(Received for publication, March 14, 1995; and in revised form, June 25, 1995)
From the
Human replication protein A (RPA; also known as human
single-stranded DNA binding protein, or HSSB) is a multisubunit complex
involved in both DNA replication and repair. While the role of RPA in
replication has been well studied, its function in repair is less
clear, although it is known to be involved in the early stages of the
repair process. We found that RPA interacts with xeroderma
pigmentosum group A complementing protein (XPAC), a protein that
specifically recognizes UV-damaged DNA. We examined the effect of this
XPAC-RPA interaction on in vitro simian virus 40 (SV40) DNA
replication catalyzed by the monopolymerase system. XPAC inhibited SV40
DNA replication in vitro, and this inhibition was reversed by
the addition of RPA but not by the addition of DNA polymerase
-primase complex, SV40 large tumor antigen, or topoisomerase I.
This inhibition did not result from an interaction between XPAC and
single-stranded DNA (ssDNA), or from competition between RPA and XPAC
for DNA binding, because XPAC does not show any ssDNA binding activity
and, in fact, stimulates RPA's ssDNA binding activity.
Furthermore, XPAC inhibited DNA polymerase
activity in the
presence of RPA but not in RPA's absence. These results suggest
that the inhibitory effect of XPAC on DNA replication probably occurs
through its interaction with RPA.
Replication protein A (RPA; ()also known as human
single-stranded DNA binding protein, or HSSB), is a eukaryotic
single-stranded DNA binding protein that contains three tightly
associated subunits of 70, 34, and 11 kDa (p70, p34, and p11,
respectively)(1, 2, 3) . It is required for
DNA replication, nucleotide excision repair, and homologous
recombination(1, 2, 3, 4, 5, 6) ,
suggesting that it has multiple functions in DNA metabolic processes.
The p34 subunit of RPA is phosphorylated at the G
/S
boundary and dephosphorylated during mitosis(7, 8) .
This phosphorylation event can also be induced by DNA
damage(9, 10) . Since DNA damage induces the
inhibition of replication, RPA and the phosphorylation of its p34
subunit may play a role in the regulation of DNA
replication(10) .
During the initiation of simian virus 40
(SV40) DNA replication, RPA interacts with SV40 large tumor antigen
(T-ag) and the DNA polymerase -primase complex (pol
-primase)(11, 12) , which appears to be essential
for DNA unwinding(12) . Human RPA cannot be replaced at the
initiation of replication by RPA from other species, suggesting that
the interaction of RPA with other replication proteins may be crucial
in this process. After unwinding, RPA is believed to both stabilize the
unwound DNA and stimulate DNA polymerase
(pol
) and DNA
polymerase
(pol
) activities, as determined by the
elongation of primed DNA templates(13) .
In nucleotide excision repair, the requirement for RPA can be bypassed by incising DNA with the E. coli UvrABC enzyme. This observation suggests that RPA is involved in an early stage of UV excision repair(14) . Although the role of RPA in repair is not yet well defined, the protein complex cannot be replaced by RPA from other species, indicating that specific interactions between RPA and other repair proteins are involved in the repair process(14) .
Xeroderma pigmentosum (XP) is a genetically recessive human disorder. Patients with XP are defective in excision repair of ultraviolet light (UV)-damaged DNA and consequently suffer from a high incidence of skin cancer. At least seven complementation group proteins (XP-A to XP-G) have been identified thus far(15, 16) . The XP group A complementing protein (XPAC) is involved in an early stage of nucleotide excision repair and is also a key protein in the recognition of UV-damaged DNA(17, 18, 19) . The XPAC gene contains a zinc finger motif that is required for XPAC function in repair(20, 21) . XPAC was recently shown to interact with rodent excision repair cross-complementing protein 1 (ERCC1) and ERCC4 (XP-F)(22, 23) .
In this report,
we show that XPAC also interacts with RPA. Further, XPAC inhibits SV40
DNA replication in vitro, and this inhibition can be reversed
by the addition of RPA. XPAC inhibited pol activity in the
presence of RPA but did not inhibit this polymerase in RPA's
absence. Taken together, these results indicate that the XPAC-RPA
interaction alters RPA's ability to stimulate pol
activity,
which, in turn, results in the inhibition of DNA replication. We
discuss how these observations support the hypothesis that the repair
and replication functions of RPA are differentially regulated.
Figure 3:
The effect of XPAC on SV40 dipolymerase
replication in vitro. Reaction mixtures (40 µl) contained
pol -primase (0.1 units each), topo I (1,000 units), 0.05 µg
of topo II, 0.4 µg of RPA, 0.1 unit of pol
, 0.4 µg of
PCNA, and 0.8 µg of A1. In lanes2-10, 0.8
µg of SV40 T-ag was included. In lanes3-6,
increasing volumes of buffer were added as described in the legend to Fig. 2. Once the reactions were complete, the reaction mixtures
were analyzed for acid-insoluble radioactivity (A) and in a
1.2% alkaline agarose gel (B). ssl represents the
position to which the single-stranded linear plasmid DNA migrated. n.t., nucleotides.
Figure 2:
The
effect of XPAC on SV40 monopolymerase replication in vitro.
Reaction mixtures (40 µl) contained the pol -primase complex
(0.3 units of pol
and 0.3 units of primase), topo I (1,000
units), 0.3 µg of human RPA, and various amounts of XPAC. With the
exception of lane1, 0.8 µg of SV40 T-ag was
included in each reaction. In lanes3-6,
increasing volumes of buffer (25 mM Hepes-KOH, pH 7.8, 25%
glycerol, 1 mM DTT, 0.5 mM EDTA, 0.01% Nonidet P-40,
and 250 mM KCl) were added, instead of XPAC, to the reactions.
Upon completion of the reactions, one-tenth of each reaction mixture
was used to measure the TCA-precipitable
dAMP incorporated into DNA (A), and the remaining DNA was isolated and analyzed for its
size distribution on a 1.2% alkaline agarose gel (B). ssl represents the position to which the single-stranded linear
plasmid DNA migrated. n.t.,
nucleotides.
Figure 1: Interaction of XPAC with human RPA. A, purified RPA and XPAC were electrophoretically separated in 12% SDS-polyacrylamide gels and visualized by Coomassie Blue staining. B, XPAC, SV40 T-ag, or bovine serum albumin-coated ELISA wells (1.0 µg/well) were incubated with various amounts of RPA for 1 h at 37 °C. Bound RPA was detected with a peroxidase-conjugated RPA monoclonal antibody (against the 70-kDa subunit of RPA).
We also examined the effect of XPAC on the
dipolymerase system, which contains, in addition to the monopolymerase
components, pol , PCNA, and activator 1 (RF-C). Again, DNA
synthesis was quantitatively inhibited by XPAC (Fig. 3) albeit
to a lesser extent than with the monopolymerase system. For example, in
the presence of 1.2 µg of XPAC, 82% of the replication activity was
inhibited in the monopolymerase system, whereas only 24% was inhibited
in the dipolymerase system (Fig. 2AversusFig. 3A). XPAC affected the sizes of the
replication products produced in the SV40 dipolymerase system in that
the size of the lagging strand increased as the concentration of XPAC
increased. There was also a significant diminution of the leading
strand synthesis (Fig. 3B).
Figure 4:
The inhibition of SV40 DNA replication by
XPAC is reversible by RPA addition. Using the reaction conditions
described in the legend to Fig. 2, reversal reactions included
0.4 and 0.8 µg of SV40 T-ag (lanes3 and 4, respectively), 0.3, 0.6, and 0.9 µg of RPA (lanes5, 6, and 7, respectively), 0.15 and
0.3 units of pol -primase (lanes8 and 9, respectively), and 500 and 1,000 units of topo I (lanes10 and 11, respectively). After incubation at 37
°C for 1 h, the products of the reaction mixtures were analyzed for
TCA-precipitable radioactive materials (A), and by 1.2%
alkaline agarose gel electrophoresis (B). n.t.,
nucleotides.
Figure 5:
The effect of XPAC on RPA's ssDNA
binding activity. Indicated amounts of either human RPA, XPAC, or a
mixture of both were combined with 250 fmol of
5`-P-labeled (dT)
and incubated for 15 min at
25 °C. The protein-DNA complexes were then separated from unbound
DNA by 5% polyacrylamide (acrylamide:bisacrylamide = 29:1) gel
electrophoresis (A). The protein-DNA complex bands were
excised and analyzed by liquid scintillation counting (B).
Figure 6:
The effect of XPAC on RPA's ability
to stimulate pol (6A) and pol
(6B). In addition to the
indicated amounts of XPAC, the reaction mixtures contained 0.05 units
of pol
-primase (A); or 0.05 units of pol
, 0.2
µg of PCNA, and 0.4 µg of A1 (B). Where
indicated, 1.0 µg of RPA was included. After incubation at 37
°C for 30 min, acid-insoluble radioactivity was
determined.
We have examined the interaction of two proteins, XPAC and RPA, that are involved in the early stages of the repair process. We reasoned that because XPAC is a UV-damage recognition protein, RPA may be recruited to damaged DNA sites though its interaction with XPAC. The resulting RPA-XPAC complex might then form multiprotein complexes at the damaged sites to promote recruitment of other repair proteins required for nucleotide excision repair. Recently, XPAC has been shown to interact with ERCC1 (22) or the ERCC1-ERCC4 (XP-F) complex(23) , a putative endonuclease complex that is necessary for 5` incision(37) . Although the XPAC-ERCC1-ERCC4 complex did not show a damaged site-specific incision(23) , it is possible that XPAC, RPA, ERCC1-ERCC4, and other repair proteins, such as the 3` incision endonuclease, XPG(37) , form a multiprotein complex at the damaged DNA site that is necessary for accurate 3` and 5` incisions.
In addition to its potential role in repair, we found
that XPAC inhibited SV40 DNA replication in vitro. This
inhibition was reversed by the addition of excess RPA but not by topo
I, pol -primase, or SV40 T-ag, indicating that the inhibition and
its reversal are physiologically relevant. The inhibition is unlikely
to be the result of competition between XPAC and RPA for DNA binding
because: (i) two known DNA binding proteins, human Rad51 protein (42) and EBNA1 protein(43) , fail to interact with RPA
or inhibit the SV40 monopolymerase replication system (data not shown),
and (ii) XPAC itself did not show any stable ssDNA binding activity in
the gel mobility shift assay; however, it did stimulate RPA's
ssDNA binding activity (Fig. 5). RPA binds as a multimer to
ssDNA more than 30 nucleotides in length(30) . It is therefore
possible that the XPAC-RPA interaction stabilizes the binding of RPA to
ssDNA binding activity, allowing stable monomeric RPA-ssDNA complexes
to form, and leading to the increased amount of RPA-DNA complex that
can be seen in Fig. 5. XPAC did not stimulate the ssDNA binding
activity of T4 phage ssDNA-binding protein (T4 gene 32), suggesting
that the stimulation of RPA's ssDNA binding activity by XPAC
occurs through their protein-protein interaction (data not shown). In
any event, this result strengthens our belief that the inhibition of
replication by XPAC is a result of its interaction with RPA rather than
its nonspecific binding to ssDNA.
XPAC binds dsDNA
weakly(19) ; however, this inhibition is unlikely to have
resulted from XPAC's interaction with dsDNA because, if this were
the case, we would expect to see the same degree of inhibition
regardless of the replication system (monopolymerase or dipolymerase)
used in the experiments. It is also unlikely that the inhibition
resulted from an interaction between XPAC and pol because: (i)
XPAC did not interact with pol
in our ELISA assay, (ii) addition
of excess pol
-primase did not reverse the inhibition of
replication (Fig. 4), and (iii) XPAC inhibited pol
activity in the presence, but not in the absence of RPA. Therefore, the
most likely explanation for this inhibition is that XPAC interacts with
RPA, altering RPA's ability to stimulate pol
.
This
belief is further supported by the fact that the inhibitory effect of
XPAC is more evident with the monopolymerase system, which relies
exclusively on pol activity, than with the dipolymerase system,
which contains both pol
and pol
( Fig. 2versusFig. 3). Pol
activity was not affected by XPAC (Fig. 6). In the monopolymerase system, pol
is responsible
for both leading and lagging strand synthesis; in the dipolymerase
system, pol
is only partly responsible for lagging strand
synthesis, while pol
is responsible for leading strand synthesis
and probably also for part of the lagging strand
synthesis(28, 38, 39) . On the other hand, we
should point out that XPAC had little effect on SV40 replication with
HeLa cell cytosolic extracts (data not shown). This lack of inhibition
in the crude extracts raises the possibility that our observations are
limited to the specific model systems used.
In view of the fact that both RPA and XPAC function in repair, our results would support the hypothesis that the XPAC-RPA complex, once formed, is used in repair rather than in DNA replication. It would be of interest to know whether the XPAC-RPA complex, which is stable enough to be isolated, can still recognize UV-damaged DNA. Since the completion of this work, two articles demonstrating specific interactions between RPA and XPAC have been published(40, 41) .