Human Replication Protein A

THE C-TERMINAL RPA70 AND THE CENTRAL RPA32 DOMAINS ARE INVOLVED IN THE INTERACTIONS WITH THE 3'-END OF A PRIMER-TEMPLATE DNA*,

Pavel E. PestryakovDagger , Klaus Weisshart§, Bernhard Schlott§, Svetlana N. KhodyrevaDagger , Elisabeth Kremmer||, Frank Grosse§, Olga I. LavrikDagger , and Heinz-Peter Nasheuer§**DaggerDagger

From the § Abteilung Biochemie, Institut für Molekulare Biotechnologie, D-07745 Jena, Germany, Dagger  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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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' right-arrow 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).

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Proteins and Nucleotides-- Recombinant human DNA polymerase beta  (pol beta ) 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). [alpha -32P]dCTP (3000 Ci/mmol) was obtained from Amersham Biosciences.

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 beta , 0.54 µM partial duplex DNA PT32, 1 µCi of [alpha -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 beta  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.

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).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).


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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.

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.)


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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.


                              
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Table I
Summary of the partial tryptic RPA digests
Origin, fragment boundaries, and potential cleavage information for the fragment were obtained or calculated from the respective data on size, antibody reactivity, and N-terminal sequence; ND, not determined.2


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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.

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.


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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.

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. 


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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.

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 beta  (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 beta  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.

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.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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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 alpha  (38, 39).

    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.

    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.

Dagger Dagger 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.

    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 beta , human DNA polymerase beta ; 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.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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