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INTRODUCTION |
Single-stranded DNA-binding proteins
(SSBs)1 contribute to DNA
metabolism, playing essential roles in different processes such as DNA
replication, repair, and recombination (1-3). SSBs bind single-stranded DNA (ssDNA) in a selective, cooperative, and
non-sequence-specific way, protecting it from nuclease attack and
preventing the formation of secondary structures on it. SSBs are
ubiquitous. They have been isolated from bacteria and their phages,
eukarya and their viruses, and archaea (1-6).
Different oligomerization states have been reported for SSBs. Thus,
monomeric (T4gp32 from bacteriophage T4 or AdDBP from adenovirus (5)),
dimeric (SSBs from filamentous phages M13 (1) and Pf3 (7)),
homotetrameric (EcoSSB from Escherichia coli (2,
8) or human mitochondrial SSB (9)), and heterotrimeric (hRPA, human RPA
(3)) SSBs have been described. Important differences have been also
reported mainly concerning the amino acid sequence and ssDNA binding
properties of the proteins of this family.
The function of the SSB is particularly important in the case of
organisms that replicate their genetic material via a protein-priming mechanism followed by strand displacement, as large amounts of ssDNA
are generated in the process (10-12). This is the case of adenovirus
and of the 
29 family of Bacillus phages. The genome of
the latter consists of a linear dsDNA molecule of about 20 kb that
contains a phage-encoded terminal protein (TP) covalently linked to
each 5' end. Replication of the viral genome starts at either DNA end
non-simultaneously by a protein-priming mechanism. After a sliding-back
step (13), the viral DNA polymerase elongates the initiation product
proceeding by strand displacement toward the other DNA end. The
displaced strand is cooperatively bound by the viral SSB. When two
growing chains, running from opposite ends, collide and separate, two
DNA molecules are generated where strand displacement is no longer
required (10-12) and where the SSB must be eliminated from the ssDNA
by the advancing DNA polymerase.
The 29 family of phages has been classified into three evolutionary
branches according to the comparison of nucleotide and amino acid
sequences of selected DNA regions and proteins (14, 15). One branch is
composed of phages 29, PZA, 15, and BS32; the second branch
consists of phages Nf, B103, and M2Y; and the third branch has phage
GA-1 as its sole member (15).
Initial studies carried out with the SSB of 29 indicated that it has
ability to protect ssDNA against nuclease degradation (16) and to bind
the ssDNA generated during viral DNA replication in vitro,
as analyzed by electron microscopy (17). Furthermore, it was determined
that 29 SSB has helix-destabilizing ability (18), and the parameters
of the complex it forms with ssDNA were defined (19).
Recent comparison of 29 SSB with the SSBs of phages Nf and GA-1,
representative examples, respectively, of the second and third branches
in which the 29 family of phages is divided, indicate that, despite
some global similarities, GA-1 SSB displays significant functional and
structural differences with respect to the other two SSBs. Thus, it
binds ssDNA with higher affinity and displays helix-destabilizing
ability with lower protein concentrations than the other two SSBs (20,
21). The complex formed by GA-1 SSB with ssDNA is clearly different
from the 29-like nucleoprotein complex, as determined by electron
microscopy in the presence of DNA (19, 20). Thus, GA-1 SSB produces an
average 6-fold reduction in the length of the ssDNA in contrast to the
2-fold reduction factor obtained upon binding of 29 SSB to ssDNA.
Besides, the nucleoprotein complex formed by GA-1 SSB shows a compact
beaded appearance (20), very different from the simple array of protein monomers of 29 SSB·ssDNA fibers (19). Further approaches oriented to the structural comparison of the three SSBs indicate that 29 and
Nf SSBs behave as monomers in solution in glycerol gradients, whereas
GA-1 SSB self-interacts sedimenting at a position corresponding to an
average hexameric complex. This differential self-interaction ability
correlates with a higher efficiency of GA-1 SSB in functional assays.
Amino acid sequence comparison of the three SSBs highlights the high
degree of homology between 29 and Nf SSBs, as well as the higher
divergence of GA-1 SSB. The latter contains an insert at its N-terminal
region that is lacking in the other two SSBs (21).
In the present report, further approaches to the characterization of
GA-1 SSB have been carried out. Thus, the self-interaction ability of
GA-1 SSB has been analyzed by visualization of the purified protein by
electron microscopy in the absence of DNA, by glycerol gradient
sedimentation, and by in vivo cross-linking of bacterial
cultures infected with phage GA-1. Furthermore, the effect of the
partial or complete deletion of the N-terminal insert of GA-1 SSB on
the self-interaction ability of the protein as well as on its
functional activity has been also examined. We show that a mutant
protein lacking the 19 N-terminal amino acids retains the structural
and functional behavior of GA-1 SSB, whereas mutants lacking 26 or 33 amino acids from the N-terminal end are greatly affected. The influence
of the N-terminal region of GA-1 SSB on the self-interaction ability of
the protein and the requirement of this ability for an efficient
functional behavior are also discussed.
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EXPERIMENTAL PROCEDURES |
Nucleotides and DNA--
Phage 29 TP-DNA was isolated as
described (22). Oligonucleotides were obtained from Isogen, and plasmid
pALTER-Ex2 from Promega. [
-32P]dATP (3000 Ci/mmol) and
[
-32P]ATP (3000 Ci/mmol) were obtained from Amersham
Biosciences. Unlabeled nucleotides and M13mp18 ssDNA were from Amersham
Biosciences. DNA sequencing was carried out in the Servicio
Interdepartamental de Investigación of the Universidad
Autónoma de Madrid.
Proteins--
Vent DNA polymerase, T4 DNA ligase, and
restriction enzymes BamHI and EcoRI were
purchased from New England Biolabs. The 29 DNA polymerase, TP, and
DBP were overproduced in E. coli and purified as described
(23-25). GA-1 SSB was purified from infected Bacillus sp.
G1R cells (26) as previously described (21).
Construction of Plasmids--
Using GA-1 DNA as template and the
appropriate oligonucleotide primers, DNA fragments corresponding to the
mutant proteins 
N19, N26, and N33 were amplified by PCR.
Vent DNA polymerase, which contains a proofreading function,
was used. The primers corresponding to the N-terminal region of each
protein contained a recognition site for BamHI, whereas the
oligonucleotide corresponding to the C-terminal region, common to all
of them, contained a recognition site for EcoRI.
PCR-generated DNA fragments were digested with BamHI and
EcoRI, and then cloned into the BamHI and
EcoRI sites of plasmid pALTER-Ex2. All the clones generated
from the PCR-amplified DNA were sequenced and found to be correct.
Purification of the Mutant Proteins--
E. coli
JM109 cells (endA1 recA1 gyrA96
thi hsdR17 (rk
,
mk+) relA1 supE44
(lac-proAB) (F' traD36
proAB+
lacIqZM15)) (27) carrying the
plasmids of interest were grown at 37 °C in LB broth (28)
supplemented with 10 mg/ml tetracycline. At a cell density
corresponding to A600 = 0.5, isopropyl-1-thio-
-D-galactopyranoside (Sigma) was added
to a final concentration of 1 mM. Further incubation was at
37 °C for 3 h. Bacteria were harvested by centrifugation and
after cell disruption by grinding with alumina (2:1, w/w), resuspended
in buffer A (50 mM Tris-HCl, pH 7.5, 1 mM EDTA,
7 mM -ME, and 5% glycerol) in the presence of 0.4 M NaCl. All purification steps were carried out at 4 °C.
The supernatant of a 1500 × g centrifugation step of 5 min was further centrifuged for 20 min at 20,000 × g.
Mutant proteins N26 and N33 were recovered in the supernatant of
this centrifugation step, whereas N19 was mainly contained in the
sediment. DNA was removed from the supernatant corresponding to
proteins N26 and N33 by addition of polyethyleneimine to 0.3%
after adjusting the absorbance at 
260 nm to 120 units/ml and centrifugation for 20 min at 20,000 × g.
The supernatant was made 0.1 M NaCl with buffer A, and the
proteins were recovered in the supernatant after centrifugation as
above. The samples were precipitated with 65% ammonium sulfate,
dissolved in buffer A, and, after dialysis against the same buffer,
applied to the following columns: in the case of N26,
phosphocellulose, heparin-agarose, Mono-Q, and hydroxyapatite; in the
case of N33, phosphocellulose, heparin-agarose, DEAE-cellulose, and
Mono-Q. Mutant protein N19 was recovered from the sediment with
denaturation buffer (25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 25% glycerol (v/v), 10 mM DTT, and 4 M guanidinium chloride). After centrifugation for 20 min at 20,000 × g, the supernatant was dialyzed against
renaturation buffer (25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 25% glycerol (v/v), 10 mM DTT, and
0.1 M NaCl) and centrifuged under the same conditions. The
supernatant containing the native protein was applied to the following
columns: phosphocellulose, heparin-agarose, and Mono-Q. In all three
cases, protein samples eluted from the last column were precipitated
with ammonium sulfate up to 65%, dissolved in buffer A, and dialyzed
against the same buffer containing 50% glycerol (v/v). In all
purification steps, proteins were followed by SDS-PAGE (10-20%
acrylamide gradients). Protein concentration was determined both by the
Lowry method and by comparison with a known amount of BSA in
polyacrylamide gel electrophoresis.
In Vivo Cross-linking of GA-1 SSB--
Both noninfected and
GA-1-infected Bacillus sp. G1R cell cultures were harvested
after 15 min of infection at 37 °C. Aliquots of 1.5 ml were
centrifuged at 4 °C, and the corresponding pellets were incubated
with increasing amounts of the cross-linking agent BS3
(Pierce) in a final volume of 200 µl. After 30 min at room
temperature, 40 µl of 1 M Tris-HCl, pH 7.5, were added to
each reaction to quench it. After centrifugation, pellets were
resuspended in 300 µl of loading buffer, samples sonicated, and
aliquots of 10 µl loaded on to 10-20% SDS-PAGE. Proteins were
electrophoretically transferred for 70 min at 100 mA and 4 °C to
PVDF membranes using a transfer buffer containing 25 mM
Tris, 192 mM glycine, and 20% (v/v) methanol. Membranes
were incubated with 1/1000 diluted rabbit antibodies against GA-1 SSB.
Antigen-antibody complexes were detected with anti-rabbit horseradish
peroxidase-linked antibody and ECL Western blotting detection reagent
(both from Amersham Biosciences). Prestained molecular weight
markers were loaded in the gel as molecular weight controls.
TP-DNA Amplification Assay--
The incubation mixture
contained, in 25 µl, 50 mM Tris-HCl, pH 7.5, 1 mM DTT, 4% glycerol, 0.1 mg/ml BSA, 20 mM
ammonium sulfate, 10 mM MgCl2, 80 µM each dCTP, dGTP, dTTP, and [-32P]dATP
(2 µCi), 3.4 ng of 29 TP-DNA, 7 ng of DNA polymerase, 25 ng of TP,
and 10 µg of DBP, all from 29. The indicated amounts of GA-1 SSB,
the mutant proteins or the corresponding buffer were added. After
incubation for 1 h at 30 °C, reactions were stopped by adding
EDTA up to 10 mM and SDS up to 0.1% (w/v), and the samples were filtered through Sephadex G-50 spin columns in the presence of
0.1% SDS. The excluded volume was subjected to alkaline agarose gel
electrophoresis as described (29), followed by autoradiography and
ethidium bromide staining.
Helix-destabilizing Assay--
The incubation mixture contained,
in 12.5 µl, 62.5 ng of primed M13mp18 ssDNA, 50 mM
Tris-HCl, pH 7.5, 4% glycerol, 0.1 mg/ml BSA, and the indicated
amounts of GA-1 SSB, the mutant proteins or the corresponding buffer.
After 30 min at 37 °C, reactions were stopped with 1.25 µl of
0.25% (w/v) bromphenol blue, 0.25% (w/v) xylene cyanol, 30%
glycerol, and 0.5% SDS. Samples were subjected to electrophoresis at
4 °C in an 8% polyacrylamide gel containing 0.1% SDS. The gel was
dried and autoradiographed.
Gel Mobility-shift Assays--
The incubation mixture contained,
in a final volume of 20 µl, a heat-denatured
-32P-labeled 29 DNA HindIIIL fragment and
the indicated amounts of 29, Nf, or GA-1 SSB in 50 mM
Tris-HCl, pH 7.5, and 4% glycerol. After incubation for 5 min at
4 °C, the samples were subjected to electrophoresis in 4%
polyacrylamide gels containing 12 mM Tris acetate, pH 7.5, and 1 mM EDTA, and run at 4 °C in the same buffer at 8 V/cm essentially as described (30). After drying, the gels were
autoradiographed and the SSB·ssDNA complexes detected as mobility of
the labeled DNA.
Glycerol Gradient Assays--
Glycerol gradients (15-30%),
containing 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 7 mM -ME, and 25 mM NaCl (loading buffer),
were formed in 5-ml Beckman polyallomer centrifuge tubes (13 × 51 mm). 100 µl of a 50 µM preparation of GA-1 SSB or each
mutant protein in loading buffer, and the corresponding amounts of
molecular weight markers, were loaded onto different gradients. After
centrifugation at 62,000 × g and 4 °C in a Beckman
SW.65 rotor for 24 h, the fraction number at which the maximal
amount of each protein appears was determined by interpolating in the
graphic elaborated with the molecular weight markers, as described
(20).
Electron Microscopy--
GA-1 SSB or the mutant proteins (10 µl of a 55 µM solution) were applied to carbon-coated
copper grids for 2 min. Grids were then washed with a few drops of
water and stained for 30 s with 2% uranyl acetate. Electron
micrographs were taken in a Jeol 1010 electron microscope at 80 kV.
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RESULTS |
Analysis of the Ability of GA-1 SSB to Self-interact by Electron
Microscopy and in Vivo Cross-linking--
The ability of GA-1 SSB to
self-interact was analyzed both by visualization of the purified
protein by electron microscopy and by in vivo cross-linking
of bacterial cultures infected with phage GA-1. After negative
staining, GA-1 SSB was visualized in the absence of DNA and chemical
cross-linking reagents by electron microscopy as described under
"Experimental Procedures." Under these conditions purified GA-1 SSB
oligomerized in vitro, as can be observed in Fig.
1A, which shows a
representative example of the most abundant structures formed by wt
GA-1 SSB. They consist of protein filaments of different lengths and a
diameter of ~10 nm. Along with these filaments, protein structures
where regions of different compacting levels coexist were detected,
like the one displayed in Fig. 1B.
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Fig. 1.
Visualization of GA-1 SSB by electron
microscopy. A, field showing filaments of different
lengths and ~10-nm diameter. B, coexistence of different
compacting levels within the same structure.
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The ability of GA-1 SSB to self-interact was also analyzed in
vivo by treatment of GA-1-infected Bacillus sp. G1R
cell cultures with the cross-linking agent BS3, followed by
Western blot analysis with antibodies raised against GA-1 SSB (for
details, see "Experimental Procedures"). Fig.
2 shows both noninfected (
I)
and GA-1-infected (+I) cell cultures after treatment with
increasing amounts of the cross-linking agent. Positions corresponding
to the mono-, di-, tri-, and tetrameric forms of the protein are
indicated. Oligomers of higher molecular mass were also detected.
Additional bands appear that could correspond to interaction of GA-1
SSB with other proteins.
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Fig. 2.
Ability of GA-1 SSB to self-interact in
vivo. Noninfected (I) and GA-1-infected
(+I) Bacillus sp. G1R cultures were harvested,
treated with the indicated amounts of BS3, and subjected to
10-20% SDS-PAGE. Western blot analysis of the gel was then carried
out. Positions corresponding to the mono-, di-, tri-, and tetrameric
forms of the protein are indicated at the right. Increasing
amounts of GA-1 SSB were used as control.
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Deletion of the N-terminal Region of GA-1 SSB Affects Its
Stimulatory Effect on Viral DNA Replication--
Previous studies
indicate that GA-1 SSB displays differential structural and functional
behavior to those of the SSBs of the related phages 29 and Nf (20,
21). GA-1 SSB contains an insert on its N-terminal region, rich in
polar and aromatic amino acids, which is not present in the SSBs of the
related phages 29 and Nf (see Fig. 3).
To analyze the effect of the partial or complete deletion of this
insert on the global activity of GA-1 SSB, the three mutant proteins
N19, N26, and N33 indicated in Fig. 3 were expressed from the
corresponding plasmids in E. coli JM109 cells and purified
as previously described. N19, N26, and N33 lack the 19, 26, or
33 amino acids, respectively, that follow the initial methionine
residue of GA-1 SSB. All three proteins were purified to near
homogeneity. The ability of the mutant proteins to stimulate viral DNA
replication was analyzed in a TP-DNA amplification assay using the
phage 29 DNA amplification system (31). The increasing amounts
indicated in Fig. 4, either of GA-1 SSB
or of the mutant proteins N19, N26, and N33, were incubated
with 29 TP-DNA, DNA polymerase, TP, and DBP, under the conditions described under "Experimental Procedures." As can be seen in Fig. 4, the stimulatory effect of N19 was similar to that of wt GA-1 SSB.
Thus, dNTP incorporation in the presence of 3.5 µM N19
was 85% of that obtained with the same concentration of the wt
protein. However, the activity of N26 and N33 was significantly
affected (compare the stimulatory effects with a concentration of 14 µM for each protein in Fig. 4). Thus, the activity of the
mutant proteins N26 and N33 was 9 and 5%, respectively, of that
of wt GA-1 SSB. Not even with a concentration of 55 µM
for the mutant proteins N26 and N33 did dNTP incorporation reach
the levels obtained with 3.5 µM wt protein. Furthermore,
addition of concentrations as high as 250 µM for the
mutant proteins N26 and N33 did not significantly improve the
levels of dNTP incorporation (data not shown). The lack of global
functional efficiency of mutant proteins N26 and N33 is discussed
below, and a comparison with the SSBs of 29 and Nf is also
established.
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Fig. 3.
Sequence alignment of
29, Nf, and GA-1 SSBs. Identical residues in
the three SSBs are white on a black
background. Similar residues of the 29 and/or Nf SSBs
with respect to the GA-1 SSB, the least related of them, are
black on a gray background.
Similarities between the 29 and Nf SSBs are not highlighted.
Numbers indicate the amino acid position. The first amino
acids that follow the initial methionine residue in the N19, N26,
and N33 mutant proteins of GA-1 SSB are indicated with
arrows. The polar and aromatic residues within the
N-terminal region are also highlighted.
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Fig. 4.
Stimulatory effect of GA-1 SSB or the mutant
proteins N19, N26,
and N33 on viral DNA replication using
the 29 DNA amplification system. The
indicated amounts of each protein were added to the reaction mixture.
Electrophoresis mobility of full-length 29 TP-DNA is also
indicated.
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The N-terminal Region of GA-1 SSB Is Essential for Its
Helix-destabilizing Activity--
The helix-destabilizing ability of
GA-1 SSB mutant proteins N19, N26, and N33 was examined on a
substrate consisting of M13 ssDNA to which a 5' radioactively labeled
17-mer oligonucleotide had been hybridized. This substrate was
incubated with increasing amounts of GA-1 SSB or the mutant proteins,
and the reaction products analyzed by native polyacrylamide gel
electrophoresis. As can be seen in Fig.
5, N19 mutant protein displayed the
same helix-destabilizing ability as the wt GA-1 SSB, displacing all the
17-mer oligonucleotide from the M13 ssDNA with a 10 µM
protein concentration. As also shown in Fig. 5, neither N26 nor
N33 mutant proteins were able to displace the oligonucleotide, even
at protein concentrations as high as 160 µM.
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Fig. 5.
Helix-destabilizing activity of GA-1 SSB and
of the mutant proteins N19,
N26, and N33. M13
ssDNA to which 5' radioactively labeled 17-mer oligonucleotide was
hybridized was incubated with increasing amounts of each protein.
Positions of the hybrid substrate and the displaced oligonucleotide are
indicated. C is the control of the heat-denatured
substrate.
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Deletion of the N-terminal Region of GA-1 SSB Does Not Fully
Abolish Its Ability to Bind ssDNA--
To determine whether the lack
of functional efficiency displayed by the mutant proteins N26 and
N33 on TP-DNA amplification and dsDNA unwinding was a consequence of
their inability to bind ssDNA, the affinity of these mutant proteins
for ssDNA was tested in a gel-mobility shift assay. For this purpose, a
radioactively labeled 273-nt-long ssDNA fragment was incubated with
increasing amounts of either the mutant proteins or the wt GA-1 SSB. As
can be seen in Fig. 6, both N26 and
N33 displayed a lower affinity for ssDNA than wt GA-1 SSB. Thus,
although GA-1 SSB shifted the band corresponding to free ssDNA with a
concentration of 1 µM, a 5-fold higher concentration of
N26 was required to produce the same effect. This protein
concentration did not suffice in the case of N33 to shift all the
DNA present in the assay. Instead, complete binding of all the DNA
present in the assay was achieved in the case of this protein with a
concentration of 8 µM (data not shown). Mutant protein
N19 displayed affinity for ssDNA similar to that of the wt GA-1 SSB
(data not shown). These results indicate that, although deletion of the
26 or 33 N-terminal amino acids affects the affinity of GA-1 SSB for
ssDNA, it does not fully abolish the ability of the protein to bind
ssDNA.
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Fig. 6.
Gel mobility shift assay of wt GA-1 SSB,
N26, and N33 mutant
proteins. A radioactively labeled 273-nt-long ssDNA fragment was
incubated with the indicated amounts of GA-1 SSB, N26, or N33 at
4 °C and subjected to electrophoresis in a 4% polyacrylamide gel as
described under "Experimental Procedures." The band corresponding
to the protein-free ssDNA is indicated (D).
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Oligomeric State of N19, N26, and N33 Mutant
Proteins--
When GA-1 SSB is subjected to ultracentrifugation in a
glycerol gradient, it sediments in the average position corresponding to a hexameric complex (20). It must be taken into account, however,
that the shape of the aggregate may affect its sedimentation behavior.
This approach was also used in the case of the mutant proteins to
determine whether the ability of GA-1 SSB to oligomerize was affected
by deletion of its N-terminal region. For this purpose, samples of
either wt GA-1 SSB (data not shown) or the mutant proteins N19,
N26, and N33 (Fig. 7) were
subjected to glycerol gradient centrifugation, and the fractions were
analyzed by SDS-PAGE. Densitometric analysis of the corresponding gels
allowed us to conclude that N19 (17.0 kDa/monomer) sediments in an
average position of 106 kDa that would correspond to an hexamer, as wt
GA-1 SSB (20). By contrast, the protein peak in the case of N26
mutant protein (16.1 kDa/monomer) is ~51 kDa. This position would
correspond to a trimeric complex of N26. Aggregation ability of
N33 mutant protein (15.2 kDa) is even more affected, with a
sedimentation peak at ~29 kDa that would correspond to a dimeric
complex. Thus, deletion of 26 or 33 but not of 19 amino acids of the
N-terminal region of GA-1 SSB results in loss of the oligomerization
ability of the protein.
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Fig. 7.
Determination of the oligomerization state
of N19 (A),
N26 (B), and
N33 (C) mutant proteins. 100 µl of each mutant protein (50 µM concentration) was
subjected to sedimentation in 15-30% (w/v) glycerol gradients in the
presence of molecular weight markers, as described under
"Experimental Procedures." After centrifugation, the fractions were
analyzed by SDS-PAGE. The fractions at which the maximal amount of each
protein appear are indicated in each panel. The markers used are:
AD, alcohol dehydrogenase (150 kDa); BSA, bovine
serum albumin (66 kDa); CA, carbonic anhydrase (29 kDa); and
L, lysozyme (14 kDa). The molecular mass estimation of each
mutant protein was carried out as indicated under
"Results."
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Visualization of the Deletion Mutant Proteins by Electron
Microscopy--
Analysis of the deletion mutant proteins by
negative-stain electron microscopy was carried out in the absence of
DNA as already described for wt GA-1 SSB. The appearance of protein
N19 in solution was identical to that of the filaments formed by
GA-1 SSB (data not shown). In contrast, in the case of the mutant
proteins N26 and N33, the ability to form protein filaments is
abolished. Instead, smaller, ring-shaped structures like the ones shown
in Fig. 8A for N26, were
detected. N33 formed the same kind of structures (data not shown). A
closer view of these structures is displayed in Fig. 8B. A
correlation between the inability of these mutant proteins to form
filaments and their loss of functionality is suggested under
"Discussion."
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Fig. 8.
A, field showing the ring-shaped
structures formed by N26 mutant protein. B, closer view
of the structures. The bar represents 100 nm.
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DISCUSSION |
When compared with the SSBs of related phages, the SSB of phage
GA-1 displays some noticeable features, one of them being its
self-association ability. Thus, on the one hand, even in the absence of
ssDNA, GA-1 is able to form filamentous and helicoidal structures that
can be visualized by electron microscopy. Under these experimental
conditions, neither of the SSBs of the related phages 29 and Nf are
able to form these high order aggregates.2
On the other hand, when subjected to
ultracentrifugation in glycerol gradients, 29 and Nf SSBs behave as
monomers in solution, whereas GA-1 SSB sediments in the average
position of an hexameric complex (20). It must be taken into account
that protein-assembly processes are strictly dependent of protein
concentration. Thus, the fact that a 10-fold lower protein
concentration was used in glycerol gradient centrifugation than in
electron microscopy may explain why no material was detected at the
bottom of the tube of the GA-1 SSB gradient that would correspond to
the filament-forming protein population. However, filament disruption
because of the more drastic experimental conditions of the
ultracentrifugation with respect to the electron microscopy experiments
cannot be ruled out. In vivo cross-linking of
Bacillus cultures infected with phage GA-1 confirms the
ability of GA-1 SSB to self-interact, although under our experimental
conditions monomers and low molecular weight oligomers were seen in
addition to high order aggregates that could correspond to the protein
filaments. Anyway, whether the filaments formed by GA-1 SSB are the
active form of the protein or not, at least they are representative of
the differential ability of the protein to self-interact, which is
lacking in the SSBs of the related phages 29 and Nf.
The importance of the self-interaction ability of GA-1 SSB is further
demonstrated by the results obtained with the deletion mutants N19,
N26, and N33 of the N-terminal region of the protein. Deletion of
the 26 and 33, but not of the 19, amino acids that follow the initial
methionine residue of GA-1 SSB results in the abolishment of the
helix-destabilizing ability of the wt protein, as well as in a drastic
reduction of its functional efficiency in DNA replication assays. These
activities are much more affected in GA-1 SSB deletion mutant proteins
than in 29 and Nf SSBs, proteins that also lack the N-terminal
region present in GA-1 SSB. Thus, both 29 and Nf SSBs are able to
displace the oligonucleotide hybridized to the M13 ssDNA molecule with
a protein concentration of 40 µM versus 10 µM GA-1 SSB (21), whereas no destabilization of the
oligonucleotide was obtained with a concentration of the mutant
proteins N26 and N33 as high as 160 µM.
Furthermore, in the 29 DNA amplification system, equivalent
stimulatory effects of DNA replication to those of GA-1 SSB were
obtained with 5-fold higher concentrations of 29 SSB (20), whereas
dNTP incorporation levels equivalent to those obtained with 3.5 µM GA-1 SSB were not reached with N26 and N33 GA-1
SSB deletion mutants, even with 250 µM protein
concentration. These effects are not just a consequence of the
inability of the mutant proteins to bind ssDNA, as they conserve this
ability, although somewhat affected with respect to that of GA-1 SSB.
Besides, as inferred from the data presented in this paper, deletion of
the 26 or 33 but not of the 19 amino acids of the N-terminal region of
GA-1 SSB results in a loss of the oligomerization ability of this
protein, as observed by ultracentrifugation in glycerol gradients.
Furthermore, mutant proteins N26 and N33 are no longer able to
form filaments, as visualized under the electron microscope. All these
experimental evidences indicate that, in contrast with 29 and Nf
SSBs, GA-1 SSB requires its differential self-interaction ability for
an efficient functional behavior, and that the N-terminal region of
GA-1 SSB comprised between amino acids 19 and 26 plays an essential role on it. Structural defects in GA-1 SSB that affect the
self-interaction ability correlate well with a drastic reduction of its
functional efficiency. At the present time mutant phages containing
GA-1 SSB proteins N19, N26, and N33 are not available.
Therefore, no experiments in vivo have been done to
determine whether oligomerization of GA-1 SSB is required for its
biological activity.
The fact that, in contrast with N26 and N33, the mutant protein
N19 behaves like the wt GA-1 SSB draws our attention to the amino
acid residues of this region, the main distinguishable feature of which
is the abundance of polar and aromatic residues. Thus, 2 of 7 of the
amino acids that are present in N19 but not in N26 are positively
charged, 1 has negative charge, and 2 more are aromatic (see Fig. 3).
Furthermore, 3 of 7 amino acids present in N26 but not in N33
have positive charge, 1 is negatively charged, and 1 is aromatic. All
these data together suggest that the residues of the N-terminal region
of GA-1 SSB could be establishing electrostatic interactions important
for the differential self-association ability of this protein. An
additional piece of evidence that would point in this direction is the
fact that, when GA-1 SSB is subjected to ultracentrifugation in
glycerol gradient in the presence of 0.2 M NaCl, it behaves
like a monomer.2 In addition, no protein filaments are
visible by electron microscopy when GA-1 SSB is in the presence of 1 M NaCl.2 This kind of behavior has already been
detected in proteins that bind ssDNA and that play important roles in
recombination events, such as T4 uvsY or RecA. Thus, T4 uvsY, essential
in the formation of the presynaptic filament of phage T4 because of its
noncooperative binding to ssDNA and establishment of specific
interactions with other proteins of the T4 recombination machinery,
forms hexamers capable of reversible association into higher aggregates
in a manner dependent on both salt and protein concentration (32). Additionally, RecA, which promotes homologous pairing and exchange of
DNA strands ubiquitously in eubacteria, has a
monomer-hexamer-higher aggregate self-association state strongly
dependent on the kind and concentration of the salt as well as on the
protein concentration (33). Nevertheless, RecA is active only as a
helical filament of indefinite length polymerized on DNA, and even in
the absence of DNA it can self-assemble into a variety of multimeric
forms (34). Resolution of the crystal structure of RecA reveals that the N-terminal region of the molecule (residues 1-30) is formed by an
-helix followed by a -strand, and protrudes from the rest of the
molecule stabilizing polymer formation (35). Interestingly, secondary
structure predictions of the N-terminal region of GA-1 SSB point to the
existence of a similar structure to that of the N-terminal region of
RecA. These evidences support the importance of an - domain in
these proteins for promoting filament formation.
The only examples of SSBs in which the effect of the deletion of a part
of the protein resembles that of GA-1 SSB and with similar
self-interaction ability to that of GA-1 SSB are the SSBs of herpes
simplex virus type I (ICP8) and the SSB of adenovirus (AdDBP). ICP8 is
a 128-kDa protein able to form protein filaments in the absence of DNA,
although it sediments as a monomer in glycerol gradients (36). Deletion
of its 60-amino acid C-terminal region does not affect the intrinsic
DNA-binding ability of the protein, but results in a total loss of
cooperativity on long ssDNA stretches (37). It has been demonstrated
that ICP8 interacts with other components of the replication machinery,
like UL9 protein helicase (38-40) or the herpes simplex virus-1
DNA polymerase and helicase-primase (41-45). On the other hand, AdDBP
is able to form small aggregates visible under the electron microscope.
Resolution of the crystal structure of AdDBP suggested that its
C-terminal extension could hook on to an adjacent monomer (46). As a
matter of fact, deletion of this C-terminal arm results in a greatly
reduced affinity for ssDNA and unwinding activity of the protein (47).
As already mentioned, adenovirus replicates its genome via a
protein-priming mechanism like the one displayed by the phages of the
29 family. Like the SSBs of these phages, AdDBP cooperatively binds
to the displaced DNA strand. However, AdDBP has additional roles during adenovirus DNA replication, like the indirect stimulation of the initiation step by increasing the binding of NFI to the replication origin (48, 49), and a direct stimulation by lowering the Km for the first dNTP (50). Taking together these
data with the differences displayed by GA-1 SSB with respect to the SSBs of the related phages 29 and Nf, it is possible that GA-1 SSB
might be implicated in additional roles to those common to the
replication systems of the three phages. Future studies concerning phage GA-1 SSB will be oriented to shed some light into possible additional roles of this protein.
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ABSTRACT
INTRODUCTION
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