase interaction was assessed by binding 50 nM
of either WT or mutant histidine-tagged gene 2.5 protein to the nickel-charged
chip and then passing 10 µl of 050 nM T7 DNA over the
chip. BIAevaluation software was used to determine dissociation constants
(KD), which were solved using the simultaneous
ka/kd data fit.
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RESULTS |
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Since gene 2.5 is an essential gene and its product is involved in DNA
synthesis in vitro, we examined the ability of gene 2.5 protein-R82C
to carry out DNA synthesis in vivo. E. coli cells expressing the WT
or mutant gene 2.5 protein were grown to midlog phase and then infected with a
T7 phage lacking gene 2.5. At specific time points, aliquots of cells were
removed and mixed with radioactively labeled thymidine. After 90 s, the
reactions were terminated. Results of such an experiment are shown in
Fig. 2. DNA synthesis peaks
30 min after infection in cells expressing WT gene 2.5. As a control, no
DNA synthesis is observed in cells harboring gene 2.5 lacking the coding
sequence for the carboxyl-terminal motif (gene 2.5 protein-
26C).
Similarly, DNA synthesis declines soon after infection in cells expressing
gene 2.5 protein-R82C. Therefore, it is likely that this mutant is lethal
because it is defective in some aspect of DNA metabolism.
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Gene 2.5 Protein-R82C Binds ssDNAOne of the primary
attributes of gene 2.5 protein is its ability to bind ssDNA
(2). In the current study, we
assessed the ability of the altered gene 2.5 proteins to bind ssDNA using an
electrophoretic mobility shift assay. Using this method, we previously
calculated the dissociation constant (KD) for WT
gene 2.5 protein to be 2.6 x 106
M (31). As shown in
Fig. 3, the mobility of a
70-mer oligonucleotide is retarded as increasing amounts of gene 2.5
protein-R82C are added. Gene 2.5 protein-R82C binds ssDNA with 10-fold
higher affinity than does the WT protein (KD =
3.0 x 107 M). Thus, the amino
acid alteration causes the protein to bind ssDNA with a higher affinity than
WT gene 2.5 protein. Since gene 2.5 protein-R82C retains this vital function,
we consider it unlikely that the alteration results in a mis-folded
protein.
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Like other ssDNA binding proteins, WT gene 2.5 protein binds ssDNA with a much higher affinity than double-stranded DNA (2). We examined the binding of gene 2.5 protein-R82C to double-stranded DNA using the electrophoretic mobility shift assay. Gene 2.5 protein-R82C bound a double-stranded 70-base pair DNA weakly and in a manner similar to the WT protein (data not shown). Thus, whereas the alteration, arginine 82 to cysteine, conferred higher ssDNA-binding affinity upon gene 2.5 protein, it did not lead to increased double-stranded DNA binding activity.
Gene 2.5 Protein-R82C Is Defective in DNA AnnealingGene 2.5 protein can anneal homologous strands of ssDNA in vitro (16, 30, 31). In this study, we looked at the ability of WT and altered gene 2.5 proteins to anneal a 310-nucleotide ssDNA fragment to single-stranded M13 DNA. As previously shown (16), WT gene 2.5 protein can efficiently anneal these homologous strands of DNA (Fig. 4A). In this reaction, an internally labeled 310-nucleotide ssDNA is mixed with M13 circular ssDNA in the presence of varying concentrations of gene 2.5 protein. The labeled DNA fragment is homologous to a region of the M13 ssDNA. Annealing of the 310-nucleotide fragment to the homologous region of M13 ssDNA does not occur after an 8-min incubation at 30 °C in the absence of gene 2.5 protein (Fig. 4A, lane 1), since we observe a single, rapidly migrating radioactively labeled species on an agarose gel. When the concentration of gene 2.5 protein in the reaction is increased, annealing of the DNA strands begins to occur. In Fig. 4A, lane 4, we observe two species, the faster migrating corresponding to the unannealed 310-nucleotide fragment and a more slowly migrating species corresponding to the annealed product. The more slowly migrating species is present even after extraction with phenol chloroform (data not shown), suggesting that the gel shift is due to the increase in size of the annealed product and not a function of gene 2.5 protein binding to the ssDNA. At even higher concentrations (Fig. 4A), all of the labeled fragment is annealed to the M13 circular ssDNA.
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As previously shown (16), DNA annealing is not observed under the same conditions when E. coli SSB protein is added to the reaction (Fig. 4B). Instead, a third species that migrates faster than the annealed product and slower than the fragment is observed upon the addition of E. coli SSB protein. Such a gel shift is noted in all protein concentrations tested (Fig. 4B, lanes 27). This species migrates more rapidly than the annealed product produced by gene 2.5 protein under the same conditions (Fig. 4B, lane 8). At pH 7.5, DNA annealing by E. coli SSB protein is dependent on the presence of a polyamine (28). Since we did not include polyamine in our assay, it is not surprising that E. coli SSB protein could not mediate this reaction under the conditions employed in this study.
Gene 2.5 protein-R82C is defective in DNA annealing
(Fig. 4C). At the
highest concentration test (45 µM), only 25% of the
fragment is converted to annealed product
(Fig. 4C, lane
6). Under the same conditions, WT gene 2.5 protein anneals 100% of the
fragment at a concentration of 15 µM
(Fig. 4A, lane
5). Like E. coli SSB protein, gene 2.5 protein-R82C has a higher
affinity for ssDNA than the WT protein. Thus, it is not surprising that we
observe the appearance of a band that probably corresponds to a protein-DNA
complex as the concentration of gene 2.5 protein-R82C in the reaction is
increased (Fig. 4C,
lanes 26). Next, we compared DNA annealing mediated by the WT
protein with annealing mediated by gene 2.5 protein-R82C over a 4-min time
period. In Fig. 5, we show that
the WT protein anneals nearly all of the labeled fragment in the reaction in
less than 3 min. In contrast, when the same concentration of gene 2.5
protein-R82C is added to the reaction, no annealed product is observed over
the 4-min time course.
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Gene 2.5 protein-R82C and E. coli SSB protein both have a higher
affinity for ssDNA than WT gene 2.5 protein (10- and 50-fold, respectively).
In addition, they are both defective in annealing homologous strands of DNA
under conditions that are optimal for the WT gene 2.5 protein annealing
activity. We asked if these two properties were related. One model might be
that the increased DNA affinity impedes the dissociation of the protein from
ssDNA, which would be required to complete the annealing reaction. To test
this hypothesis, we examined the ability of another altered gene 2.5 protein,
gene 2.5 protein-26C, to facilitate annealing. Previously, we showed
that gene 2.5 protein-
26C has a higher affinity for ssDNA than does the
WT protein, with a dissociation constant of 3.6 x
108 M
(30). This protein facilitates
the annealing of ssDNA at even lower concentrations than does the WT protein
(Fig. 4D) and does so
at a slightly higher rate (Fig.
5). These results agree with previous studies that showed another
carboxyl terminus-deleted protein, gene 2.5-
21C, can facilitate DNA
annealing
(11).2
Annealing occurred at a 10-fold lower concentration than it did in reactions
with WT gene 2.5 protein, demonstrating that the higher affinity for ssDNA did
not lead to defective annealing. Therefore, other explanations for the defect
in annealing exhibited by gene 2.5 protein-R82C must pertain.
Gene 2.5 Protein-R82C Condenses ssDNAWT gene 2.5 protein
condenses circular M13 ssDNA in the presence of magnesium
(2), resulting in compact
structures when viewed by electron microscopy. The M13 circular ssDNA molecule
appears as a collapsed structure when viewed by electron microscopy
(Fig. 6A), in contrast
to the open configuration observed with E. coli SSB protein
(Fig. 6B). Unlike WT
gene 2.5 protein, E. coli SSB protein binds DNA and opens the circle
both in the presence and absence of magnesium
(Fig. 6, compare B and
C with D and G). Since E. coli SSB protein
does not facilitate DNA annealing under the same conditions as does gene 2.5
protein, it is conceivable that the annealing activity is related to the
ability of gene 2.5 protein to condense M13 ssDNA. Therefore, we examined
interaction between the DNA annealing-defective protein, gene 2.5
protein-R82C, and M13 ssDNA using electron microscopy. Gene 2.5 protein-R82C
generates a structure with M13 ssDNA similar to WT gene 2.5 protein. The M13
circle appears in an open form in the absence of magnesium and as a condensed
structure in the presence of magnesium
(Fig. 6, E and
H). It also appears that more gene 2.5 protein-R82C is
bound to M13 DNA than the WT protein, most likely the consequence of the
higher affinity gene 2.5 protein-R82C has for ssDNA. We examined another
higher affinity variant of gene 2.5 protein, gene 2.5 protein-26C. This
variant also condenses ssDNA in the presence of magnesium
(Fig. 6I).
Interestingly, it does not readily form open structures in the absence of
magnesium but rather collapses the ssDNA upon itself
(Fig. 6F). Since both
gene 2.5 protein-R82C, which cannot anneal homologous strands of DNA as
efficiently as does the WT protein, and gene 2.5 protein-
26C, which
does anneal homologous strands, appear to condense M13 ssDNA in the presence
of magnesium, we conclude that the two properties are not related.
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Gene 2.5 Protein-R82C Is a DimerWT gene 2.5 protein is a dimer in solution (2). Using gel filtration analysis, we find that gene 2.5 protein-R82C elutes from the column at the same volume as does the WT gene 2.5 protein (Fig. 7). The column was calibrated, and a standard curve was generated by determining the elution volume of a series of molecular weight markers, specifically RNase A (13.7 kDa), chymotrypsinogen (25 kDa), ovalbumin (43 kDa), and bovine serum albumin (67 kDa). Using the standard curve, the molecular weight of gene 2.5 protein-R82C was estimated to be 58,200, which is consistent with the protein forming a dimer in solution. Thus, the altered proteins can form dimers, further suggesting that single amino acid substitution does not lead to misfolding of the protein.
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Gene 2.5 Protein-R82C Physically Interacts with T7 DNA
PolymerasePrevious studies have shown that gene 2.5 protein
physically and functionally interacts with T7 DNA polymerase
(3), an interaction that
requires the carboxyl terminus of the protein
(14). We followed this
interaction using surface plasmon resonance
(31). In these experiments, we
bound WT or altered gene 2.5 protein on a nickel-NTA coated chip and then
passed T7 DNA polymerase over the bound protein. When the interaction was
assessed in 100 mM NaCl, T7 DNA polymerase bound both WT gene 2.5
protein and gene 2.5 protein-R82C (Fig.
8A). The dissociation constant for these interactions was
calculated to be 2.97 x 106 M
and 1.28 x 106 M for the WT and
altered protein, respectively. By contrast, T7 DNA polymerase does not bind
gene 2.5 protein-26C, which lacks the acidic carboxyl terminus
(Fig. 8A). When the
salt concentration was raised to 200 mM NaCl, T7 DNA polymerase did
not bind to WT gene 2.5 protein or gene 2.5 protein-R82C
(Fig. 8B). We conclude
that gene 2.5 protein-R82C interacts with T7 DNA polymerase with approximately
the same affinity as WT gene 2.5 protein.
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DISCUSSION |
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Proteins that increase the rate and efficiency of annealing of ssDNA are
found in bacteriophages, prokaryotes, and eukaryotes. One group of these
proteins includes E. coli RecA, bacteriophage T4 UvsX gene product,
and the eukaryotic Rad51 protein (reviewed in Ref.
19). These proteins not only
bind to ssDNA but also bring together the DNA strands and in some cases
mediate a search for homology in a reaction that often requires energy. A
second group of proteins, known as the single strand annealing proteins,
includes the protein from bacteriophage
, the E. coli
RecT protein, and the eukaryotic Rad52 protein
(17). These proteins bind to
ssDNA and anneal homologous strands of DNA, often functioning in
RecA-independent recombination pathways. Many members of the single strand
annealing family form multimeric rings both in the presence and absence of
ssDNA (26,
3438).
Finally, the family of ssDNA-binding proteins, best illustrated by E.
coli SSB protein and T4 gene 32 protein, can also facilitate annealing of
DNA (28,
29). These proteins most
likely achieve this function by eliminating secondary structure, thus allowing
homologous regions to base pair on the two strands. On first consideration,
one might equate gene 2.5 protein with E. coli SSB protein and T4
gene 32 protein. However, it binds ssDNA with a 10-fold lower affinity
(2). More strikingly, it
increases the efficiency of annealing of homologous DNA much more readily than
these proteins (16). In
addition, efficient homologous DNA annealing by E. coli SSB protein
in vitro requires either low pH or the presence of a polyamine
(28). Therefore, the mechanism
of DNA annealing by gene 2.5 protein is unclear. It has many properties in
common with members of the RecA family, although it clearly differs from this
family in that ATP is not required to carry out the reaction
(16). It also bears a number
of similarities with the single strand annealing proteins, but gene 2.5
protein has not been shown to form the multimeric rings that characterize this
superfamily (26). We propose
that gene 2.5 protein mediates annealing by first binding to each strand of
DNA. Next, interactions between ssDNA-bound gene 2.5 molecules bring the two
strands of DNA in close proximity of one another, allowing a passive search
for homology to occur. Finally, gene 2.5 protein dissociates from the DNA,
leaving annealed duplex DNA.
By analyzing gene 2.5 protein-R82C, we had hoped to shed some light on this
mechanism. If our model is correct, then two features of gene 2.5 protein are
essential for annealing: ssDNA binding activity and pairing of gene 2.5
protein-bound DNA strands. It is clear that ssDNA binding is required, since
gene 2.5 proteins that do not bind DNA are also defective in annealing
(30). This is not the case in
gene 2.5 protein-R82C, since it binds ssDNA 10-fold more tightly than WT
protein. This tighter binding itself might be a problem, but this is not so,
since gene 2.5 protein-26C binds ssDNA even more tightly, yet it
mediates annealing. Likewise, dissociation from the newly formed
double-stranded DNA molecule is not a problem, since gene 2.5 protein-R82C
displays the same preference for ssDNA as the WT protein. Thus, a defect in
the second property, pairing of gene 2.5 protein when bound to ssDNA, seems
more plausible. We initially hypothesized that the condensed structure of
circular M13 ssDNA bound to gene 2.5 protein observed by electron microscopy
(2) was correlated with this
property. However, it appears that this phenomenon seems to be related to
ssDNA binding, since gene 2.5 protein-R82C condenses ssDNA in a similar manner
(Fig. 6). There are a number of
structural elements that could be involved in the pairing of ssDNA-bound gene
2.5 protein. It is conceivable that the carboxyl-terminal domain is involved,
since it has been shown to be important in interactions with other T7 DNA
replication proteins (12,
14,
15). However, the
carboxyl-terminal truncated version of gene 2.5 protein, gene 2.5
protein-
26C, anneals DNA (Fig.
4). The dimer interface might also be involved. However, a
previous study has shown that two different gene 2.5 proteins with alterations
in the dimer interface retain the ability to efficiently anneal DNA, whereas a
third does so at a slightly slower rate
(31). Whether gene 2.5 protein
dimerizes when bound to DNA is still unknown. If it does so, then the
structural motifs involved in that process may hold the key to the defect in
gene 2.5 protein-R82C.
Recently, a three-dimensional structure of the DNA annealing domain of
human Rad52 was solved (24,
25). The domain crystallized
as an undecameric ring. Interestingly, the monomer does not have the same
oligonucleotide/oligosaccaride binding fold that is found in gene 2.5 protein
and other ssDNA-binding proteins. However, a structure-based alignment shows
significant similarities between gene 2.5 protein and the annealing domain of
human Rad52.3 The
proposed DNA binding cleft of gene 2.5 protein consists of helix
A and sheets
2A,
4, and
5 on one side and strands
3 and
3A on the other side
(27). This cleft aligns quite
well with a similar region on the hRad52 structure consisting of helix
3 and sheets
3,
5, and
5 on one side and sheets
1 and
2 on the
other.3 In addition,
the aromatic residues tyrosine 111 and tyrosine 158, which are positionally
conserved among ssDNA-binding proteins
(27), are structurally
conserved in hRad52 (tyrosine 65 and tyrosine 126, respectively). The
conserved tyrosine residues from gene 2.5 protein form a trinucleotide binding
motif that is also found in E. coli SSB protein and human RPA 70
(27). A variant of gene 2.5
protein where tyrosine 158 is changed to a cysteine bound ssDNA with 10-fold
lower affinity than the WT protein
(30). Interestingly, these
residues are also highly conserved in other eukaryotic Rad52 and Rad22
proteins (17,
24). Therefore, despite the
lack of sequence homology and the general structure, there are a number of
structural similarities between gene 2.5 protein and hRad52 that suggest
functional homology, and it is conceivable that they work by similar
mechanisms.
The single amino acid change described in this paper, arginine 82 to cysteine, is lethal to bacteriophage T7. The altered protein has two distinct differences when compared with the WT protein, increased ssDNA binding affinity and a defect in DNA annealing activity. Since gene 2.5 protein has multiple functions in bacteriophage T7 replication, it is difficult to pinpoint which of theses changes is responsible for the lethal phenotype. The binding affinity of gene 2.5 protein-R82C is 10-fold higher than the WT protein, which could account for the lethal phenotype. However, we feel this is unlikely, since bacteriophage T7 grows in the host E. coli cells that express E. coli SSB protein, a nonspecific ssDNA-binding protein with an affinity for ssDNA higher than gene 2.5 protein (2). Whereas we cannot exclude the possibility that higher ssDNA binding affinity affects T7 growth, we feel it is more likely that the defect in DNA annealing accounts for the lethal phenotype.
If indeed the lethality is due solely to the defect in mediating homologous
base pairing, then it would suggest that the annealing activity is essential
for T7 survival. DNA annealing is important in both DNA repair and
recombination. Bacteriophage T7 has a high rate of recombination, and
mutations in gene 2.5 reduce recombination frequencies
(7). Although it is not known
whether recombination is essential for phage growth, it is likely that extreme
breakage of the T7 chromosome without subsequent annealing to form recombinant
molecules could be lethal. Homologous base pairing is also essential to one
step in T7 DNA replication, the formation of concatemers. The bacteriophage T7
genome has a terminal redundancy of 160 nucleotides
(39), which allows the ends to
be replicated via concatemer formation (reviewed in Ref.
40). Annealing of these
terminally redundant ends by gene 2.5 protein could be vital for concatemer
formation and therefore for DNA replication. In fact, purified gene 2.5
protein is required to reconstitute T7 concatemer formation in vitro
(41). Thus, it is likely that
this is the reason that a plasmid expressing the annealing-defective gene 2.5
protein-R82C cannot complement the growth of bacteriophage T72.5.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: Dept. of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. Tel.: 617-432-1864; Fax: 617-432-3362; E-mail: ccr{at}hms.harvard.edu.
1 The abbreviations used are: ssDNA, single-stranded DNA; WT, wild-type; NTA,
nitrilotriacetic acid; SSB protein, single-stranded binding protein.
2 S. Tabor and C. C. Richardson, unpublished observation.
3 T. Hollis, personal communication.
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ACKNOWLEDGMENTS |
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REFERENCES |
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