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INTRODUCTION |
RPA1 (replication
protein A) is the most abundant eukaryotic single strand-specific
DNA-binding protein with copy numbers of up to
105/mammalian cell nucleus (1). Mammalian RPA, like RPA in
other eukaryotic organisms, is a heterotrimeric protein with subunits of apparent molecular masses of 70, 32, and 14 kDa, respectively known
as RPA70, RPA32, and RPA14 (2). Within the three subunits, RPA may
contain at least four potential binding sites for single-stranded DNA
as recently suggested based on weak homologies to the bacterial single
strand binding protein (3). Two of the sites in the central part of the
RPA70 subunit are characterized by cocrystallization of the central
RPA70 binding domain with an oligonucleotide ligand (4), and recent
cross-linking experiments suggest that additional sites are localized
in carboxyl-terminal regions of RPA70 and RPA32 (5).
The high affinity for single-stranded DNA is one property of RPA,
another is its ability to interact with a variety of proteins involved
in DNA replication (6-8), DNA repair (9, 10), recombination (11, 12),
and, possibly, transcription (13). Thus, RPA binding to single-stranded
DNA and its interaction with DNA polymerase
-primase and with other
replication factors are required for the establishment and the
propagation of replication forks (14). In recombination, RPA stimulates
DNA strand exchange between Rad51-coated single-stranded DNA and the
double-stranded DNA substrate (11); and in nucleotide excision repair,
binding of RPA may help to stabilize the locally unwound DNA and to
localize the repair endonucleases at damaged DNA sites (15).
RPA32 is phosphorylated when cells transit from the G1
phase into the S phase of the cell cycle and remains so throughout S
phase (16, 17). In addition, dramatic increases of RPA32 phosphorylation occur as a response to DNA damage in x-ray- and UV-irradiated cells (18-21) as well as during apoptosis (22). The
enzymes responsible for RPA32 phosphorylation in intact nuclei have not
yet been unambiguously identified although in vitro
experiments show that RPA32 serves as a substrate for the
cyclin-dependent kinase Cdc2-cyclin B as well as for the
DNA-dependent protein kinase (DNA-PK). The enzymes
recognize serine and threonine residues as phosphorylation sites in the
amino-terminal section of RPA32 and cause a retardation in the
electrophoretic mobility of RPA (23-25).
The physiological consequences of RPA32 phosphorylation are not clear
yet. Mutations of the major phosphorylation sites of RPA32 cause no
detectable phenotype in yeast (3). Mutant RPA, lacking the
amino-terminal phosphorylation sites, binds well to single-stranded DNA
(24), supports SV40 DNA replication in vitro, and appears to
be active in nucleotide excision repair (26). Phosphorylation of RPA is
suppressed in human cells lacking a functional DNA damage surveillance
ATM gene product as well as in yeast cells without the MEC1
gene, a counterpart of the mammalian ATM gene (18, 27). The
MEC1 gene product is known to be involved in S phase arrest of cells
with damaged DNA (28). It is therefore possible that the
phosphorylation of RPA32 has a function regulating the switch from
replicative DNA synthesis to repair synthesis (26, 29). This is
consistent with a report that damage-induced inhibition of DNA
replication correlates with RPA32 phosphorylation in an in
vitro system and can be reversed by the addition of purified unphosphorylated RPA (19).
In our previous studies with proliferating cultured human cells we
observed that phosphorylated RPA32 may be less stably associated with
the large RPA70 subunit than unmodified RPA32 (22). To further
investigate this possibility we have performed the experiments reported
in this communication. We could show that trimeric RPA complexes tend
to desintegrate when subunit RPA32 is phosphorylated, whereas
unmodified trimeric RPA remains perfectly stable under the same
conditions. The results contribute to discussions on the role of RPA32
phosphorylation in proliferating cells and could explain earlier data
of others who detected by immunological means that individual RPA
subunits partition to different parts of the nucleus at some cell cycle
stages (30).
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EXPERIMENTAL PROCEDURES |
Cell Culture, Preparation of Chromatin Proteins, and Sucrose
Gradient Centrifugation--
Human HeLa S3 cells (31) and Jurkat T
lymphoma cells (32) were cultivated as described. For cell
fractionation, nuclei were isolated using the digitonin procedure of
Adam et al. (33) with 10 mM NaF and 20 mM
-glycerophosphate and the modifications described
earlier (34). Chromatin was prepared as originally described by Hancock
(35) with the modifications detailed in Ritzi et al. (31).
For the preparation of chromatin-associated proteins, isolated
chromatin was resuspended in hypotonic buffer (0.25 mM
EDTA, pH 8) containing NaCl at concentrations specified in the text.
Unsoluble nucleoprotein was removed by centrifugation at 12,000 × g. Supernatants were analyzed by centrifugation through linear 25 to 5% sucrose gradients in TE buffer (20 mM
Tris-HCl, 5 mM EDTA, pH 7.8; with either 0.1 or 0.4 M NaCl) using the Beckman SW 40 rotor at 35,000 rotations/min and 4 °C for 60 h. The gradients were
fractionated from the top into 0.6-ml aliquots. Samples from each
fraction were analyzed by Western blotting for RPA.
Analysis of RPA--
Recombinant human RPA was isolated from
bacterial extracts as described previously (36). The purified protein
was used as an antigen for the preparation of antisera in rabbits.
RPA-specific antibodies were affinity-purified according to standard
procedures (37). The antibodies were used for immunoblotting (Western
blotting) after denaturing polyacrylamide gel electrophoresis (38)
essentially as described by Towbin et al. (39). Immunoblots
were visualized by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech).
In Vitro Phosphorylation--
Active cyclin A/CDK1, cyclin
B/CDK1, and cyclin E/CDK2 were isolated from Xenopus egg
extracts according to Strausfeld et al. (40). For in
vitro phosphorylation, 4 µg of recombinant human RPA was
incubated with 500 ng of purified cyclin-dependent kinases
in 15 µl of kinase buffer (20 mM Hepes, pH 7.5, 10 mM MgCl2, 100 mM NaCl, 2 mM dithiothreitol, 0.02% Tween 20) with 0.2 mM
ATP and, if required, 5 µCi of [
-32P]ATP. Control
phosphorylation assays were performed with 5 µg of histone H1 (Roche
Molecular Biochemicals) instead of RPA as substrate. Aliquots from each
assay mixture were analyzed by denaturing polyacrylamide gel
electrophoresis and immunoblotting or autoradiography. Using frog
enzymes in these reactions appears to be justified, since
cyclin-dependent kinases are highly conserved as
exemplified by the fact that human CDK1 can complement yeast strains
mutated in the cdc2 gene (41).
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RESULTS |
Phosphorylated RPA32 Occurs in the Chromatin-bound Fraction of HeLa
Cell RPA--
About one third of RPA in cultured human cells is bound
to chromatin while the remaining fraction of RPA occurs free in the nucleosol (34). To investigate whether phosphorylated RPA32 is present
in the chromatin-bound fraction, in the nucleosol, or both, we
extracted proteins from isolated HeLa cell nuclei under different salt
conditions and probed the extracts with RPA-specific antibodies using
an immunoblot ("Western") procedure. The antibodies, prepared in
rabbits against recombinant human RPA as an antigen, recognize the two
largest RPA subunits, but not RPA14 (34) (Fig. 1A). Using these antibodies as
a tool, we were able to identify subunits RPA70 and RPA32 in
unfractionated total cell extracts (Fig. 1B, lane
1). We also detected these subunits in nuclear extracts and found
that RPA32 subunits in the chromatin-bound fraction, but not in the
nucleosolic fraction, had retarded electrophoretic mobilities
(indicated as bands p1 and p2 in Fig.
1B).

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Fig. 1.
RPA in nuclear extracts. A,
characterization of RPA antibodies. Recombinant human RPA was analyzed
by polyacrylamide gel electrophoresis and visualized by Coomassie Blue
staining or Western blotting using affinity-purified antibodies.
B, cell fractionation. A total of about 107 HeLa
cells from a semiconfluent plate was washed and suspended in 5 ml of
phosphate-buffered saline. An aliquot was directly resuspended in
Laemmli buffer (whole cells). The remainder was used to prepare nuclei,
which were lysed by resuspension in 5 ml of hypotonic buffer (0.25 mM EDTA, pH 8). Unsoluble nucleoprotein was pelleted, and
the supernatant was used as soluble fraction 1. The nucleoprotein
pellet was washed first with 5 ml of 0.25 mM EDTA (soluble
fraction 2) and then with 5 ml of 1% Nonidet P-40 in 0.25 mM EDTA (Nonidet P-40 wash). The final chromatin pellet was
resuspended in 5 ml of hypotonic buffer. Equal aliquots were analyzed
by Western blotting. Chromatin-bound RPA32 appeared in three bands,
p0 (unphosphorylated), p1, and p2 with
probably one and two (or more) phosphate groups, respectively.
C, phosphatase treatment. Protein extracts from Jurkat cells
were incubated in phosphatase buffer for 30 min with or without
alkaline phosphatase. RPA was analyzed by Western blotting.
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Identical results were obtained using fractionated nuclear extracts
from cells of the human Jurkat lymphoma cell line (data not shown). To
verify that the retarded electrophoretic mobilities were due to
phosphorylation, we treated the nuclear extracts with alkaline
phosphatase and found that the slower moving bands were converted to a
single electrophoretic band corresponding to unmodified RPA32 (Fig.
1C).
We conclude that most phosphorylated RPA32 occurs in the
chromatin-bound fraction of RPA and could be in direct contact with DNA
(34). This would be consistent with the results of biochemical experiments that had shown previously that DNA-bound RPA is a better
substrate for protein kinases than free RPA (42).
To further investigate the chromatin-bound RPA fraction, we treated
aliquots of isolated chromatin with buffers of increasing salt
concentrations. We found that low salt concentrations cause a release
of RPA with unmodified RPA32 subunits, whereas higher salt
concentrations were necessary to also release RPA with phosphorylated RPA32 (Fig. 2). The data of Fig. 2 also
indicate that more RPA70 subunits than RPA32 subunits appeared to be
mobilized at 0.1 M NaCl. This has been noted before (see
Fig. 5B in Ref. 34) and could mean that the subunits of
chromatin-bound RPA separate upon release from their binding sites on
chromatin. That would be surprising as isolated trimeric RPA complexes
are known to be extremely stable under a variety of ionic conditions
(2).

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Fig. 2.
Mobilization of chromatin-bound RPA.
HeLa cell chromatin was prepared according to Hancock (35) with the
modifications described in Ritzi et al. (31). Equal aliquots
of chromatin were resuspended in hypotonic buffer with different salt
concentrations as indicated. After 5 min on ice, insoluble
nucleoprotein was removed by centrifugation, and the supernatants were
investigated by denaturing polyacrylamide gel electrophoresis and
Western blotting. C, control without centrifugation.
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Separation of RPA Subunits--
To further investigate the
possibility that phosphorylated RPA could dissociate into its subunits,
HeLa cell chromatin was treated with 0.4 M NaCl, a salt
concentration that mobilizes a large fraction of non-histone chromatin
proteins, including most of the bound RPA (Fig. 2). Chromatin proteins
were then fractionated through sucrose gradients at 0.4 M
NaCl. Controls were investigated in parallel tubes of the same
sedimentation run. One control was recombinant human RPA, and a second
control was the soluble nucleosolic fraction that contains unmodified
RPA as shown in lane 2 of Fig. 1B. We confirmed
that recombinant human RPA remained stable under the experimental
conditions of 0.4 M NaCl, since all three RPA subunits
sedimented together in a symmetrical peak with a sedimentation rate
just ahead the bovine serum albumin marker with 4.2 S (Fig. 3A). Unphosphorylated RPA in
the nucleosolic fraction had the sedimentation properties of
recombinant RPA (Fig. 3B), suggesting that all three RPA
subunits remained in trimeric complexes although the properties of the
antibodies precluded a determination of the smallest subunit, RPA 14 (see Fig. 1A).

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Fig. 3.
Analysis of RPA by sucrose gradient
centrifugation. A, recombinant human RPA; B,
nucleosolic soluble RPA (see Fig. 1B, lane 2); C,
chromatin-bound RPA, released from isolated chromatin at 0.4 M NaCl (see Fig. 2, lane 5). Sucrose gradients
in hypotonic buffer contained 0.4 M NaCl. Sedimentation is
from the left to the right. Gradient fractions in A were
analyzed by Laemmli gel electrophoresis and Coomassie Blue staining.
Gradient fractions in B and C were analyzed by
Western blotting.
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In contrast, the RPA fraction, prepared from chromatin at 0.4 M salt, behaved quite differently: the RPA70 subunits and
the phosphorylated RPA32 subunits sedimented as independent peaks (Fig.
3C). Again, we could not determine the distribution of the RPA14 subunit in the gradient. We can therefore not say whether RPA14
remained bound to RPA70, to RPA32, or to both subunits. In either case,
the data clearly show that the large and the phosphorylated middle
subunit of RPA dissociate when extracted from chromatin at 0.4 M NaCl. The sedimentation analysis revealed an unusual distribution of released RPA70. While RPA70 subunits appeared in
symmetric peaks when they remain in intact trimeric RPA (Fig. 3,
A and B), released RPA70 exhibited a leading
shoulder (Fig. 3C), suggesting that RPA70 tends to form
aggregates with itself or with unidentified chromatin proteins upon
release from RPA32. In the particular experiment, leading to Fig.
3C, a minor fraction of unphosphorylated RPA32 sedimented
together with the main RPA70 peak in the gradient. This is an
interesting internal control showing again that the association of
unphosphorylated RPA32 and RPA70 was not disrupted under the
experimental conditions.
To demonstrate that phosphorylation was responsible for the
dissociation of RPA, we dialyzed released chromatin proteins against buffer with 0.1 M NaCl in the presence or absence of
phosphatase. The structure of RPA was then determined by sucrose
gradient centrifugation and Western blotting as shown above. We found
that the removal of phosphate groups resulted in reformation of RPA
complexes as shown by a cosedimention of subunits RPA70 and RPA32
exactly as normal trimeric RPA (Fig.
4A). However, when not
dephosphorylated, RPA32 failed to combine with RPA70 during dialysis
against 0.1 M NaCl (Fig. 4B). We conclude that
phosphorylation is a major determinant causing the separation of RPA70
and RPA32 in chromatin-bound RPA. Immunoprecipitation of
chromatin-bound RPA showed indeed that phosphorylated RPA32 could be
immunoprecipitated with polyclonal antibodies to the total RPA complex
(data not shown), but a monoclonal RPA70 antibody failed to
coimmunoprecipitate the phosphorylated RPA32, in agreement with
previous reports (20, 22).

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Fig. 4.
Reformation of RPA. Chromatin-bound RPA
was prepared as in Fig. 3C and dialyzed for 8 h at
4 °C against hypotonic buffer with 0.1 M NaCl in the
presence (A) or absence (B) of phosphatase (see
Fig. 1C). Sucrose gradient centrifugation was performed as
in Fig. 3 except that the gradient contained 0.1 M
NaCl.
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While necessary, RPA32 phosphorylation may not be sufficient for RPA
desintegration, and we cannot exclude the possibility that an
additional chromatin function may be required for the separation of
RPA70 and RPA32. As a first attempt to investigate this possibility we
used recombinant human RPA as substrate for cyclin-dependent protein kinases in in vitro
phosphorylation experiments.
In Vitro Phosphorylation of RPA--
Previous work had shown that
isolated RPA serves as an in vitro substrate for DNA-PK and
for cyclin-dependent protein kinases (CDK) (24, 26, 43).
Since DNA-PK has a major function in DNA damage recognition and repair
(20), and the present research deals with undamaged cycling cells, we
decided to investigate the effects of CDK on the stability of isolated
RPA. For that purpose, we used CDK1 in association with cyclin A and
cyclin B, or cyclin E/CDK2 as kinase activities and recombinant RPA as a substrate. While cyclin A/CDK1, cyclin B/CDK1, and cyclin E/CDK2 efficiently phosphorylated the common substrate histone H1, only cyclin
A/CDK1 and cyclin B/CDK1 could phosphorylate RPA32 causing a
electrophoretic mobility shift characteristic for phosphorylated RPA32
(Fig. 5A). The kinetics of the
reaction showed that incubation times of 30-60 min were sufficient to
obtain a maximal transfer of phosphate groups to RPA and that longer
incubation had no additional effects. We also observed a low degree of
phosphate transfer to the large subunit, RPA70 (Fig.
5B).

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Fig. 5.
Phosphorylation of RPA in
vitro. A, comparisons. Recombinant RPA was incubated
for 60 min in phosphorylation buffer without enzyme (C,
control), with cyclin A/CDK1 (A), with cyclin B/CDK1
(B), or with cyclin E/CDK2 (E). Aliquots from
each reaction mixture were investigated by polyacrylamide gel
electrophoresis and Western blotting (upper panel) or
autoradiography (central panel). Lower panel, a
parallel control experiment was performed with histone H1 instead of
RPA as substrate. We show the relevant section of the autoradiogram.
B, Kinetics. A phosphorylation assay was performed with
cyclin A/CDK1 and RPA. Equal aliquots were investigated at the times
indicated. We show the autoradiogram of a polyacrylamide gel.
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To determine whether CDK-mediated phosphorylation caused a separation
of RPA subunits, we performed sucrose gradient centrifugation at 0.1 and 0.4 M NaCl as described above under Fig. 3. In four independent experiments, we obtained ambiguous results: most RPA subunits remained together in one complex as shown by a cosedimentation of the middle and the large RPA subunits, of which some were degraded during incubation (Fig. 6). However, a
minor fraction of RPA32 sedimented slower than the main peak of RPA.
Interestingly, the fraction of slower sedimenting RPA32 appeared to be
enriched for the phosphorylated P1 and P2 forms of RPA32 (Fig. 6),
showing a partial dissociation from the RPA complex. Similar results
were obtained by centrifugation at 0.1 M NaCl (data not
shown). In addition, we observed in these experiments a wide
distribution of RPA70 reaching into fractions close to the top of the
sucrose gradient. The reason for this is not known, but could be due to an adherence of CDK-treated RPA to the walls of the centrifuge tubes. A
trailing of RPA or its subunits was not observed in control experiments
using RPA, incubated with phosphorylation buffer in the absence of CDK
(not shown) or with RPA in nuclear extracts (see Figs. 3 and 4).

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Fig. 6.
Structure of in vitro
phosphorylated RPA. RPA was incubated with cyclin A/CDK1
under phosphorylation conditions for 60 min. The reaction was stopped
by addition of 10 mM EDTA and 0.4 M NaCl
(final). The mixture was directly applied to a sucrose gradient in
hypotonic buffer with 50 mM Tris-HCl, pH 7.5, and 0.4 M NaCl and centrifuged as in B. We show the
results of a Western blotting experiment. A control experiment was
performed with RPA incubated in the absence of the protein kinase. The
results were similar to those of Fig. 3A (not shown).
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To summarize the in vitro phosphorylation experiments, we
note that recombinant human RPA serves as a substrate for cyclin A/CDK1
and for cyclin B/CDK1, but even incubation times of up to 60 min lead
to an incomplete phosphorylation of RPA32 as shown by the fact that
only a minor fraction of CDK-treated RPA showed the electrophoretic
retardation characteristic for highly phosphorylated RPA32. The reasons
for this behavior of RPA in biochemical phosphorylation assays remain
to be investigated. However, the interesting point here is that
whenever a high degree of in vitro phosphorylation was
achieved, the large and the middle subunit of RPA tended to dissociate.
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DISCUSSION |
Phosphorylation of the RPA32 subunit has been observed in normally
proliferating cells at the time of genome replication and in cells with
damaged DNA during DNA repair synthesis (see Introduction). Here we
have investigated RPA32 phosphorylation in proliferating transformed
human HeLa and Jurkat T lymphoma cells in tissue culture. We confirmed
our previous results showing that the majority of RPA occurs in the
nucleosol, while only about one-third of RPA is associated with
chromatin (34). The physiologic reason for this distribution is not
clear. RPA in the soluble nuclear fraction could function as a back-up
system for situations with high demands for RPA such as repair
synthesis. Another possibility is that soluble RPA forms a nucleosolic
pool or reservoir where phosphorylated RPA from replicated DNA is
dephosphorylated and recycled. In this regard it is interesting to note
that most soluble RPA is unphosphorylated, whereas chromatin-bound RPA
contains phosphorylated RPA32. This suggests that RPA32 is
phosphorylated as a consequence of the functions that RPA performs at
replication forks. These functions could include an orchestration of
the "polymerase switch" occurring when DNA polymerase
has
finished the synthesis of short RNA-DNA primers and DNA polymerase
takes over to continue chain elongation synthesis (14, 44). While the
large RPA subunit probably resides on and protects the exposed
single-stranded DNA template, subunit RPA32 contacts the 3'-ends of DNA
primers (29). After the polymerase switch RPA32 changes its position
and can no longer be detected at the 3'-ends of the more advanced
products of DNA synthesis. RPA cycling at replication forks may require
changes of the RPA70/RPA32 interface and flexible subunit interactions.
These conformational changes could be regulated by phosphorylation and
may facilitate the separation of the large and the middle RPA subunits
at the experimental conditions described above (Fig. 3). This does not
necessarily imply that subunits RPA70 and the RPA32 physically separate
when bound to their sites at replication forks. It is in fact quite
possible that RPA desintegration is induced upon its release from
chromatin at 0.4 M NaCl. We note, however, that a reduction
of salt concentration by dialysis from 0.4 to 0.1 M NaCl
leads to a reformation of stable trimeric RPA only when dialysis is
accompanied by dephosphorylation of RPA32 (Fig. 4). Furthermore, Murti
et al. (30) have shown by immunostaining of fixed intact
HeLa cells that RPA70 binds to spindle poles and RPA32 to chromosomes
during mitosis. Cardoso et al. (45) found that RPA70, but
not RPA32, localizes at replication foci in mouse myotube cells. These
earlier studies are consistent with the possibility that a separation
of the RPA subunits could occur in vivo, at least in some
cell types and during certain cell cycle stages.
While our data clearly indicate that the mode of interaction between
RPA70 and RPA32 is strongly affected by phosphorylation, we cannot say
yet which enzymes are responsible for RPA32 phosphorylation and how
their function is regulated. Biochemical studies had shown that DNA-PK
as well as cyclin A- or cyclin B-dependent kinases phosphorylate RPA32 in vitro at defined serine and threonine
side chains in an amino-terminal section of RPA32 (17, 46). DNA-PK has
a major function in DNA damage repair (2). Therefore, CDKs are more
likely candidates for RPA phosphorylation in normally proliferating
cells as studied in this communication. For this reason, we have used
cyclin A/CDK1 and cyclin B/CDK1 for in vitro phosphorylation
experiments with recombinant RPA as a substrate. Niu et al.
(25) have shown that cyclin B/CDK1 efficiently transfers one phosphate
group to a serine residue at amino acid position 29 of RPA32 and other
phosphates with reduced efficiency to additional unmapped RPA32 sites.
This leads to two electrophoretic bands of phosphorylated RPA32, termed
P1 with one, and P2 with two or maybe more, phosphate groups per
molecule. In agreement with these results, we find that a treatment of
RPA with either cyclin A/CDK1 or cyclin B/CDK1 predominantly produces
the P1 form of phosphorylated RPA32 (Fig. 5). However, even after long
incubation times, in vitro phosphorylation never reached the
level of RPA32 phosphorylation in vivo as judged from the
relative abundance of the phosphorylated RPA forms P1 and P2. Thus,
CDKs are either more active in vivo, or the DNA-PK together
with other unidentified protein kinases are responsible for the major
phosphorylation of chromatin-bound RPA in vivo. In either
case, only a partial fraction of the most highly phosphorylated form of
CDK-treated RPA desintegrated in vitro (Fig. 6), supporting
the notion that phosphorylation plays a major role in changing the
interaction of the RPA subunits. A possible explanation for the
ambiguous results could be the use of Xenopus CDKs to
phosphorylate human RPA in vitro, although CDKs are known to
be highly conserved from yeast to human and are functionally
exchangeable (41). However, it could be possible that there exist
important differences in the phosphorylation pattern of RPA32 in
vivo and in vitro. Further work has to identify the
amino acid residues in RPA32 whose phosphorylation is responsible for
the changes in RPA complex formation that we have observed.
The important point emerging from the experiments reported in this
communication is that phosphorylation changes the interaction and
affinity between the large and the middle RPA subunits, resulting in
their separation at least under in vitro conditions, but
possibly also in intact nuclei.