From the Department of Pathology, Brigham and
Women's Hospital, Harvard Medical School,
Boston, Massachusetts 02115, the § Imperial Cancer
Research Fund, Clare Hall Laboratories, South Mimms,
Herts, EN6 3LD, United Kingdom, and the ¶ Charles A. Dana
Division of Human Cancer Genetics, Dana-Farber Cancer Institute,
Boston, Massachusetts 02115
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ABSTRACT |
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The largest subunit of the replication protein A
(RPA) contains an evolutionarily conserved zinc finger motif that lies
outside of the domains required for binding to single-stranded DNA or forming the RPA holocomplex. In previous studies, we showed that a
point mutation in this motif (RPAm) cannot support
SV40 DNA replication. We have now investigated the role of this motif
in several steps of DNA replication and in two DNA repair pathways.
RPAm associates with T antigen, assists the unwinding of
double-stranded DNA at an origin of replication, stimulates DNA
polymerases and
, and supports the formation of the initial
short Okazaki fragments. However, the synthesis of a leading strand and
later Okazaki fragments is impaired. In contrast, RPAm can
function well during the incision step of nucleotide excision repair
and in a full repair synthesis reaction, with either UV-damaged or
cisplatin-adducted DNA. Two deletion mutants of the Rpa1 subunit (eliminating amino acids 1-278 or 222-411) were not functional in
nucleotide excision repair. We report for the first time that wild type
RPA is required for a mismatch repair reaction in vitro. Neither the deletion mutants nor RPAm can support this
reaction. Therefore, the zinc finger of the largest subunit of RPA is
required for a function that is essential for DNA replication and
mismatch repair but not for nucleotide excision repair.
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INTRODUCTION |
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Human replication protein A (RPA)1 is a stable complex of three subunits of Rpa1 (70 kDa), Rpa2 (34 kDa), and Rpa3 (13 kDa). It was first purified from HeLa and 293 cell extracts as an essential component of SV40 DNA replication (1-3). Binding to single-stranded DNA with high affinity is a hallmark of human replication protein A, and purification of this protein largely takes advantage of this property (3, 4).
Rpa1 is the most well characterized subunit among the complex. Rpa1 alone confers the high affinity for single-stranded DNA (5, 6), yet only the whole heterotrimeric complex is active in supporting DNA replication (4, 7). Rpa1 can be subdivided into 3 domains: an N-terminal domain of protein-protein interaction, a central domain for DNA interaction, and a C-terminal domain for complex formation with Rpa2-3 (8, 9).
RPA is highly conserved throughout evolution. Homologous heterotrimeric single-stranded DNA-binding proteins have been identified in nearly all eukaryotes examined (10-17). All genes reveal significant homology between species at the amino acid level. All of the known Rpa1 homologs contain a conserved putative C4-type zinc finger motif in the C-terminal third of the protein (7, 11, 18, 19). In fact, a point mutation that disrupts the putative zinc finger eliminates DNA replication, confirming the importance of this highly conserved motif (8, 20).
RPA is essential for other cellular DNA metabolism involving
single-stranded DNA intermediates. This includes DNA repair (21, 22)
and homologous recombination (10, 23-25). hRPA's role in DNA
replication is largely based on the study of in vitro SV40 DNA replication model. With its interaction with ssDNA, T antigen, and
DNA polymerase -primase complex, hRPA assists T antigen to unwind
the origin (2, 26-28) and DNA polymerase
-primase complex to
synthesize the first Okazaki fragment (29). hRPA is also important in a
later elongation step where it stimulates both DNA polymerase
and
DNA polymerase
activity and cannot be substituted for by
Escherichia coli SSB (30). Besides the interaction with repair proteins (XPA, XPG, and XPF see below) and replication proteins,
hRPA has also been implicated in interactions with transcription factors p53 (31, 32), Gal4, VP16 (33), RNA polymerase holoenzyme (34),
and recombination protein RAD52 (35-37).
Since DNA repair reactions generally involve DNA synthesis to form a repair patch, it is perhaps not surprising that hRPA takes part in DNA repair and can modulate the repair synthesis step (38). However, the most important function of RPA is at an earlier stage of nucleotide excision repair (21, 39), and analysis of nicking of UV-irradiated plasmid DNA during excision repair revealed that RPA is necessary for incision formation (40). RPA is an essential factor for dual incision of damaged DNA with purified human protein components (41, 42). The Rpa1 and Rpa2 subunits of hRPA interact with the nucleotide excision repair protein XPA, probably to increase the efficiency of damage recognition, and this would be expected to modulate the excision reaction (22, 43, 44). In addition, RPA can also modulate the efficiency of cleavage by the incision enzyme XPG and ERCC1-XPF (45). A role for hRPA in general mismatch repair in eukaryotes has not been defined. E. coli SSB protein is required in a reconstituted system of mismatch repair with purified bacterial proteins (46). It therefore seems reasonable that hRPA could be involved in eukaryotic mismatch repair.
To help dissect the role of RPA in DNA replication and repair, we have taken advantage of three defined mutants in the Rpa1 subunit (8). All of the Rpa1 mutants form a stable heterotrimeric complex and show a similar extent of binding to single-stranded DNA under physiological conditions. The present work examines their ability to function during defined steps in DNA replication, nucleotide excision repair, and mismatch repair. The experiments reveal requirements for intact domains in Rpa1 that differ depending on the DNA transaction, suggesting that specific interactions take place during each of the three DNA replication and repair processes examined.
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MATERIALS AND METHODS |
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Protein Purification--
The plasmid expressing wild type and
various RPA mutants were described in a previous report (8). The
proteins were named according to the Rpa1 mutant present in the
complex. The proteins were expressed and purified as described (4). The
baculovirus expressing DNA polymerase /primase subunits were
generous gifts from Drs. Teresa Wang and Ellen Fanning. DNA polymerase
/primase was expressed in Hi-5 cells with baculovirus infection and
purified by immunoaffinity column chromatography with monoclonal
antibody SJK-237-71 specific for the large subunit p180 (47). T Ag was also purified from baculovirus-infected Hi-5 cells by immunoaffinity column chromatography with monoclonal Ab Pab 419 (48). Polymerase
was purified from calf thymus in four steps (DEAE-cellulose, phenyl-Sepharose, S-Sepharose, and Mono-Q
chromatography).2 The
conditions in DEAE-cellulose and phenyl-Sepharose columns were as
described (49). The active fractions from phenyl-Sepharose eluate were
pooled and loaded onto an S-Sepharose column (5 mg/ml bed)
pre-equilibrated with buffer D (20 mM potassium phosphate, pH 7.2, 0.5 mM EDTA, 0.1 mM EGTA, 0.5 µg/ml
leupeptin, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM DTT, 20% glycerol). After the column was washed with
2-3 bed volumes of buffer D containing 0.1 M KCl, DNA
polymerase
activity was eluted stepwise with a KCl gradient from
0.1 to 1 M in the same buffer. Active fractions were pooled
and dialyzed against buffer E (25 mM Tris-HCl, pH 8.0, 0.025 M NaCl, 1 mM EDTA, 1 mM DTT,
0.1 mM phenylmethylsulfonyl fluoride, 10% glycerol, 0.01%
Nonidet P-40, 2 µg/ml leupeptin) containing 20% sucrose. The
dialyzed enzyme was loaded onto a Mono-Q column pre-equilibrated with
buffer E. After washing with 5 bed volumes with buffer E, polymerase
activity was eluted with a NaCl linear gradient from 0.025 to 0.5 M in the same buffer. PCNA-stimulated DNA polymerase
activity eluted at 0.2 M NaCl fractions just prior to the
protein peak. The resulting polymerase can be stimulated 10-fold by
PCNA on a poly(dA)/oligo(dT) template and has no detectable activity on
a primed M13 ssDNA template unless PCNA and RF-C were both present.
Baculoviruses expressing subunits of human RF-C and calf thymus RF-C
protein were the generous gift of V. Podust and E. Fanning (Vanderbilt
University) (50).
SV40-based DNA Replication--
Replication of a plasmid
(pSV010) containing SV40 origin of DNA replication was carried out with
293 cell extracts depleted of RPA (1) and supplemented with various
bacterially expressed recombinant RPA holocomplexes as described
previously (4). The products were linearized with HindIII
and analyzed on a 0.8% alkaline agarose gel. The gel was fixed in 8%
(w/v) trichloroacetic acid after electrophoresis and dried. The
incorporation of [-32P]dCMP was determined using
the DE81 paper and scintillation counting (51).
Pulse Labeling for Early DNA Product--
The assay is
essentially based on previous reports (52, 53). Reaction mixtures (50 µl) contained 7 mM MgCl2, 0.5 mM
DTT, 4 mM ATP, 40 mM creatine phosphate, 30 mM HEPES (pH 7.8), 0.2 units of creatine kinase, 0.6 µg
of supercoiled SV40 origin-containing plasmid (pSV010), 0.84 µg of T
Ag, 1.4 µg of recombinant RPA preparations as indicated, and
RPA-depleted 293 cell lysate. Reaction mixtures were preincubated for
45 min at 37 °C in the absence of T Ag to lower T Ag-independent
labeling of form II DNA and then further incubated for 15 min after the
addition of T Ag. Reaction mixtures were pulse-labeled for 1 min by the
addition of 3.5 µl of a solution containing [-32
P]dCTP (final concentration in the complete reaction, 1 µM, 300 cpm/fmol) dATP, dGTP, and dTTP (final
concentration of 100 µM each) and CTP, GTP, and UTP
(final concentration of 200 µM each). The reactions were
terminated by addition of 30 µl of stop solution (60 mM
EDTA, 0.3% (w/v) SDS, 2 mg/ml of Pronase) and incubated at 37 °C
for another 30 min. The products were extracted with phenol/chloroform
followed by ethanol precipitation, boiled for 4 min in an equal volume
of formamide loading buffer, and analyzed by electrophoresis through
10% polyacrylamide gels containing 8 M urea. The gel was
fixed in 10% acetic acid, 10% methanol for 15 min, dried, and
analyzed by radiography.
Origin Unwinding Assay--
The origin unwinding reaction was
carried out in 20-µl volumes containing unwinding buffer (30 mM HEPES (pH 8.0), 7 mM MgCl2, 4 mM ATP, 0.5 mM DTT, 0.1 mg/ml bovine serum
albumin, 40 mM creatine phosphate, and 0.8 units of
creatine phosphokinase) in the presence of 72 ng of T Ag, 0.2 µg of
Sau3A-digested carrier DNA, 50-60 ng of radiolabeled
HindIII-SphI fragment of pSV010, and the
indicated amounts of recombinant RPA (54, 55). Reaction was carried out
at 37 °C for 2 h and terminated by adding 10 µl of stop
solution (described above) followed by continued incubation at 37 °C
for 30 min. The products were extracted with phenol/chloroform followed by ethanol precipitation and then analyzed by electrophoresis through
6% acrylamide gels.
Enzyme-linked Immunosorbent Assay-- The assay was carried out as described previously (56). Briefly, the wells of a 96-well microtiter plate were coated overnight with excess (1 µg) recombinant RPA proteins or E. coli single-stranded DNA-binding protein. The wells were then blocked by incubation for 2 h with 3% bovine serum albumin in phosphate-buffered saline. The indicated amounts of purified T Ag were added in 50-µl volumes of blocking solution for 2 h. After washing, bound T Ag was detected by Pab 419 monoclonal antibody (diluted 1:1000 in blocking solution), peroxidase-conjugated rabbit anti-mouse antibody (diluted 1:1000 in blocking solution), and the chromogenic substrate 2,2-azino-bis-(3-ethyl-benzothiazoline-6-sulfonic acid) (Sigma).
DNA Polymerase Assays--
DNA polymerase stimulation
reactions were carried out in 20-µl volumes containing 30 nM M13 ssDNA, 90 nM sequencing primer (U. S. Biochemical Corp.), 20 mM Tris acetate (pH 7.3), 5 mM magnesium acetate, 20 mM potassium acetate,
1 mM DTT, 0.1 mg/ml bovine serum albumin, 1 mM
ATP, 0.1 mM dATP, dGTP, TTP, and 0.025 mM dCTP
(1000 cpm/pmol). DNA polymerase
was added after a 10-min
preincubation period with RPA, and the reaction was continued for
another 10 min in 37 °C. The incorporation of
[
-32P]dCMP was determined by DE81 paper method (51).
DNA polymerase
stimulation reactions were carried out in 25-µl
volumes as described (57). The incorporation of
[
-32P]dCMP was determined by DE81 paper, and the
products were analyzed on a 1% alkaline agarose gel. The gel was
processed as mentioned above.
Nucleotide Excision Repair Assays
Repair Synthesis in UV-damaged DNA--
The plasmids used were
derivatives of pUC vectors, the 3.0-kb pBluescript KS+
(Stratagene), and the 3.7-kb pHM14 (58). pBluescript KS+
was UV-irradiated (450 J/m2). Both plasmids were treated
with E. coli Nth protein, and closed circular DNA was
isolated from cesium chloride and sucrose gradients (59). Reaction
mixtures (50 µl) contained 48 µg of human CFII fraction protein
depleted of RPA and PCNA (40), 25 ng of recombinant PCNA, 250 ng of
irradiated pBluescript KS+, 250 ng of non-irradiated pHM14,
45 mM HEPES-KOH (pH 7.8), 70 mM KCl, 7.4 mM MgCl2, 0.9 mM dithiothreitol
(DTT), 0.4 mM EDTA, 20 µM each of dGTP, dCTP,
and TTP, 8 µM dATP, 74 kBq of [-32P]dATP
(110 TBq/mmol), 2 mM ATP, 22 mM phosphocreatine
(di-Tris salt), 2.5 µg of creatine phosphokinase, 3.4% glycerol, 18 µg of bovine serum albumin and were incubated at 30 °C for 3 h. Plasmid DNA was purified from the reaction mixtures, linearized with
BamHI, and loaded on a 1% agarose gel containing 0.3 µg/ml ethidium bromide. Data were analyzed by autoradiography with
intensifying screens, densitometry, and liquid scintillation counting
of the excised bands.
Dual Incision Assay of Cisplatin-damaged DNA-- Covalently closed circular DNA containing a single 1,3-intrastrand d(GpTpG)-cisplatin cross-link (Pt-GTG) was prepared as described (60). This DNA substrate was used to analyze the dual incision process of nucleotide excision repair which leads to the excision of characteristically sized platinated oligomers 24-32 nucleotides in length. Each 150-µl reaction mixture contained 144 µg of HeLa cell CFII fraction protein, depleted of RPA and PCNA (40), and 4.8 µg of the indicated recombinant RPA in buffer containing 45 mM HEPES-KOH (pH 7.8), 70 mM KCl, 7.4 mM MgCl2, 0.9 mM dithiothreitol (DTT), 0.4 mM EDTA, 2 mM ATP, 22 mM phosphocreatine (di-Tris salt), 2.5 µg of creatine phosphokinase, 3.4% glycerol, and 18 µg of bovine serum albumin. After 5 min at 30 °C, either Pt-GTG DNA (750 ng) or control DNA (750 ng) without the cisplatin cross-link was added and incubation continued for 30 min at 30 °C. The DNA was purified and digested with XhoI and HindIII for 4 h at 37 °C. The reactions were stopped by adding formamide buffer containing bromphenol blue and xylene cyanol. The DNA was denatured at 95 °C for 5 min prior to loading on a 12% acrylamide gel and run until the blue dye migrated ~30 cm from the wells. DNA was transferred by capillary action for 90 min onto a Hybond-N+ membrane soaked in 10 × Tris borate buffer. The membrane was fixed in 0.4 M NaOH for 20 min followed by a 2-min wash in 5 × SSC. The fixed membrane was incubated at 42 °C for 16 h in hybridization bottles containing 40 ml of 130 mM potassium phosphate (pH 7.0), 250 mM NaCl, 7% SDS, 10% PEG 8000, and 100 pmol of 32P-labeled 27-mer oligonucleotide which is complementary to the excised platinated oligomers (60). The membranes were washed for 10 min in 2 × SSC buffer containing 0.1% SDS before exposure of the membrane to x-ray film.
Mismatch Repair Assay
Construction of Phagemid Mutants and Heteroduplex-- The mismatch-containing substrates used in these studies were constructed from derivatives of phagemid pBS-SK in which the polylinker between the ApaI and BamHI sites had been replaced by different mutant polylinkers. Heteroduplex substrates were constructed and purified using unpublished procedures similar to those described by others (61). The polylinker regions of the substrates used the following Sequence 1
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(Sequence 1) |
Mismatch Repair Assay-- The repair assay contains 50 µg of S100 extract (from 293 cells); 30 mM HEPES (pH 7.8); 7 mM MgCl2; 4 mM ATP, 200 µM CTP, GTP, and UTP; 100 µM dNTP; 40 mM creatine phosphate; 100 µg of creatine phosphokinase/ml; 15 mM sodium phosphate (pH 7.5); and 100 ng of the mismatch substrate (62). The reaction mixture was incubated at 37 °C for 2-3 h. After the 2-h incubation, 50 µl of stop buffer (0.67% SDS; 0.025 M EDTA) and proteinase K (0.3 mg/ml final concentration) were added. After an additional 15 min incubation at 37 °C, the reaction mixture was extracted with phenol/chloroform and chloroform, and the DNA was precipitated, digested with appropriate enzymes, and run on a 1.2% agarose gel at 65 V for 2.5 h. The fractionated DNA was stained by SYBR green and visualized on a fluoroimager (Molecular Dynamics). The repair results in the restoration of an XhoI or NsiI site, and the repair efficiency observed ranged from 5 to 15%. For antibody neutralization experiments, the extract was incubated with various antibodies for 15 min at 37 °C prior to the addition of the mismatch substrate. For RPA rescue experiments, the RPA and the mismatch substrate were added simultaneously.
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RESULTS |
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The DNA Binding Subdomains and the Evolutionarily Conserved Zinc
Finger Motif in RPA Are Indispensable for Replication--
We have
previously described three human RPA complex mutants, m1-616 tRPA (two
of the four cysteines in the putative zinc finger of Rpa1 (amino acid
500 and 503) are changed to serine, renamed here as RPAm),
278-616 tRPA (deletion of amino acids 1-277 of Rpa1), and 222-411 tRPA (deletion of amino acids 222-411 of Rpa1) (8). The recently solved crystal structure of the DNA binding domain of Rpa1 indicates that it is composed of two structurally homologous subdomains comprising amino acids 198-291 and 305-402 (63, 64). Salt-resistant binding of single-stranded DNA by 278-616 tRPA implies that the second
subdomain of Rpa1 is capable of binding DNA with high affinity, perhaps
in cooperation with the putative DNA binding subdomains in Rpa2 and
Rpa3 (65).
222-411, on the other hand, has lost both DNA binding
subdomains and cannot bind single-stranded DNA in high salt. However,
this form of RPA binds DNA under the low salt conditions that are
necessary for DNA replication. RPAm has no mutation in the
essential DNA binding domain. All the mutant forms of tRPA, however,
fail to support SV40 DNA replication.
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T Antigen-RPA Interaction--
T antigen (T Ag) is a central
molecule of the SV40 DNA replication that binds to the origin of DNA
replication and recruits RPA to the origin as it launches the
replication process. To study the protein-protein interaction between T
Ag and RPA, a modified enzyme-linked immunosorbent assay was employed
(56). Increasing amount of T Ag was allowed to interact with a fixed
level of immobilized RPA protein or E. coli SSB. The results
indicated that all the RPA mutants are able to interact with T Ag (Fig.
2). 222-411 tRPA bound to T Ag to the
same extent as wild type RPA, whereas RPAm and 278-616
tRPA retain about 80 and 50% activity, respectively. Therefore the
inability of the mutants to support DNA replication is not explained by
a failure to interact with T Ag.
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Zinc Finger Motif, but Not the DNA Binding Domains, Is Dispensable
for Origin Unwinding--
T Ag recognizes the SV40 origin and unwinds
the DNA with the assistance from RPA. A radiolabeled SV40
origin-containing duplex linear DNA will be unwound to generate
single-stranded DNA products that can be detected as slower migrating
species relative to the duplex DNA (Fig.
3A). When increasing amounts
of wild type RPA (1-616 tRPA) (lanes 1-4) or zinc finger
mutant RPAm (lanes 5-8) were added to the
reaction in the presence of T Ag and competitor DNA, the amount of
unwound products increased. However, 278-616 tRPA (lanes
9-12) showed only low activity, and 222-411 tRPA (lanes
13-17) had no detectable activity (compare with (lane
18, no RPA)). The unwound products were excised from the gel and
quantitated by scintillation counting (Fig. 3A).
RPAm had at least 80% activity compared with that of wild
type RPA. In comparable experiments, both 1-616 tRPA and zinc-finger
mutant RPAm unwind up to 50% of the input double-stranded
DNA. Thus the zinc finger in RPA is not essential for origin unwinding.
E. coli SSB supports T Ag-mediated origin unwinding (66),
but 278-616 tRPA, which also binds single-stranded DNA under high salt
conditions, failed to do so. Thus the salt-resistant DNA binding noted
with 278-616 tRPA is physiologically non-functional, suggesting that both DNA binding subdomains of Rpa1 were essential to bind DNA in a
manner that was essential for origin unwinding.
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Zinc Finger of Rpa1 Is Dispensable for Stimulating DNA Polymerase
and
--
RPA is known to physically interact with DNA
polymerase
-primase complex and stimulate lagging strand DNA
synthesis. This stimulatory effect is species-specific, and E. coli SSB does not support this stimulation (30, 56). Various
levels of RPA proteins and E. coli SSB were added to
reactions containing primed M13 ssDNA and DNA polymerase
-primase
complex (Fig. 4A). Both wild type RPA and the zinc-finger mutant RPAm stimulated DNA
synthesis by polymerase
by 8-fold, and higher concentrations of RPA
inhibited polymerase
activity completely. This is consistent with a
previous report (67). However, neither 278-616 tRPA nor
222-411
tRPA, which bind to ssDNA with high and low affinity, respectively,
stimulated DNA polymerase
. Consistent with previous reports,
E. coli SSB did not stimulate DNA synthesis by DNA
polymerase
(data not shown).
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Formation of the Earliest Okazaki Fragments--
Synthesis of an
RNA primer and the first Okazaki fragment is carried out by the DNA
polymerase -primase complex following unwinding of the origin. Most
of the Okazaki fragments detected in the denaturing agarose gel in Fig.
1A are synthesized during the elongation phase of a 60-min
reaction. If DNA synthesis is initiated (but the products are not
significantly elongated) one expects to detect the initial Okazaki
fragments in a short pulse-labeled SV40 DNA replication reaction
(53).
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The Zinc Finger Mutant, RPAm, Is Competent in Nucleotide Excision Repair-- The activities described so far for the zinc finger mutant RPAm are intriguing. Although not able to form detectable replication products, it is competent in a variety of related functions and replication sub-reactions. The zinc finger is dispensable for DNA binding, origin unwinding, and initiation of DNA replication but appears to be required for elongation of DNA replication products. It was of interest to determine whether the zinc finger and deletion mutants could function in DNA repair. We first tested their activity in nucleotide excision repair.
Nucleotide excision repair DNA synthesis reactions were carried out with UV-irradiated and non-irradiated templates. Equal amounts of template were present as shown in the ethidium bromide-stained gel (Fig. 6A, upper panel), where UV-irradiated versus non-irradiated templates can be discriminated by their different sizes. No incorporation of radiolabeled dNTP (repair synthesis) is detected in non-irradiated template. A template-specific repair on the irradiated template is seen with both wild type protein 1-616 tRPA (lower panel, lanes 2-4) and zinc finger mutant RPAm (lower panel, lanes 5-7). RPAm has about 50-60% the activity of wild type RPA (data not shown). None of the other two mutants 278-616 tRPA (lanes 8-10) and
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Wild Type RPA but None of the Mutant Forms of RPA Rescue Mismatch Repair in RPA-depleted Cell Lysate-- In E. coli, methylation is used as a strand discrimination system to choose the template in mismatch repair (71). In higher eukaryotes, it is still unclear how cells decide which strand is to be repaired in a mismatch. However, by introducing a nick, one can artificially bias the in vitro system to correct the strand with a break (72, 73). We used a circular heteroduplex template containing a site-specific strand-specific nick and a single base (G/T) mismatch to examine the role of RPA in mismatch repair (Fig. 8A). The presence of the mismatch within overlapping recognition sites for two restriction endonucleases (XhoI and NsiI) renders the DNA resistant to digestion by either enzyme. Correction of the mismatch permits the assessment of strand specificity of repair. Thus the nick-driven repair will use the circular strand as template and repair the nicked strand thereby restoring the XhoI site and eliminating the NsiI site. Repair using the opposite strand as template will, on the other hand, restore the NsiI site and eliminate the XhoI site. Thus the repair process can be monitored by linearizing the substrate DNA with AlwNI, digesting with either XhoI or NsiI, and measuring the formation of a smaller fragment of 2.1 kb that is defined by the largest distance between the AlwNI site and the NsiI/XhoI sites.
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DISCUSSION |
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In this study, we investigated the function of RPA in DNA repair and replication using three recombinant RPA mutants as well as wild type RPA. Among the three mutants, the zinc finger mutant is particularly interesting. Zinc finger structures are known to be important in DNA-protein and protein-protein interactions (75, 76). The zinc finger is highly conserved among different RPA homologs, implying that it is important for some of the functions of RPA.
The studies presented here demonstrate that the zinc finger point
mutant is able to physically interact with T Ag, assist origin
unwinding, stimulate DNA polymerase and
in DNA synthesis, and
support the formation of the earliest Okazaki fragments. Neither of the
other two mutants retained any of the functions described above except
for interacting with T Ag and polymerase
stimulation. The likeliest
explanation for why RPAm is inactive in SV40 DNA
replication but active in the various steps up to early Okazaki
fragment formation is that the mutant can support the events related to
initiation but not elongation. Since RPAm is able to
stimulate polymerase
on a primed M13 ssDNA template, the deficit
could be in the loading of polymerase
at replication forks. The
pulse labeling experiment allowed us to examine the smallest initial
Okazaki fragments. Our hypothesis is supported by the fact that a
strong-stop nascent DNA product of 20 bases that could correspond to a
pause associated with polymerase switching is not seen in the
RPAm supported reactions. The situation is similar to a
reaction without PCNA when only 1-2% DNA synthesis is seen and most
of it is due to formation of short Okazaki fragments confined to the
origin of DNA replication (5).
A less likely explanation for the failure of RPAm to support DNA replication is that the slightly decreased activity of this mutant protein in several of the sub-reactions add up to the complete absence of detectable products in the replication reaction. The nearly wild type activity of RPAm in nucleotide excision repair argues for the first explanation that the protein is totally inactive in a specific elongation step required for DNA replication, since elongation steps would not likely be required for excision repair.
Based on the results of SV40 DNA replication, it was speculated that RPAm (since it executes all steps up to Okazaki fragment formation) may be a dominant negative inhibitor of wild type RPA in a competitive assay. However, we did not detect any dominant negative effect of RPAm over wild type RPA. It is conceivable that RPAm fails to enter into an initiation complex in the presence of wild type RPA accounting for the absence of a dominant negative effect. Future experiments will address this issue.
In addition to DNA replication, nucleotide excision repair and mismatch
repair assays were performed to assess further the functional
properties of the mutant RPA proteins studied here. Among other things,
the results presented here provide the first demonstration of the
biochemical requirement of RPA in the in vitro mismatch
repair reaction. In addition, none of the mutant forms of RPA studied
here were capable of supporting mismatch repair. This was in contrast
to nucleotide excision repair, which is also RPA-dependent.
In this case, RPAm was able to support nucleotide excision
repair in vitro, whereas neither of the deletion mutants
were able to support nucleotide excision repair. In this regard
RPAm exhibited an intriguing phenotype. It is unable to
replace wild type RPA in mismatch repair or in DNA replication but is
fully competent in nucleotide excision repair. The zinc finger area may
be important for certain interactions but not others; for example, it
could be essential for recruiting mismatch repair proteins and
replication factors but not nucleotide excision repair proteins. DNA
polymerase has been implicated in mismatch repair in addition to
DNA polymerase
(73, 77, 78). It is likely that the long stretch of
DNA formed in DNA replication and mismatch repair requires switching
from polymerase
to
and hence is affected by the mutation in the
zinc finger motif of Rpa1. Filling in of a short gap during nucleotide
excision repair could be executed by a single DNA polymerase,
especially polymerase
and so is still supported by
RPAm. Alternatively, RPA may be required in nucleotide
excision repair to displace the short excised fragment produced by dual
incision rather than to support DNA synthesis, whereas in mismatch
repair and DNA replication RPA may be required at the elongation stage of DNA synthesis.
Our studies and those from Wold's group (20) have consistently failed
to detect replication products from RPA-depleted cell lysate
supplemented with the zinc finger mutant or the other two DNA domain
deletion mutants. However, another group reported that RPA with a
deletion of 50 amino acids including the zinc finger motif was
functional in SV40 DNA replication but inhibited both DNA polymerase
and
(79). The discrepancy between these two results could be
because the two mutants are not identical; ours is a point mutation,
whereas the other is a 50-residue deletion across the zinc finger.
Perhaps the existence of the unfolded zinc finger domain in
RPAm is more deleterious to protein function than the
absence of the domain in the deletion derivative (20).
The two RPA deletion mutants studied here bound to single-stranded DNA
under physiological salt concentrations, and 278-616 tRPA bound to
DNA with high affinity (8). Both retained sufficient secondary
structure to allow interaction with Rpa2 and Rpa3 to form the RPA
holocomplex and support interaction with T Ag. However, both were
inactive in the repair and replication assays. Perhaps the deletions in
these Rpa1 molecules affect their mode of interaction with
single-stranded DNA or their interaction with replication or repair
proteins resulting in this null phenotype. An interesting feature of
the data on these two deleted forms of RPA is they suggest that
single-stranded DNA binding per se is not sufficient for RPA
to promote the excision step in nucleotide excision repair.
The mutation analysis presented here provides strong evidence that RPA
is not merely a single-stranded DNA-binding protein or a complex that
brings Rpa2 and Rpa3 to the replication machinery. The failure of Rpa1
alone to support DNA replication despite binding DNA well (7) could be
explained by Rpa2 and Rpa3 executing unknown but essential functions.
However, all the mutant forms of RPA characterized here contain Rpa2
and Rpa3 and thus bring these subunits to regions of single-stranded
DNA. This is particularly relevant in the case of RPAm
since it supports origin unwinding, stimulates DNA polymerase and
, as well as supports DNA replication up to the formation of the
first Okazaki fragments suggesting that the molecule binds to
single-stranded DNA appropriately. Consistent with this,
RPAm also supports nucleotide excision repair. However, its
failure in replication beyond the initiation of DNA synthesis and its failure in mismatch repair strongly suggest that RPA is also required as a molecular matchmaker, coordinating activities of proteins involved
in strand elongation or DNA repair and that the zinc finger region of
Rpa1 could be important in this regard.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant CA60499 (to A. D.), Career Development Award DAMD 17-94-J-4064 from the United States Armed Forces Medical Research Command (to A. D), and National Institutes of Health Grant GM 50006 (to R. D. K.).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.
To whom correspondence should be addressed: Dept. of
Pathology, Brigham and Women's Hospital, Harvard Medical School, 75 Francis St., Boston, MA 02115. Tel.: 617-278-0468; Fax: 617-732-7449; E-mail: adutta{at}bustoff.bwh.harvard.edu.
1 The abbreviations used are: RPA, replication protein A; RF-C, replication factor C; PCNA, proliferating cell nuclear antigen; T Ag, T antigen; DTT, dithiothreitol; ssDNA, single-stranded DNA; h, human; Ab, antibody; kb, kilobase pair(s).
2 T. Tsurimoto, personal communication.
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