(Received for publication, September 28, 1994; and in revised form, December 21, 1994)
From the
Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein that is essential for DNA metabolism. Human RPA is composed of subunits of 70, 32, and 14 kDa with intrinsic DNA-binding activity localized to the 616-amino acid, 70-kDa subunit (RPA70). We have made a series of C-terminal deletions to map the functional domains of RPA70. Deletion of the C terminus resulted in polypeptides that were significantly more soluble than RPA70 but were unable to form stable complexes with the other two subunits of RPA. These data suggest that the C-terminal region of RPA70 may be important for complex formation. The DNA-binding domain was localized to a region of RPA70 between residues 1 and 441. A mutant containing residues 1-441 bound oligonucleotides with an intrinsic affinity close to wild-type RPA complex. This mutant also appeared to bind with reduced cooperativity. We conclude that the C terminus of RPA70 and the 32- and 14-kDa subunits are not involved directly with interactions with DNA but may have a role in cooperativity of RPA binding. RPA70 deletion mutants were not able to support DNA replication even in the presence of a complex of the 32- and 14-kDa subunits, suggesting that the heterotrimeric complex is essential for DNA replication. The putative zinc finger in the C terminus of RPA70 is not required for single-stranded DNA-binding activity.
DNA replication in eukaryotic cells is a complex process requiring the coordinated action of multiple proteins to carry out the regulated synthesis of chromosomal DNA. Because of this complexity, viral model systems like the papova virus SV40 have been essential for studying the molecular mechanism of chromosomal DNA replication (for recent reviews, see (1, 2, 3, 4, 5) ). The SV40 genome codes for only a single replication protein, large T antigen; all other replication proteins are supplied by the host cell. Using this model system, a number of cellular proteins required for SV40 DNA replication have been identified and their functions in DNA replication partially elucidated(6, 7, 8, 9, 10) .
One of the proteins essential for DNA replication is the
multisubunit single-stranded DNA binding protein, replication protein A
(RPA, ()also known as human SSB; (11, 12, 13) ). Studies using the in
vitro SV40 replication system have demonstrated that RPA has
multiple functions during DNA
replication(9, 14, 15, 16) . RPA is
required for both initiation and elongation phases of DNA replication.
Initiation of SV40 replication requires the concerted action of three
proteins: RPA, DNA polymerase
primase complex, and T
antigen(1, 2, 3, 4, 5, 17) .
T antigen first binds to the origin of replication and, in the presence
of RPA, catalyzes the localized unwinding of the origin. DNA polymerase
primase complex interacts with the resulting nucleoprotein
complex and synthesizes RNA primers that lead to nascent DNA synthesis.
RPA specifically interacts with both T antigen and DNA polymerase
(18) . These specific protein-protein interactions have
been shown to be essential for priming by DNA polymerase
primase complex on physiological templates and are required
for specific initiation of DNA replication (9, 15, 16) . RPA is also required for
elongation; RPA stimulates the activities of multiple enzymes that
function at the replication fork including DNA polymerases and DNA
helicases(14, 19, 20, 21, 22, 23) .
In addition to being essential for multiple stages of DNA replication,
RPA is involved in DNA repair and
recombination(24, 25, 26) . RPA from calf
thymus has also been shown to unwind single-stranded DNA (ssDNA) under
low salt conditions(27) . RPA interacts specifically with
several other proteins including the tumor suppressor p53 and several
transcriptional activators such as GAL4 and
VP16(28, 29, 30) . The functional importance
of these interactions is not known; however, it has been suggested that
these interactions could be important for coordinating DNA metabolism
with other cellular processes.
RPA binds tightly to ssDNA (11, 12, 13) with an apparent binding
constant of approximately
10(31, 32, 33, 34) .
Studies of the human RPA (hRPA) have shown that binding is dependent
upon both the sequence and length of the DNA being
bound(31, 34) . Human RPA binds to ssDNA with low
cooperativity (31, 34, 35) and has a binding
site size of 30 nucleotides with between 20 and 30 nucleotides of ssDNA
directly interacting with RPA(34) . hRPA may also have other
DNA binding modes. A recent chemical cross-linking study suggested that
under certain conditions hRPA may bind with a binding site size of
8-10 nucleotides (36) . The binding properties of hRPA
are similar to those of Drosophila melanogaster RPA and bovine
RPA(33, 37) . In contrast, the RPA homologue from Saccharomyces cerevisiae has been shown to bind to ssDNA with
high cooperativity and with a much larger binding site(32) .
While DNA binding seems to be essential for RPA function, it is not
known how variations in the interaction of RPA with DNA affect DNA
synthesis.
Human RPA is a stable complex of three subunits of 70,
32, and 14 kDa (12, 13) and is highly conserved
throughout evolution. Homologous heterotrimeric single-stranded
DNA-binding proteins have been identified in all eukaryotes examined
including S. cerevisiae, Xenopus laevis, D.
melanogaster, and Crithidia
fasiculata(25, 27, 33, 37, 38, 39, 40, 41) .
Amino acid sequence comparison has shown that the homologues of RPA70
from S. cerevisiae, C. fasiculata, and X. laevis are 44, 58, and 90% similar, respectively, to human RPA70 (21, 38, 42) . All known RPA70 homologues
contain a conserved putative C-type zinc-finger motif in
the C-terminal third of the
protein(21, 38, 42) . However, in spite of
this high degree of homology, only some RPA homologues can substitute
for human RPA in SV40 DNA replication. RPA from mouse and Drosophila support specific SV40 initiation, while RPA from
yeast and trypanosomes RPA do
not(16, 39, 40, 43) , suggesting
that there are species-specific interactions essential for normal RPA
function.
All experimental and genetic evidence to date suggests that all three subunits of RPA are needed for function. Individual subunits do not function in DNA replication(14, 21, 44, 45, 46) , and, in S. cerevisiae, the genes encoding all three subunits are essential for viability(25, 47) . Most antibodies to RPA inhibit DNA replication even when they only interact specifically with one subunit(14, 44) . The specific functions of the three subunits of RPA are currently not well understood. The 32- and 14-kDa subunits are capable of forming a soluble complex together(46, 48) . This subcomplex is unable to support DNA replication but may be essential for the proper folding of the 70-kDa subunit and/or assembly of the RPA complex(46) . The 32-kDa subunit of RPA also becomes phosphorylated in a cell cycle-dependent manner(49) . It has been suggested that the phosphorylation could be a regulatory event in either DNA replication or DNA repair(50, 51, 52, 53) ; however, the specific role of phosphorylation of RPA is currently not known(54, 55) . There is currently no specific role attributed to the 14-kDa subunit except for a structural function in RPA complex assembly (46) , although sequence comparisons have been used to suggest that the 14-kDa subunit may be involved in protein-protein interactions (45) .
The 70-kDa subunit of
RPA has several distinct functions. The intrinsic DNA-binding activity
of RPA has been localized to the 70-kDa subunit (14, 56) . In addition, isolated RPA70 has been shown
to interact with DNA polymerase primase complex and to
stimulate DNA polymerase
activity(18, 21) .
RPA70 has also been shown to specifically interact with p53 and several
transcriptional activators (28, 29, 30) but
not with SV40 T antigen(18) . Biochemical analysis of the
functions of RPA70 have been hampered by the very low solubility of the
isolated subunit(21, 46) . The development of Escherichia coli plasmids capable of expressing recombinant
human RPA (rhRPA) (46) allowed us to generate and express a
series of C-terminal deletion mutants of RPA70. Characterization of
these mutants has resulted in an initial mapping of the functional
domains of RPA70. A small region at the C terminus of the 70-kDa
subunit seems to be important for the formation of the heterotrimeric
RPA complex. The DNA binding domain of RPA70 has been mapped to the
region between residues 1 and 441.
HI buffer contains 30
mM HEPES, pH 7.8, 1 mM dithiothreitol, 0.25 mM EDTA, 0.5% (w/v) inositol, and 0.01% (v/v) Nonidet P-40. HI was
supplemented with different concentrations of salt as indicated in the
text. 1 Tris acetate/EDTA (TAE) gel buffer contained 40 mM Tris acetate and 2 mM EDTA, pH 8.5(58) .
pET expression plasmids used were obtained from W. Studier and co-workers(57) . RPA expression plasmids (p11d-tRPA and p3d-RPA14/32) were described previously(46) .
Whole cell lysates were made by collecting cells by
centrifugation at 4,000 rpm for 20 min in a Beckman JS4.2 rotor. Cells
were then resuspended in one-tenth original volume with HI buffer with
1% SDS. 500 µl of suspended cells were transferred to a 1.5-ml
microcentrifuge tube, boiled for 10 min to lyse cells, and spun at
10,000 g for 5 min to remove cell debris.
Sedimentation constants of
442 and
327 were determined by glycerol gradient
sedimentation as described previously(34) . Samples or
standards were brought to a final volume of 50-100 µl with HI
buffer containing 200 mM KCl and loaded onto a 5-ml
15-35% glycerol gradient in HI buffer containing 200 mM KCl. The gradients were centrifuged at 48,000 rpm in a Beckman
SW55 Ti rotor for 24 h at 4 °C. The gradients were fractionated
from the bottom of the tube using a gradient fractionator (Hoefer
Scientific Instruments). The fractions were analyzed by
SDS-polyacrylamide gel electrophoresis followed by silver staining.
Figure 1:
Schematic
of individual C-terminal deletion mutants and summary of properties.
The leftside of figure shows schematic of wild-type
RPA70 and the six deletion mutants (507,
442,
373,
327,
250, and
169). Thickbars indicate residues contained in each polypeptide. Ticks indicates positions every 100 amino acids, and the position of the
putative zinc finger motif is indicated by a solidbox (Zn?). Lines under schematic indicate predicted
location of functional domains. Righthand side
summarizes the properties of each mutant determined during these
studies: solubility (- denotes insoluble; + 25-50%
soluble; ++ >50% soluble) and complex formation and DNA
binding (-, no activity; +,
activity).
The
expression plasmids were tested for their ability to express mutant
RPA70. E. coli BL21(DE3) cells were transformed with
individual expression plasmids, grown and induced with
isopropyl-1-thio--D-galactopyranoside. Lysates from
induced cell were analyzed by SDS-polyacrylamide gel electrophoresis
followed by staining with Coomassie Blue or immunoblot analysis. All
six expression plasmids generated polypeptides of the appropriate size
after induction (Fig. 2). Immunoblot analysis demonstrated that
all six polypeptides cross-reacted with antibodies to RPA70 (data not
shown, also see Fig. 4). The level of expression varied
consistently between the six mutants.
442,
373, and
327
were present in lysates from induced cells at 2-4-fold higher
levels than the other mutants.
Figure 2:
Expression of the various deletion mutants
of RPA70 in E. coli.E. coli BL21(DE3) cells were
transformed with either p11d-RPA70 (RPA70) or pET-11d vectors
containing the various mutated RPA70 genes (507,
442,
373,
327,
250, and
169; see Table 1). 10
µg of whole cell lysates from uninduced (U) or induced (I) cells were separated by electrophoresis on an 8-14%
SDS-polyacrylamide gel and visualized by Coomassie Blue staining. Arrows indicate the position of the induced wild-type RPA70
and the various deletion mutants of RPA70. The position of standards in
kDa are indicated.
Figure 4:
Solubility analysis of C-terminal deletion
mutants of RPA70. Equal volumes of the supernatant (S) or
pellet (P) fractions of full-length RPA70 and the various
deletion mutants (507,
442,
373,
327,
250, and
169) were separated on an 8-14% SDS-PAGE gel, transferred
onto nitrocellulose, and probed with monoclonal antibodies to RPA70.
The arrows indicate the position of RPA70 and the deletion
mutants of RPA70. The asterisk indicates the position of a
proteolytic fragment of RPA70 observed in multiple
lanes.
Figure 3:
Southwestern analysis of the C-terminal
deletion mutants of RPA70. Approximately 20 µg of pellet fractions
from induced cultures containing p11d-RPA70 (RPA70) or pET-11d
containing mutant RPA70 (507,
442,
373,
327,
250, and
169; see Table 1) were separated on an
8-14% SDS-polyacrylamide gel and subjected to Southwestern
analysis as described under ``Experimental Procedures.'' Solidarrows indicate the position of the wild-type
RPA70 and the various deletion mutants of RPA70 that bound to DNA; openarrow indicates the position of
169. E.
coli proteins capable of binding ssDNA are indicated by the asterisks. India ink staining of nitrocellulose indicated that
all lanes contained equal amounts of RPA70 or RPA70 deletion proteins
(data not shown).
In addition to the full-length
C-terminal mutant proteins, other polypeptides able to bind ssDNA were
observed that were unique to individual lysates. For example, a
polypeptide of approximately 56 kDa was observed in the lane containing
wild-type RPA70 (Fig. 3, lane1). We believe
that these polypeptides are proteolytic fragments of individual RPA
deletion mutants that maintain ssDNA-binding activity. The sensitivity
of individual mutants to proteolysis by endogenous proteases varied
significantly (also see Fig. 4, below). In Fig. 3, major
bands of 72-,
18-, and
14-kDa were present in all lanes.
These polypeptides were due to endogenous E. coli ssDNA
binding proteins present in the lysates used in these experiments.
We next examined the solubility properties of the C-terminal
deletion mutants of RPA70. Plasmids capable of directing the expression
of each mutant were transformed individually into E. coli BL21(DE3) cells. Individual transformants were induced with
isopropyl-1-thio--D-galactopyranoside, lysed, and
separated into soluble (supernatant) and insoluble (pellet) fractions.
Equal amounts of pellet and supernatant fractions were separated on
8-14% SDS-polyacrylamide gels and analyzed by immunoblotting (Fig. 4). Full-length RPA70 has been shown previously to be
insoluble when expressed in E. coli with >90% of the
protein being found in the pellet fraction(21, 46) .
In contrast, we found that the solubility of this series of deletion
mutants varied significantly and could be divided into three classes
based upon solubility. Deletion mutant
507 was highly insoluble (Fig. 4, lanes3 and 4), deletions
373 and
250 were only partially soluble with less than 50% of
the expressed protein present in the soluble fraction (Fig. 4, lanes7, 8, 11, and 12),
and deletions
442,
327, and
169 were mostly soluble (Fig. 4, lanes5, 6, 9, 10, 13, and 14). Identical results were
obtained from gels stained with Coomassie Blue (data not shown). These
results suggest that residues between
442 and 616 play a critical
role in determining the solubility of RPA70. We also observed that the
monoclonal antibodies used in these studies reacted much better with
all of the deletion mutants than with full-length 70-kDa subunit; even
though equal amounts of proteins were loaded in each lane, the signal
from full-length RPA70 was very weak (Fig. 4, lanes1 and 2). This observation indicates that after
electrophoresis and transfer to nitrocellulose, full-length RPA70 has a
different conformation than the deletion mutants. This is consistent
with the C terminus of RPA70 being important in determining the
structure of the polypeptide.
In these studies, we observed a number
of cross-reactive bands that resulted from proteolytic degradation (Fig. 4). The level of proteolysis and the specific proteolytic
fragments varied between individual mutants with the exception of an
18-kDa band that was observed with several different mutants (Fig. 4, lanes6, 8, 13, and 14). This fragment is derived from the from the N-terminal
region of RPA70. (
)
Figure 5:
Purified C-terminal deletion mutants.
0.5-1 µg of purified rhRPA, 442, and
327 were
electrophoresed on an 8-14% SDS-polyacrylamide gel and visualized
by staining with silver. Arrows indicate the position of the
three subunits of RPA (RPA70, RPA32, and RPA14) and the two deletion mutants (
442 and
327). The positions of molecular weight standards in kDa
are indicated.
The deletion mutants 442 and
327 have
30 and
50% of their amino acid residues deleted,
respectively. Such large deletions could have a large effect on the
folding of these proteins. We have shown that both proteins were
soluble bound DNA and could be purified following a procedure similar
to that of rhRPA. This suggested that these deletion mutants had a
structure that was related to the structure of RPA70 in the rhRPA
complex. However, to define the solution structure of both mutants more
precisely, purified
327 and
442 were subjected to glycerol
gradient sedimentation and gel permeation chromatography, and their
sedimentation constants and Stokes radii were determined (Table 3). These values were used to calculate the molecular
weight of both deletion mutants in solution (Table 3). The
calculated molecular weights for both
327 and
442 were very
close to the value predicted by their amino acid sequences. We conclude
that both
327 and
442 exist as a monomers in solution. The
frictional coefficient calculated for both mutants were close to 1.6 (Table 3). Assuming that both mutants are shaped like prolate
spheroids, the axial ratio for
327 and
442 would be predicted
to be approximately 12:1 and 10:1, respectively. These values are
similar to that of the rhRPA complex, which has a frictional
coefficient of 1.60 and a predicted axial ratio of 12:1. These
hydrodynamic data suggest that while
327 and
442 are smaller,
both have a general shape similar to heterotrimeric RPA complex.
We have shown previously that binding of RPA to
oligodeoxythymidine 30 nucleotides in length (oligo(dT))
is a simple bimolecular reaction(34) , and the RPA
concentrations needed to obtain an observable fluorescence signal are
high enough (50-100 nM) to cause stoichiometric
binding(34, 35) .
442 and
327 both bind
oligo(dT)
with stoichiometry of 1:1 (see below). Each
mutant was titrated with oligo(dT)
, and changes in
fluorescence were monitored (data not shown). In a stoichiometric
titration of a bimolecular binding reaction, the moles of
oligo(dT)
necessary to reach saturation (i.e. give the maximum decrease in fluorescence signal) should equal the
moles of protein capable of binding DNA. Comparing the amount of active
protein to the total protein present for titrations of
442 and
327 with oligo (dT)
indicated that the mutants were
35 and 62% active, respectively (Table 3). These values were
similar to those obtained previously for both native and recombinant
RPA complexes (34, 35) . We also determined the
binding site size of
442 and
327 in a second series of
stoichiometric titrations using poly(dT) 3,000 nucleotides in length.
Saturation of such a long DNA fragment occurs when the protein coats
the DNA and thus, when a protein is titrated with poly(dT), the ratio
of moles of nucleotide/active protein at saturation corresponds to the
binding site size of the protein. Such titrations indicated that the
binding site sizes of
442 and
327 were
21 and
18
nucleotides, respectively (Table 3). These results indicate that
both
442 and
327 are only partially active and both have
smaller binding site sizes than rhRPA (Table 3).
Equilibrium
binding of 442 and
327 to oligonucleotides was examined in
gel mobility shift assays. In these assays, short oligonucleotides were
incubated with increasing concentrations of protein, and the resulting
complexes were analyzed on agarose gels. RPA
oligonucleotide
complexes are stable to separation by electrophoresis and migrate with
altered mobility(31) . Fig. 6shows titrations in which
oligo(dT)
was titrated with either rhRPA,
327, or
442. As has been observed in previous studies(31) , two
bands of altered mobility were observed when rhRPA binds to
oligo(dT)
. At low concentrations of rhRPA, a single
complex with altered mobility was observed (Fig. 6A, lanes4-8) and as the protein concentration was
increased, a second complex with lower mobility appeared (Fig. 6A, lanes8-12). These
complexes represent singly and doubly liganded complexes, respectively (34) . When oligo(dT)
was titrated with
442,
two complexes with altered mobility were also observed (Fig. 6B, lanes4-11); however,
with more than 100 fmol of
442, a third complex was observed (Fig. 6B, lane12). We believe this
to be a triply liganded complex. This conclusion is consistent with the
21-nucleotide binding site of
442.
327 also has a smaller
binding site size than that of rhRPA, yet only two complexes with
altered mobility were observed when oligo(dT)
was titrated
with
327 (Fig. 6C). In addition, much higher
concentrations of protein were required for binding of this mutant (Fig. 6C). This indicated that the binding affinity of
327 is much lower than that of rhRPA and that saturating
concentrations of protein had probably not been added in this assay. In
these experiments, we observed that in reactions containing greater
than 1,000 fmol of protein, the protein
DNA complexes migrated as
smears (e.g.Fig. 6C, lane11). This is probably due to RPA-RPA interactions or
aggregation at high protein concentrations. Aggregation of human RPA
has been observed previously at high protein
concentrations(34) .
Figure 6:
Gel mobility shift assay of recombinant
proteins using labeled oligo (dT). 2 fmol of radiolabeled
oligo(dT)
was incubated with indicated amounts of either
rhRPA (A),
442 (B), or
327 (C).
After incubating at 25 °C for 20 min, the reaction mixture was
separated on a 1% agarose gel in 0.1% TAE as described under
``Experimental Procedures.'' The positions of the free and
bound oligo(dT)
are as indicated. D, quantitation
of titrations shown in A-C. Each band was excised, and
the radioactivity was measured by liquid scintillation counting.
Fraction-free DNA was plotted versus concentration of active
protein, and the resulting binding isotherms for rhRPA (
),
442 (
), and
327 (
) binding to oligo(dT)
are shown. The data were fit to the Langmuir binding equation for
bimolecular binding reactions using nonlinear least square fitting
software (Kaleidograph, Abelbeck Software) as described
previously(34) . Best fit curves are shown for rhRPA (solidline, K
= 5.1
10
),
442 (dottedline, K
= 2.4
10
),
and
327 (dashedline, K
= 1.7
10
).
The titrations shown in Fig. 6were quantitated by excising individual bands and
determining the amount of radioactive DNA present. Individual binding
isotherms were obtained by plotting the fraction of free DNA versus active protein present (Fig. 6D). Apparent binding
constants were then determined by fitting the data to the Langmuir
binding equation. We have shown previously that under these conditions
RPA binds to DNA with low cooperativity, and that individual binding
events can be considered unlinked(34) . Therefore, the multiple
binding events of RPA binding to oligo(dT) can be analyzed
as a series of independent bimolecular binding reactions to obtain a
good estimate of the apparent binding constant ((34) ; see also Fig. 6D). The apparent association constants determined for
rhRPA,
442, and
327 binding to oligo(dT)
are
shown in Table 4. The affinity for oligo(dT)
of
442 was only 4-fold lower than that of rhRPA complex, while the
affinity of
327 was 60-fold lower than of rhRPA complex.
Binding of 442 and
327 to oligo(dT)
and
oligo(dT)
was also examined. When oligo(dT)
was titrated with rhRPA, only a single band with reduced mobility
was observed when up to 490 fmol of rhRPA was added (data not shown;
see also (31) ). In contrast, two bands with reduced mobility
were observed in a similar titration of oligo(dT)
with
442 (data not shown). Titration of oligo(dT)
with
327 resulted in a single band of lowered mobility (data not
shown). When binding to oligo(dT)
was examined, a single
complex with lowered mobility was observed with all three proteins
(data not shown). The complexes formed by
442 were exactly as
predicted by its binding site of 21 nucleotides; the maximum number of
molecules of
442 bound to oligo(dT)
,
oligo(dT)
, and oligo(dT)
were 3, 2, and 1,
respectively. The binding site of
327 is slightly smaller than
442 but no higher order complexes were found with either
oligo(dT)
or oligo(dT)
. We believe that the
explanation for this observation is that
327 has a binding
affinity significantly lower than either rhRPA or
442 (see below)
and that concentrations of
327 higher than were used in these
assays were needed to saturate the oligonucleotides used.
All of the
binding titrations of oligo(dT) and oligo(dT)
were quantitated and analyzed as describe above. The resolved
binding constants are summarized in Table 4. While the affinity
of
442 for oligo(dT)
was approximately 4-fold less
than rhRPA, the affinities for oligo(dT)
and
oligo(dT)
were essentially identical to those of rhRPA
complex. These results suggest all of the residues needed to interact
with DNA are contained in this mutant and that the 32- and 14-kDa
subunits of RPA do not contribute significantly to the binding of RPA
to oligonucleotides. In contrast to
442, the smaller mutant,
327, was found to have a much lower affinity for oligo(dT)
(Table 4). Thus, deletion of residues 327-441
resulted in at least a 10-fold decrease in the affinity for DNA. In
spite of this decrease, the affinity of
327 for ssDNA is still
high, indicating that residues 1-326 of RPA70 contain a core DNA
binding domain.
Figure 7:
Activity of 442 and
327 in SV40
replication. SV40 replication assays were carried out as described
previously, and the products were analyzed by electrophoresis on 1%
agarose gels followed by autoradiography(46) . Cellular
proteins necessary for SV40 replication were supplied as partially
purified protein fractions. Each reconstituted replication reaction
contained 20 µg of cellular fraction II (contains DNA polymerases
and
and replication factor C), 5.6 µg of fraction
cellular fraction IBC (contains proliferating nuclear cell antigen and
protein phosphatase 2A)(46, 56) , 0.7 µg of
purified SV40 T antigen, 1.5 units of topoisomerase I (Promega Corp.),
and indicated amounts of rhRPA, rhRPA32
14,
442, and
327. Plasmid pUC.HSO(17) , which contains the SV40 origin
of replication, was used as the template for these
reactions.
RPA70 has multiple functions; it has intrinsic ssDNA binding
activity, and it interacts directly with the other two RPA subunits,
DNA polymerase primase complex and several transcriptional
activators. The goal of these studies was to map the functional domains
of the 70-kDa subunit of RPA. A series of C-terminal deletion mutants
were constructed and characterized. A schematic showing the deletions
constructed is shown in Fig. 1. This figure also summarizes the
properties of the individual mutants. Initial characterization
indicated that the solubility of individual mutants varied
significantly. Full-length RPA70 and
507 were both highly
insoluble, while all of the smaller mutants were soluble. Deletion of
the C terminus also resulted in polypeptides that were unable to form a
stable complex with the 32- and 14-kDa subunits, suggesting that the C
terminus of RPA70 is important for heterotrimeric complex formation.
The low solubility of the full-length subunit suggests interactions
between the C terminus and the 32- and 14-kDa subunits are primarily
hydrophobic (Fig. 1). This hypothesis is supported by an
examination of the amino acid sequence of RPA70; the residues between
440 and 616 have predominantly hydrophobic character on a
Kyte-Doolittle hydrophobicity plot (data not shown). Such hydrophobic
interactions would also be consistent with the high stability of the
heterotrimeric RPA complex (12, 13) and the proposal
that RPA has an ordered assembly pathway in which RPA70 must interact
with a subcomplex of the 32- and 14-kDa subunits(46) .
All
of the deletion mutants were tested for DNA binding activity. These
experiments indicated that all of the deletion mutants except for
169 were capable of binding to ssDNA (Fig. 3). Two soluble
deletion mutants,
442 and
327, were purified, and their DNA
binding properties were examined quantitatively in gel mobility shift
assays. The binding constants obtained (Table 4) indicated that
both deletion mutants bound ssDNA with high affinity. The apparent
association constants determined for
442 are nearly identical to
those of rhRPA complex. We conclude that the
442 contains the
entire DNA-binding domain of RPA and that the 32- and 14-kDa subunits
of RPA do not affect the binding of RPA to oligonucleotides (see also
the discussion of cooperativity below).
327 bound DNA with an
affinity approximately 10-fold lower than rhRPA. The finding that
169 did not interact with ssDNA in Southwestern blots indicated
that residues between 169 and 326 are essential for DNA interaction. We
conclude that the DNA binding domain of RPA is between residues 1 and
441 and that there is a core DNA-binding domain contained between
residues 1 and 326 (Fig. 1). The N-terminal boundary of the DNA
binding domain is not well defined, but proteolytic mapping studies of
RPA indicate that a
17-kDa polypeptide from the N-terminus is not
necessary for DNA binding activity.
The binding site
size of 442 and
327 were both approximately 20 nucleotides.
This is significantly smaller than the 30-nucleotide binding site size
of the RPA complex. The decrease is consistent with their smaller size
and monomeric state in solution. The finding that there is little
difference in the binding affinity between
442 and RPA suggests
that part of the 30-nucleotide binding site of RPA is the result of
steric occlusion by either the C-terminal residues of RPA70 or the 32-
and 14-kDa subunits.
442 bound to oligo(dT)
and
oligo(dT)
with association constants equivalent to those
of rhRPA complex, yet it bound to oligo(dT)
with an
association constant one-quarter of that of rhRPA (Table 4). One
possible explanation for this difference is that
442 binds to DNA
with reduced cooperativity. The stoichiometry of binding for
442
to oligo(dT)
, oligo(dT)
, and oligo(dT)
is 1:1, 2:1, and 3:1, respectively, while the stoichiometry of
rhRPA to these oligonucleotides is 1:1, 1:1, and 2:1, respectively.
Thus cooperative interactions would be expected for
442 binding to
oligo(dT)
and for both
442 and rhRPA binding to
oligo(dT)
. We have shown previously that under the
conditions used in these studies, rhRPA binds to ssDNA with a low but
measurable level of cooperativity (
=
10-20)(35) . This level of cooperativity causes the
association constant of rhRPA for oligo(dT)
to be
10-fold higher than that for oligo(dT)
(Table 4). If
442 had the same cooperativity of
binding as rhRPA, then the association constant for oligo(dT)
should have been
10 greater than that of oligo
(dT)
, and the association constant for oligo(dT)
should have been similar to that of rhRPA. This was not observed.
These data are consistent with
442 having significantly less
cooperativity in binding than rhRPA and would suggest that either the C
terminus of RPA70 or the 32- and 14-kDa subunits are responsible for
cooperativity of RPA binding to DNA.
Human RPA70 contains a putative
C-type zinc-finger motif at positions
481-503(21) . This sequence has been found to be
conserved in all RPA70 homologue genes whose sequences are
known(38, 42, 47) .
442 does not contain
this motif, yet it binds oligonucleotides with an affinity similar to
that of rhRPA complex. We conclude that the putative zinc-finger is not
necessary for DNA binding activity. The single-stranded DNA binding
protein from bacteriophage T4, gene 32 protein, has a zinc metal
binding site. Inactivation of this metal binding region reduces the
binding affinity for long single-stranded DNA due to a reduction in the
cooperativity of binding (61) . Our data imply that
442
binds with reduced cooperativity. Thus, it remains possible that the
C
-type zinc-finger motif could have a role in cooperative
binding of RPA.
The purified C-terminal deletion mutants of RPA70
were also tested for the ability to support SV40 DNA replication.
Neither 442 nor
327 were able to support SV40 replication
when added alone or in combination with a complex of the 32- and 14-kDa
subunits of RPA. This suggests that the ability to bind DNA is not
sufficient for DNA replication and confirms that a heterotrimeric RPA
complex is necessary to support DNA replication. We also found that
neither mutant inhibited replication when mixed with rhRPA, even when
present at a 10-fold molar excess. Since
442 binds DNA with an
affinity close to that of rhRPA, 10-fold excess should have been
sufficient to preferentially bind to most of the ssDNA in the
replication reaction; however, replication still occurred. These
results suggest several possible models for the function of RPA in DNA
replication. One explanation for the lack of inhibition by
442 is
that although it binds to oligonucleotides with high affinity, its
binding properties of are different enough to prevent it from competing
effectively with rhRPA complex. Alternatively, specific protein-protein
interactions may be needed to load RPA onto ssDNA during the initiation
or elongation stages of replication. If these interactions were absent
or weak with
442, it would not compete with RPA during
replication. Thus, although both RPA-protein and RPA-DNA interactions
are essential for DNA replication, it is not known whether both types
of interactions are needed simultaneously, sequentially, or
independently. The absence of inhibition of
442 suggests that
ssDNA binding is not a prerequisite for required RPA-protein
interactions. Additional characterization of the interactions between
RPA and other replication proteins and RPA and DNA will be needed to
distinguish between these models of RPA function.