From the § Abteilung Biochemie, Institut für
Molekulare Biotechnologie, D-07745 Jena, Germany,
Novosibirsk Institute of Bioorganic Chemistry, Siberian
Division of Russian Academy of Sciences, 630090 Novosibirsk, Russia,
GSF, Institut für Molekulare Immunologie,
D-81377 München, Germany, and ** National University
of Ireland, Galway, Ireland
Received for publication, February 5, 2003
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ABSTRACT |
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Although the mechanical aspects of the
single-stranded DNA (ssDNA) binding activity of human replication
protein A (RPA) have been extensively studied, only limited information
is available about its interaction with other physiologically relevant
DNA structures. RPA interacts with partial DNA duplexes that resemble DNA intermediates found in the processes of DNA replication and DNA
repair. Limited proteolysis of RPA showed that RPA associated with
ssDNA is less protected against proteases than RPA bound to a partial
duplex DNA containing a 5'-protruding tail that had the same length as
the ssDNA. Modification of both the 70- and 32-kDa subunits, RPA70 and
RPA32, respectively, by photoaffinity labeling indicates that RPA can
bind the primer-template junction of partial duplex DNAs by interacting
with the 3'-end of the primer. The identification of the protein
domains modified by the photoreactive 3'-end of the primer showed that
domains located in the central part of the RPA32 subunit (amino acids
39-180) and the C-terminal part of the RPA70 subunit (amino acids
432-616) are involved in these interactions.
The human single-stranded DNA
(ssDNA)1-binding protein,
replication protein A (RPA), is a stable heterotrimer consisting of three subunits with apparent molecular masses of 70, 32, and 14 kDa
(RPA70, RPA32, and RPA14, respectively). Homologous ssDNA-binding proteins like RPA that are essential for cell viability have been found
in all eukaryotic species investigated to date. RPA is one of the key
players in various essential processes of DNA metabolism including the
initiation and elongation of DNA replication, nucleotide and base
excision repair, and homologous recombination (reviewed in Refs. 1 and
2).
RPA functions are based on its DNA binding activity and its
protein-protein interactions (1-4). The major ssDNA binding activity is located in RPA70. A total of three DNA-binding sites are now distinguished in this subunit, which are referred to as the DNA-binding domains A-C (DBD-A, DBD-B, and DBD-C, respectively) (5). A fourth
DNA-binding domain, domain D (DBD-D), resides in the RPA32 subunit
(6-9). Although the RPA14 subunit contains a structural motif found in
other DBDs, there is no biochemical evidence that this subunit binds to
DNA (7, 9).
The interaction of RPA with various ssDNA substrates has been
investigated in detail. Several ssDNA-binding modes were observed and
characterized both biochemically and structurally (reviewed in Refs.
1-3). In the first mode RPA binds to ssDNA with an occluded binding
site of 8-10 nucleotides (nt) in a cooperative manner with low
affinity. A second one is characterized by high affinity binding, an
occluded binding site of 30 nucleotides, and a low cooperativity
(10-13). It has now become an accepted view that RPA binds to ssDNA in
a multistep pathway (6, 14, 15). The first step involves the
cooperative binding to DNA by DBD-A and DBD-B and corresponds to the
less stable "8-nt" binding mode. On a gapped template binding is
performed in a 5' Among the different DNA structures partial DNA duplexes are mimicking
in vivo intermediates that occur during DNA replication and
DNA repair (3, 18-20). Because these structures are more complex than
exclusive ssDNA templates, it is the central question how RPA interacts
with these DNA structures. In previous reports, photoaffinity labeling
studies using DNA duplexes with 5'-protruding ends suggested that
binding of RPA subunits is dependent on the template configuration, and
the protein:DNA ratio. RPA70 subunit binds to the single-stranded part
of the DNA duplex, whereas the RPA32 subunit is located near 3'-end of
the primer (19, 21). These studies were confirmed in vivo by
investigating replicating SV40 minichromosomes. Depending on the extent
of primer elongation, both subunits were photocross-linked to the
3'-end of the growing primer (20, 22). In this report we have
investigated conformational changes, which accompany the binding of RPA
to partial DNA duplexes and assigned protein domains playing important
roles in such interactions.
Proteins and Nucleotides--
Recombinant human DNA polymerase
DNA Hairpins, Primer-Template Systems, and ssDNAs--
The
designations and sequences of hairpin oligonucleotides are as follows
(underlined is the palindrome part of the hairpin, which were designed
according to Ref. 18): HP-T0 (single prominent nucleotide);
5'-AGGCCGTGCTCTGAATTCCTGGATGTTCGAAAGCTTTCGAACATCCAGGAATTCAGAGCACGGCC-3'; HP-T4 (5-nt ssDNA extension),
5'-(T)4AGGCCGTGCTCTGAATTCCTGGATGTTCGAAAGCTTTCGAACATCCAGGAATTCAGAGCACGGCC-3'; HP-T8, (9-nt ssDNA extension),
5'- (T)8AGGCCGTGCTCTGAATTCCTGGATGTTCGAAAGCTTTCGAACA
TCCAGGAATTCAGAGCACGGCC-3'; HP-T16 (17-nt ssDNA extension),
5'-(T)16AGGCCGTGCTCTGAATTCCTGGATGTTCGAAAGCTTTCGAACATCCAGGAATTCAGAGCACGGCC-3'; HP-T32 (33-nt ssDNA extension),
5'-(T)32AGGCCGTGCTCTGAATTCCTGGATGTTCGAAAGCTTTCGAACATCCAGGAATTCAGAGCACGGCC-3'.
The partial duplex system is as follows: PT32,
5'-(T)32AGTGTTATAGCCCCTACC-3' and
3'-ACAATATCGGGGATGG-5'.
The deoxyoligonucleotides d(AT4), d(AT8),
d(AT16), and d(AT32) were used to compare
ssDNAs and partial duplex DNAs.
Primer of the partial duplex system was annealed to the template at a
molar ratio of 1:1 in 10 mM Tris-HCl, pH 7.5, 10 mM KCl, by heating the mixture at 95 °C for 5 min and
then slowly cooling it down to room temperature. Hairpins were
self-annealed in the same manner.
Limited Proteolysis of RPA and Identification of Proteolytic
Fragments--
5 µg of RPA was treated at 37 °C with the
indicated amount of trypsin (2.5-100 ng) in a reaction mixture (10 µl) containing 30 mM HEPES-KOH, pH 7.8, 1 mM
EDTA, 70 mM MgCl2, 10 mM DTT. Where indicated a DNA hairpin (HP-T0, HP-T4, HP-T8, HP-T16, or HP-T32) was
present in concentrations equimolar to that of RPA. After 20 min of
incubation, reactions were terminated by addition of Laemmli sample
buffer and boiling for 15 min. The proteolyzed products were separated
by 14% SDS-PAGE (29) and analyzed by Coomassie Brilliant Blue
G-250 staining, immunoblotting, and automated N-terminal sequencing.
Molecular masses of the products were determined on the basis of their
electrophoretic mobility using protein molecular mass markers as a reference.
Immunoblotting--
Protein samples were separated by 14%
SDS-PAGE (29) and transferred to polyvinylidene difluoride membrane.
RPA subunits and proteolytic fragments were detected using monoclonal
antibodies to RPA70 and RPA32, respectively. Secondary antibody
conjugates, ECL detection, and bromo-4-chloro-3-indolyl phosphate/nitro
blue tetrazolium detection kits were used according to the
manufacturer's protocols.
Automated N-terminal Sequencing of Proteolytic
Fragments--
Protein samples were separated by 14% SDS-PAGE (29),
transferred to polyvinylidene difluoride membrane, and stained with Coomassie Brilliant Blue G-250. The protein bands of interest were excised, and the first 8-10 N-terminal amino acids were
determined using a Procise 494 HT Protein Sequencer (Applied
Biosystems, Weiterstadt, Germany).
Calculation of Proteolytic Fragments--
The "WinPep"
software (30) was used to calculate the polypeptides of RPA70 and RPA32
derived from limited proteolysis and characterized by N-terminal
sequencing and SDS-gel electrophoresis. The protein sequences of
RPA70 and RPA32 were obtained from the Swiss-Prot protein Sequence
Database (entries RFA1-HUMAN, P27694, and
RFA2-HUMAN, P15927).
Primer Labeling and Elongation in the Presence of Photoreactive
dNTP Analogs--
Primers were 3'-end-labeled at 37 °C in a
reaction mixture (300 µl) containing 50 mM HEPES-KOH, pH
7.8, 50 mM KCl, 10 mM MgCl2, 0.32 µM pol Photochemical Cross-linking and Limited Proteolysis of
Protein-DNA Adducts--
RPA was labeled with a photoreactive primer
in a reaction mixture (200 µl) containing 50 mM
HEPES-KOH, pH 7.8, 50 mM KCl, 10 mM
MgCl2, 0.5 µM radioactively labeled
photoreactive partial duplex DNA, and 0.5 µM RPA.
Reaction mixtures were incubated for 5 min at room temperature, placed
in micro-Eppendorf tubes on ice, and irradiated through the open top
for 10 min using the Stratalinker UV cross-linker (Stratagene,
Amsterdam, The Netherlands) equipped with lamps producing UV light of
312 nm. After cross-linking EDTA, MgCl2, and DTT were added
to final concentrations of 1, 70, and 10 mM, respectively.
Partial proteolysis of the samples was started by adding trypsin
(17,400 units/µg) to a trypsin:RPA weight ratio of 1:250 and
subsequent incubation at 37 °C. Aliquots (20 µl) of the reaction
mixture were quenched with Laemmli loading buffer at the indicated
times and boiled for 15 min. Photochemically cross-linked and partially
digested protein-DNA samples were separated by 13.5% SDS-PAGE. Gels
were stained with silver (31, 32), dried, and exposed to x-ray films
(Kodak Biomax, Sigma) or quantified using the PhosphorImager Storm 860 (Amersham Biosciences).
Proteolytic Profile of RPA Digested by Trypsin--
In a previous
report (17) limited proteolysis was used to analyze the protein
conformation and to determine structural domains of RPA. It was shown
that the heterotrimer undergoes a significant conformational change
upon binding to extended ssDNA regions. We have used the same technique
to address the question of whether the binding of RPA to partial DNA
duplexes (DNA hairpins) containing 5'-protruding ends shows some
peculiarities in comparison to the binding of ssDNA. Proteolysis was
performed using trypsin as a protease and RPA being either free in
solution, bound to ssDNA or DNA-hairpins with ssDNA template strands of
various lengths. As shown earlier (17), digestion of RPA by trypsin
results in a limited number of distinct fragments. In our experiments
we have made titrations of RPA with increasing amounts of the protease. As can be seen in Fig. 1 the proteolytic
pattern in this case was almost identical to that obtained when free
RPA was digested with a fixed amount of protease for increasing times
(17).
In some instances it was not possible to resolve fragments from each
other during denaturing one-dimensional gel electrophoresis. Therefore,
sometimes single Coomassie Brilliant Blue-stained band contained two
distinct peptides (Fig. 1; peptides designated "1" and "2,"
RPA32, and "6," as well as "9a" and "9b") that
could be distinguished by region-specific antibodies. Several tryptic peptides have not been identified previously, so we employed
immunoblotting and automated Edman sequencing to map precisely the
origins of the products. We used five different monoclonal antibodies
directed against RPA70 that recognize different parts of the subunit.
70A and 70B bind within the first 173 amino acids (aa), which cover the
N-terminal part and some of the following interdomain linker. 70C
interacts with the region of amino acids 174-330, which compose the
central domain of the 70-kDa subunit. RAC-1 and RAC-4 are both
antibodies specific for the C-terminal domain of RPA70 and recognize
regions spanning amino acids 441-525 and 525-616, respectively (Fig.
2 and data not shown). In addition,
N-terminal sequences of the proteolytic fragments were determined. The
polypeptides were then identified by calculation using
WinPep software (30) and selecting the
polypeptides with the best matches to the experimentally obtained data,
molecular masses, N-terminal sequences, and antibody reactivities. The results of the peptide mapping are summarized in
Table I and in Fig. 3. (A more detailed
explanation of the proteolytic degradations is presented in the
Supplemental Material Figs. S1-14. Supplemental Figs. S3-7 in
particular address the partial tryptic digestion in the absence of
DNA.)
Analysis of the RPA70 peptide map revealed especially exposed
proteolytic sites in this subunit. As was shown earlier, major cleavage
sites are located in the linker between the N-terminal domain (NTD70)
and DBD-A after Lys-167 and Lys-163, dividing RPA70 into peptides
designated "3," "10," and "11" (Figs. 1,
4A, and 5A) (17).
However, additionally determined fragments indicate that this site is
not the only one accessible during this initial stage of degradation.
The cleavage sites after Lys-367, Arg-390, and Lys-354, which are all
located within the DBD-B (Figs. 1 and 4A), lead to the
peptides "5a," "8," and 6, respectively, which are present in
small amounts. A minor cleavage site after Lys-431, which lies in the
linker between DBD-B and DBD-C, creates peptides "4" and 9a, and
the tryptic digestion after Arg-489 and Lys-473 within DBD-C yields the
minor proteolytic fragments 1 and 2. This number of exposed sites and
available proteolytic pathways explain in part the high rate of RPA70
degradation (Fig. 5A). The
relatively large tryptic peptides 3 and 5a are proteolyzed
further utilizing the latter cleavage sites. The kinetics of digestion
suggests that product 3 is further degraded within the DBD-B region
giving rise to fragments 8, 9a, or 9b. Peptide 5a seems to be cleaved in the region of the interdomain linker into fragment 10 and further to
11, which contain the N-terminal part of the RPA70 subunit. Peptides 8 and 6 are further reduced to the C-terminal DBD-C-containing fragment
9a.
In accordance with earlier published data, RPA32 is degraded primarily
because of the potential cleavage at the Lys-38 which removes the
N-terminal part and gives rise to fragment 7 (Figs. 1 and
4A) (8, 17). This product is further cleaved at Arg-180 so
that the proteolytically stable core of RPA32, peptide "12," is
obtained (data not
shown).2
Proteolysis of RPA in the Presence of DNA Hairpins with
5'-Protruding ssDNA Tails--
The addition of equimolar amounts of
partial DNA-hairpins to RPA changed the pattern of the RPA70 subunit
proteolysis (Figs. 4, A-F, and 5; summarized in Table I; a
detailed description of the proteolytic degradations is given in the
Supplemental Material (Figs. S1-14); Figs. S8-12 show an extensive
comparison of the changes in the polypeptide pattern in the
presence of partial duplex DNAs). As in the case of ssDNA, the presence
of partial DNA hairpins in the reaction mixture leads to a decrease of
the proteolytic sensitivity of RPA70 (17). The access to cleavage sites
might be blocked either by the bound DNA or by changes of the RPA
conformation that are induced upon DNA binding. When we used a partial
DNA hairpin with only a single 5'-prominent nucleotide (HP-T0) the
access to the initial cleavage sites within the DBD-B and the linker
between DBD-B and DBD-C were greatly reduced as indicated by the
significantly lower amounts of fragment pairs 5a and 8, 4 and 9a, as
well as fragment 6 (Fig. 4, A and B; groups of arrows "a," for explanations see Table I and
Figs. 3 and 5). The same initial cleavage sites seems to be completely
blocked in the presence of a hairpin with a 9-nt ssDNA tail (HP-T8)
(Fig. 4D and Supplemental Material Figs. S8-12). The
proteolytic sites that map within the linker between the N-terminal
domain and DBD-A are not affected to the same extent. The major
cleavage in the presence of partial DNA hairpins still occurs after the
N-terminal amino acid ~167 which is similar to that with ssDNA and
results in fragment 3 (region aa 168-616, containing DBD-A, -B, and
-C).
The proteolytic pattern of fragment 3 changes in the presence of DNA
hairpins (Fig. 4, B-F, summarized in Fig. 5B and
Table I). Sites in DBD-B become less accessible, and polypeptide 3 is
more stable, whereas a novel fragment 5b (aa 168-489, containing DBD-A
and DBD-B) appears as a product of cleavage in DBD-C after Lys-489. Yet
another band with an apparent molecular mass of 33 kDa seems to be
closely related to fragment 5b, and we predict that it is generated by
cleavage of fragment 5b within DBD-C after Lys-473. Further digestion
of 5b apparently results in fragment 9b upon cleavage within the DBD-B
after Lys-354, and the new tryptic polypeptide consists of DBD-A plus
parts of DBD-B. Although we can detect these novel fragments on
intermediate stages of degradation in the presence of both HP-T0 and
HP-T4, significant levels are detected only if the protruding tail
extends to 9 nucleotides in HP-T8 (Fig. 4D, groups of
arrows "b"). Moreover, upon the addition of
hairpins with even longer ssDNA tails, HP-T16 and HP-T32, these fragments only appear at higher trypsin concentrations indicating that
cleavage in DBD-C at Lys-489 is affected by the length of the
protruding DNA strand (Fig. 4, D-F, groups of
arrows "c").
In earlier reports it was shown that the presence of single-stranded
(dT)30 caused RPA32 and its proteolytic fragment 7 to become more sensitive to trypsin. Both the pattern and the rate of
proteolysis were altered (17). However, in the case of all employed
partial DNA hairpins, we observed the same proteolytic pathway, and no
decrease in stability of either RPA32 or its major proteolytic fragment
was detected (Fig. 4, A-F, arrows
"d"). Intriguingly, in the presence of both DNA
hairpins HP-T16 and HP-T32, which have 17- and 33-nt ssDNA tails,
respectively, fragment 7 appears even at later digestion stages than in
the absence of DNA. These findings suggest that the conformation of the
RPA heterotrimer bound to DNA hairpins significantly differs from that
of RPA bound to ssDNA of comparable length and of free RPA in solution.
Comparison of the Stability of RPA-bound ssDNA and
Partial Duplex DNA Constructs--
To investigate the differences in
the conformation of free and DNA-bound RPA as well as the influences of
various DNA substrates, we performed partial tryptic digest of the
protein complex in the absence and in the presence of ssDNA or partial
duplex DNA, and we compared the proteolysis products (Fig. 6). The
single-stranded oligonucleotides with lengths ranging from 5 to 33 nt
exactly matched the length and sequence composition of protruding ends of the partial duplex DNAs. The partial proteolysis experiments revealed that ssDNA with a length of 9 nucleotides allowed some protection against tryptic digest compared with the digest of RPA in
the absence of any DNA. However, this protection was very weak (Fig.
6, compare lanes 2 and
5; see also Supplemental Material Figs. S13 and 14). Only
ssDNA with a length of 17 nt or longer was able to produce significant
and clear protection against tryptic digests under these conditions,
with the polypeptide 3 being clearly detectable (Fig. 6, lanes
6 and 7). Further protection of RPA was achieved in the
presence of identical molar amounts of ssDNA with a length of 33 nt
which is accompanied by a slight decrease in the amounts of the
proteolytic fragments 8, 9a, 9b, and a sustained increase in the
stability of fragment 3.
In contrast to these results, partial duplex DNA with a protruding tail
of 5 nt was already sufficient to significantly decrease the
proteolytic sensitivity of RPA70 (Fig. 6, compare lanes 2 and 9). Duplex DNA with a 9-nt protruding tail stabilized
RPA to an extent that is comparable with that of exclusive ssDNA
substrates with lengths between 17 and 33 nt (Fig. 6, compare
lane 10 with lanes 6 and 7). Longer
protruding ssDNA parts of partial duplex DNAs (17 or 33 nt) so
efficiently protected some sites in RPA70 sensitive to tryptic cleavage
that specific proteolytic degradation products of free RPA70 in the
range of 18-28 kDa (fragment 8, 9a, and 9b) were diminished
(lanes 11 and 12, respectively). In addition to the disappearance of some fragments, the polypeptides 5b
and especially 3 are stabilized, which means that the N-terminal domain
of RPA70 is still cleaved off in RPA bound to DNA. The observed
differences suggest that the conformation of the RPA heterotrimer bound
to DNA hairpins significantly differs from that of RPA associated with
ssDNA of comparable length or free RPA in solution.
Identification of Protein Domains Interacting with a
Primer-Template Junction--
We have demonstrated in an earlier study
(33) the cross-linking of the RPA32 subunit to the 3'-end of the primer
using partial DNA duplexes with 5'-protruding tails. To test whether
the low affinity DNA-binding domains DBD-D and possibly DBD-C are
involved in this type of interaction, we have employed a combination of photocross-linking and limited proteolysis. To this end a partial DNA
duplex with a 5'-protruding ssDNA template tail of 32 nt was used. A
radioactively labeled nucleotide and a photoreactive analog of dTTP
(NAB-4-dUTP) were enzymatically incorporated in the 3'-end of the
primer. After photocross-linking RPA to this structure, we used trypsin
to obtain a proteolytic profile of labeled DNA protein species at
different times at a trypsin:RPA ratio of 1:250 (Figs.
7A and 4F). The
tryptic fragments were separated on a denaturing 13.5% PAGE and
stained with silver. Then the dried gel was subjected to
autoradiography. In accordance with earlier published data (15, 21),
the two larger subunits of the heterotrimer were cross-linked with
RPA32 being labeled to a significantly more extent (Fig.
7A, lane 3). Partial proteolysis revealed than
only a limited number of radioactively labeled DNA cross-linked
fragments were generated upon addition of trypsin (Fig. 7A).
Apart from the full-length subunits a total of five proteolytic
fragments were detected by autoradiography. The same fragments were
labeled when we used "zero-length" cross-linking reagent
(4-S-dTTP) to elongate the DNA probe (data not shown). The ssDNA
protruding tail of the elongated primer-template PT32 system differs in
one single nucleotide from the protruding template strand of the HP-T32 hairpin, but this change hardly influences the RPA-DNA complex because
the RPA digestion patterns in the presence of these two DNA structures
were alike as revealed by a silver stain of the gel used for
autoradiography (data not shown). Notably, overstaining of the gel by
silver permitted us to detect trace amounts of peptides that were not
stained by Coomassie Brilliant Blue, namely fragments 4, 5a, and
8. Silver staining is known to be significantly more sensitive than
Coomassie Brilliant Blue staining so the data obtained stress
the predominance of a proteolytic pathway that does not consist of
cleavages within DBD-B and the linker between DBD-B and DBD-C.
Therefore, we compared the proteolytic stages of the established
digestion pattern of RPA bound to HP-T32 to that of the radioactively
labeled protein-DNA conjugates. These data in combination with the
molecular masses of the generated radioactive products allowed us to
assign the peptides present in the labeled conjugates. It should be
noted that the addition of the covalently attached DNA resulted in an
increase in the apparent molecular mass. This shift of the apparent
mass could be deduced by comparing the molecular masses of DNA-protein
conjugates containing the full-length RPA70, RPA32, and pol
The RPA70-derived peptides 3 (DBD-A, DBD-B plus DBD-C; spanning aa
168-616), 8 ("long" DBD-C; aa 390-616), and 9a ("short" DBD-C; aa 432-616) were shown to carry a labeled DNA indicating that
it is the C-terminal part of this subunit that is cross-linked to the
3'-end of the primer. One should keep in mind that there is another
possibility to assign the origin of the radioactive conjugate with the
apparent molecular mass of 31 kDa other than to peptide 9a (short
DBD-C), namely to peptide 9b (DBD-A; aa 168-354). We assume, however,
that peptide 9b is less favorable because within the same proteolytic
experiment we cannot observe a labeling of its bigger ancestor 5b (Fig.
7A). The absence of labeled products corresponding to
fragments 5b, 10, and 11, which consist of aa 168-489 (DBD-A plus
DBD-B), aa 1-167, and aa 1-163 (NTD70), respectively, indicates that
the central part of RPA70, containing DBD-A and DBD-B, and the
N-terminal part of the subunit are not cross-linked to the primer (Fig.
7B).
In addition to the tryptic RPA70 polypeptides 3, 8, and 9a, two
radioactively labeled proteolytic fragments have descended from RPA32
(Fig. 7A, summarized in Fig. 7B). The
polypeptides 7 (DBD-D plus CTD32; aa 39-270) and 12 (DBD-D; aa
39-180) are conjugated to DNA and have the molecular masses of 38,5 and 24 kDa, respectively. Both of the products contain the central part of the RPA32 subunit including the DNA-binding domain DBD-D.
The heterotrimeric RPA complex and its interactions with ssDNA
have been thoroughly investigated (reviewed in Refs. 1 and 2). Recent
studies (6, 9, 14, 15, 17, 34-36) have unraveled the roles that
different domains of RPA play in ssDNA binding and have revealed
changes in RPA conformation upon DNA binding as well as different
binding modes. A sequential binding of RPA domains is probably
responsible for the conformational changes of the RPA heterotrimer that
are induced upon DNA binding. Data obtained earlier from proteolysis
studies indicated the existence of a limited number of cleavage sites
accessible for the protease within the heterotrimer. Upon binding with
ssDNA, the pattern of proteolysis changed suggesting both a direct
blocking of the cleavage site by DNA and occlusion of some sites due to
conformational changes (17).
Our data corroborate previous investigations showing the presence of
several easily accessible proteolytic sites. The major initial cleavage
site for RPA70 in the absence of DNA is located in the interdomain
linker between NTD70 and DBD-A (17). This unstructured region is rather
large and is obviously favorably exposed to the protease regardless of
protein conformation. Additional initial cleavage site positions
determined in our work differ from those that have been mapped earlier.
We found that they are located in the DNA-binding domains DBD-B and
DBD-C. Although the sites in DBD-B are located in exposed extended
loops and helices, a very weak proteolytic cleavage at DBD-C occurs in
the zinc finger motif (comprising cysteines at positions 481, 486, 500, 503; for an explanation see Figs. 3, 5, 7B, and 8 as well as
the Supplemental Material) implying that at least part of this motif is
exposed to the solution (9, 14, 34). Cleavage within the linker between
domains DBD-B and DBD-C, which had been reported in earlier publications, was also observed, but it is underrepresented during the
initial stages of proteolysis. Perhaps this region of RPA is not
accessible to trypsin under the conditions used here.
The presence of DNA hairpins, even those with the shortest 5'-extended
ssDNA part, resulted in a significant protection of initial cleavage
sites located in DBD-B (see Fig. 8). Our
favorite explanation is a block of this cleavage site by steric
hindrance of the DNA. We want to stress that the length of the
available ssDNA of an extension by one nucleotide is not sufficient for binding per se. We assume that the dsDNA destabilizing
activity of RPA is not likely to provide additional 7 nucleotides for
the binding event to take place because this RPA activity is greatly reduced in the presence of magnesium (37), which was added to our
incubation buffers. However, it is worth mentioning that an increase in
the length of the 5'-protruding tail of the hairpin up to 5 nt results
in a further stabilization of RPA70 subunit and its major tryptic
fragments. de Laat et al. (18) working with hairpins similar
to those used in the present study reported a significant increase in
DNA binding affinity of RPA in the presence of a hairpin and ssDNA
stretches. Nevertheless, they were not able to detect significant dsDNA
destabilizing activity working at even lower concentrations of
Mg2+ and salt. The higher affinity of RPA toward partial
duplex DNA than to ssDNA was indirectly reproduced because the presence
of a DNA hairpin with 20 nucleotides dsDNA and a 5-nucleotide ssDNA protruding tail (AdT4) was sufficient to significantly
protect RPA against proteolysis. In contrast to this result,
RPA was not able to bind efficiently to the oligodeoxynucleotide
AdT4, and RPA was not protected against tryptic
digestion.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3' direction that determines the overall polarity
of RPA (16). The following steps include the successive 3'-directed DNA
binding of DBD-C adjacent to DBD-B and of DBD-D adjacent to DBD-C
resulting in the stable "30-nt" binding mode (6, 14). All these
transitions are accompanied by changes in the conformation of the RPA
heterotrimer as has been revealed by electron microscopy and limited
proteolysis (11, 17).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(pol
) was purified as described previously (23). RPA was
expressed in Escherichia coli and purified as outlined
elsewhere (24, 25) except for the absence of EDTA in all purification
buffers. Sequencing grade modified trypsin (17400 units/mg) was
purchased from Promega (Mannheim, Germany). The 10-kDa protein ladder
and prestained molecular mass markers were from Invitrogen and from
Sigma, respectively. The RPA70-specific mouse monoclonal antibodies
70A, 70B, and 70C have been described elsewhere (26). Rat monoclonal
antibodies directed against the C terminus of RPA70 (RAC-1 and RAC-4)
as well as RPA32 (RBF-7) were produced according to standard procedures
(27). Secondary horseradish peroxidase or alkaline
phosphatase-conjugated antibodies were from Dianova (Hamburg, Germany)
and Promega. The ECL chemiluminescence kit for immunoblotting was from
Amersham Biosciences; 5-bromo-4-chloro-3-indolyl phosphate/nitro blue
tetrazolium substrates for immunoblotting were from Sigma.
Oligonucleotides were synthesized at the Abteilung Biochemie
(Institut für Molekulare Biotechnologie, Jena, Germany). The
photoreactive nucleotide analogs NAB-4-dUTP (28) and 4-S-dTTP were kind
gifts of Dr. D. Kolpashchikov and Dr. V. Bogachev, respectively
(Novosibirsk Institute for Bioorganic Chemistry, Novosibirsk, Russia).
[
-32P]dCTP (3000 Ci/mmol) was obtained from Amersham
Biosciences.
, 0.54 µM partial duplex DNA
PT32, 1 µCi of [
-32P]dCTP, and 2.5 µM
unlabeled dCTP. After 5 min of incubation photoreactive NAB-4-dUTP or
4-S-TTP was added at a final concentration of 5 µM, and
the mixture was further incubated for 15 min at 37 °C. Reactions
were stopped by heating the mixture for 5 min at 95 °C and rapidly
cooling down on ice for 5 min. After such a treatment denatured pol
was precipitated and removed by centrifugation for 15 min at
15,000 × g. Resulting mixtures were heated at 95 °C
for 5 min and cooled down slowly to room temperature to allow the
radioactively labeled photoreactive primer to reanneal to the template strand.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Partial proteolysis of RPA with trypsin.
5 µg of RPA were treated with trypsin at 37 °C at the indicated
trypsin:protein mass ratios (lanes 3-9). After 20 min of
incubation reactions were terminated and products separated by 14%
SDS-PAGE followed by Coomassie Brilliant Blue G-250 staining.
Untreated RPA was analyzed for comparison (lane 2). The
10-kDa protein ladder (Invitrogen) was used as the molecular mass
marker (lane 1). Designations of individual fragments are
indicated on the right.
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Fig. 2.
Probing proteolyzed RPA with different
monoclonal antibodies. RPA was incubated at 37 °C with trypsin
at a trypsin:protein weight ratio of 1:750. After 20 min of incubation
reactions were terminated, and products (lanes 3-7) along
with the 10-kDa protein ladder (lane 1) or a prestained
marker (lane 2) were separated by 14% SDS-PAGE and analyzed
either by Coomassie Brilliant Blue G-250 staining (lanes
1-3) or by Western blots probed with different specific
antibodies (lanes 4-7). Designations of the antibodies and
regions of RPA that are recognized by each set of antibodies are
schematically shown above the picture. Those of individual
tryptic peptides are indicated on the left.
Summary of the partial tryptic RPA digests
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Fig. 3.
Schematic map of proteolytic peptides
generated upon RPA treatment with trypsin. Positions of tryptic
peptides within the RPA70 and RPA32 subunits are shown on A
and B, respectively. Open bars represent subunits
with structural domains shown by gray boxes, and
unstructured linkers are shown in white (NTD70,
N-terminal domain of RPA70; DBD, DNA-binding domain;
CTD32, C-terminal domain of RPA32). Black bars
correspond to individual tryptic fragments with their identification
number given on top of each bar. Molecular masses of the
peptides in kDa are indicated on the left of the
corresponding bar.
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Fig. 4.
Digestion of RPA by trypsin in the presence
of partial DNA hairpins with 5'-protruding tails. Each panel
represents the profile of proteolytic digestion of RPA by trypsin in
the absence (A) or presence of an equimolar amount of
partial DNA-hairpins with ssDNA tails of increasing length (hairpin
HP-T0, B; HP-T4, C; HP-T8, D; HP-T16,
E; and HP-T32, F). For each set 5 µg of RPA was
treated with increasing amounts of trypsin at 37 °C at the indicated
trypsin:protein weight ratios. After 20 min of incubation reactions
were terminated, and products were separated by 14% SDS-PAGE. Gels
were analyzed by Coomassie Brilliant Blue G-250 staining. Lanes
designated RPA, trypsin, and markers
contain untreated RPA, 0.5 µg of trypsin without RPA added, and the
10-kDa protein ladder, respectively. Designations of individual tryptic
peptides are indicated on the right of each panel.
Arrows with lowercase letters indicate fragments
whose presence or quantity considered to be DNA-related. A detailed
explanation and comparison of these proteolytic degradation products
are presented in the Supplemental Material (Figs. S8-12).
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Fig. 5.
Digestion pathways of RPA70 by trypsin in the
absence and in the presence of partial DNA hairpins with extended
5'-protruding tails. A depicts the digestion pathway of
RPA70 by trypsin in the absence of DNA and B in the presence
of DNA hairpins with extended 5'-protruding tails. Each bar
represents a peptide with structural domains shown in gray,
and unstructured linkers are shown in white
(NTD70, N-terminal domain of RPA70; DBD,
DNA-binding domain). The designation of each peptide is shown on the
left of the bar. Arrows indicate positions of
available tryptic cleavage sites for each peptide. The most common
cleavage sites are indicated by black, the least frequent
ones are marked by white arrowheads, and those
with intermediate accessibility are marked in gray
arrowheads. Peptides that are the products of a specific cleavage
event are shown next to the arrow representing this
cleavage. For some peptides we have omitted the schematic bar and put
the fragment name instead. The asterisk in A
indicates the cleavage site that is sensitive to the addition of DNA
hairpins even with a single 5'-prominent nucleotide. The double
asterisks in B identify those cleavage sites that are
only sensitive to DNA hairpins with long (17 and 33 nucleotides)
protruding ssDNA tails.
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Fig. 6.
Protection of RPA70 cleavage sites by ssDNA
and partial DNA duplexes. The presence of DNA stabilizes RPA
against proteolytic degradation. After the addition of equal
molar amounts of dATP (lane 3), single-stranded
oligonucleotides with increasing lengths (d(AT4),
d(AT8), d(AT16), and d(AT32),
lanes 4-7, respectively marked as ssDNA) or partial DNA
duplexes with increasing lengths of protruding ssDNA ends (HP-T0
(contains a single dAMP protruding end), HP-T4, HP-T8, HP-T16, and
HP-T32, lanes 8-12, respectively marked as hairpins), RPA
was incubated with trypsin. Then the proteolytic products were analyzed
by SDS-gel electrophoresis and Coomassie Brilliant Blue staining of the
polypeptides. Untreated RPA and RPA incubated with trypsin in the
absence of DNA (lanes 1 and 2, respectively)
served as controls.
(78, 41.5, and 49 kDa, respectively) to the known native determined
molecular masses of the free polypeptides, and this amounted to about
10 kDa (Fig. 7A, lanes 1 and 3). The results of the combined photocross-linking and proteolytic digestion assays are summarized in Fig. 7B.
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Fig. 7.
Limited proteolysis of RPA cross-linked to a
primer 3'-end of a partial DNA duplex. A, RPA was
UV-induced cross-linked to the photoreactive radioactive
primer-template probe as explained under "Experimental
Procedures." Limited proteolysis of the samples was started by
adding trypsin to a trypsin:RPA weight ratio of 1:250 and was carried
out at 37 °C for the indicated times (lanes 3-14).
Photochemically cross-linked and partially digested protein-DNA samples
were separated by 13.5% SDS-PAGE. The dried gel was subjected to
autoradiography. Designations of the cross-linked and digested
fragments are shown on the right. In lane 1, the
products of a photocross-linking reaction of 0.5 µg of DNA pol to
the substrate were loaded which served as control to show that the
enzyme was fully removed from the assay before cross-linking.
Lane 2 represents the photoreactive radioactive
primer-template irradiated with UV in the absence of RPA and then
incubated with trypsin for 90 min at 37 °C. B, schematic
sketch of RPA fragments cross-linked to the 3'-end of the primer using
a partial DNA duplex with a 5'-protruding tail of 32 nucleotides. Each
bar represents a peptide with structural domains shown in
gray, and unstructured linkers in white.
(NTD70, N-terminal domain of RPA70; DBD,
DNA-binding domain; P, N-terminal phosphorylation site of
RPA32, and CTD32, C-terminal domain of RPA32). The molecular
masses of the corresponding DNA-protein adducts are presented on the
left. The asterisks either at the end of the
polypeptide or at the number of the proteolytic fragment indicate a
covalent attachment of DNA to protein.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (15K):
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Fig. 8.
Sequential binding of RPA70 domains as
observed by protection of RPA70 cleavage sites. The domains of
RPA70 are protected against tryptic digest. First the
protease-sensitive site in domain DBD-B is protected after binding to
short oligonucleotides. Long single-stranded oligonucleotides or
hairpins with a short protruding ssDNA extension also protect the
protease-sensitive site in domain DBD-C, which interacts with the
3'-end of the hairpin or the 3'-end of a primer on partial duplex DNA
with short protruding ssDNA. On partial duplex DNA with a long
protruding ssDNA domain, DBD-D is interacting with the 3'-end.
The presence of a hairpin with a protruding tail of 9 nt causes an elevated rate of cleavages in the region of the zinc finger motif within DBD-C concomitant to a decreasing cleavage efficiency at DBD-B sites (summarized in Fig. 8 and the Supplemental Material). In contrast, the presence of hairpins with 17 and 33 nt of the extended strands noticeably hamper the cleavages of the DBD-C region. The lengths of the ssDNA tails of the latter two hairpins are theoretically sufficient to accommodate the binding of two major domains DBD-A and DBD-B, and even three (DBD-A, DBD-B, and DBD-C) or all four domains (DBD-A, DBD-B, DBD-C, and DBD-D; see model in Fig. 8) (6, 14). We assume that changes in the proteolysis pattern of RPA70 reflect its gradual conformational changes that occur upon sequential binding of RPA70 DBDs to the single-stranded part of the oligonucleotides adjacent to the hairpin. Accordingly, the protection of DBD-B and exposure of DBD-C cleavage sites is caused by binding of the central part of RPA70 to a short ssDNA tail in an 8-nt binding mode, whereas the successive protection of all cleavage sites matches the additional binding of DBD-C and sprawling of the heterotrimer.
These studies were extended, and we compared RPA bound to exclusive ssDNA or to partial duplex DNA. These results revealed that there is significant difference in the association of the protein complex to these substrates. Short and long protruding ssDNA stretches adjacent to duplex DNAs are more efficiently bound by RPA than the same length of exclusive ssDNA. Thus, trypsin is less able to cleave RPA70. In addition, the partial duplex DNAs with long protruding ends (17 and 33 nt) induce the appearance of the proteolytic fragment 5b, which was hardly or not detected with exclusive ssDNA substrates. In contrast to these results, proteolysis of RPA32 is only slightly affected by the presence of DNA hairpins with an adjacent dT tail. A minor decrease in efficiency of the initial cleavage, which is responsible for removing the first ~38 N-terminal aa, can be observed in the presence of DNA hairpins with extended protruding ends. We suggest that in template-primer systems DBD-D is not bound to the ssDNA tail of the hairpin even when the size of its tail favors binding of RPA in the stable 30-nt mode. DBD-D probably has a higher affinity to the primer-template junction of the hairpin DNAs and therefore prefers binding to a 3'-primer end to the interaction with ssDNA.
This explanation is supported by several lines of evidence, e.g. RPA32 has some affinity of to such a primer-template junction. In our labeling experiments with individual soluble RPA subunits fused to MBP, we observed cross-linking of the respective RPA32-MBP fusion protein to the 3'-photoreactive primer end (33). Although this effect was observed at an excess of protein, it is likely to be specific because the signal was stable above background, and the ssDNA binding activity of intact RPA32 even in the RPA32·RPA14 complex is hardly ever seen (8). Photocross-linking of RPA32 to the 3'-end of a primer was also observed in experiments using the RPA heterotrimer bound to partial DNA duplexes with 5'-protruding ssDNA tail. The extent of this modification was shown to depend on the protein:DNA ratio as well as the size of the available ssDNA (21). These experiments revealed that the RPA70 subunit orients the heterotrimer upon binding to DNA and positions the RPA32 subunit in close proximity to the primer end. Notably, if the size of the length of the ssDNA template strand was not sufficient for RPA to bind in the 30-nt mode, RPA70 cross-linked to the 3'-end of the primer, and the yield of RPA32 cross-linking is reduced. Additional confirmation of an affinity of the RPA32 subunit to the 3'-end of primers was obtained from studies of SV-40 replicating minichromosomes where contacts of RPA32 and RPA70 with the growing RNA-DNA primers were visualized by cross-linking (20, 22). To extend the characterization of the binding mode of RPA, we have analyzed in this study which domains of RPA are responsible for the indicated subunit interactions with a primer-template junction using a combination of photocross-linking experiments with limited proteolysis. We have used partial DNA duplexes containing a 3'-photoreactive primer and an ssDNA tail of 32 nucleotides which is sufficient for the stable 30-nt binding mode. By using this technique we were able to identify the C-terminal domain of RPA70 and the central domain of RPA32 as the regions that are cross-linked to the 3'-end of the primer even by a zero length cross-linker group (single residue of 4-S-dTMP). In the context of DNA binding activities, these regions contain the known ssDNA-binding domains DBD-C and DBD-D.
Our results suggest a model that includes the different observed
binding modes. The extended 5'-template strand of a primer-template DNA
is stably bound by various DBDs, depending on its available size. If
the ssDNA extension is less than or equal to 8 nt in length, only DBD-A
and DBD-B bind to DNA by RPA exhibiting a globular shape and
positioning DBD-D further away from the 3'-end of the primer. When the
ssDNA stretch is less than 17 nt but more than 8 nt in length, DBD-A
and DBD-B are bound to the ssDNA part inducing an extended contracted
conformation of RPA, and DBD-C is contacting the 3'-end of the primer.
With a single-stranded part, which is longer than 17 nt, three domains
DBD-A, DBD-B, and DBD-C are associated with ssDNA, and RPA is in an
extended elongated conformation. All three are likely to bind the ssDNA
part with DBD-C oriented toward the 3'-end of the primer. In contrast
to the other DNA-binding domains, DBD-D does not seem to contact the
template strand in any of the above three cases, regardless of its
size, but specifically interacts with the primer-template junction in
the latter two cases. This interaction is probably restricted in the
conformation-dependent manner because DBD-D can be
displaced from the 3'-end of the primer by the C-terminal DBD-C (21,
22). These interactions of DBD-C and DBD-D might provide the
mechanistic framework of how RPA is able to signal about available
primer-template junctions, to serve as an anchor for other proteins
that require primer-template junctions, and to work and to monitor the
growth of the primer during DNA synthesis. One of the consequences of
RPA sensitivity to primer-template junction might be the regulation of
the early events of DNA synthesis by acting as a "fidelity clamp"
for DNA polymerase (38, 39).
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ACKNOWLEDGEMENTS |
---|
We thank A. Willitzer, U. Stephan, and A. Schneider for technical assistance and Dr. I. Petrousseva for purified RPA, as well as Drs. V. Bogachev and D. Kolpashchikov for photoreactive reagents. In addition, we thank Drs. E. Bochkareva and A. Bochkarev for communicating results prior to publication.
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FOOTNOTES |
---|
* This work was supported by Deutsche Forschungsgemeinschaft Grant 436-RUS-113/299/6-2, SFB604, and the Russian Foundation for Basic Research Grants 00-04-22002, 01-04-48854, 01-04-48895, and 02-04-48404.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains Figs. S1-14.
¶ Present address: Carl Zeiss Jena GmbH, Carl-Zeiss-Promenade 10, D-07745 Jena, Germany.
To whom correspondence should be addressed: Dept. of
Biochemistry, the Cell Cycle Control Laboratory, National University of
Ireland, Galway, Ireland. Tel.: 353-91-512-409; Fax:
353-91-512-504; E-mail: h.nasheuer@nuigalway.ie.
Published, JBC Papers in Press, February 24, 2003, DOI 10.1074/jbc.M301265200
2 E. Bochkareva and A. Bochkarev, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
ss, single-stranded;
RPA, human replication protein A;
RPA70, RPA 70-kDa subunit;
RPA32, RPA
32-kDa subunit;
RPA14, RPA 14-kDa subunit;
ds, double-stranded;
aa, amino acid(s);
nt, nucleotide(s);
DBD(s), DNA-binding domain(s);
NTD70, RPA70 N-terminal domain;
CTD32, RPA32 C-terminal domain;
MBP, maltose-binding protein;
pol , human DNA polymerase
;
NAB-4-dUTP, 5-[N-(2-nitro-5-azidobenzoyl)-trans-3-aminopropenyl-1]-2'-deoxyuridine-5'-triphosphate;
4-S-TTP, 4-thio-2'-deoxyuridine-5'-triphosphate;
DTT, dithiothreitol.
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