(Received for publication, October 31, 1994; and in revised form, December 10, 1994)
From the
XPA is a zinc finger DNA-binding protein, which is missing or altered in group A xeroderma pigmentosum cells and known to be involved in the damage-recognition step of the nucleotide excision repair (NER) processes. Using the yeast two-hybrid system to search for proteins that interact with XPA, we obtained the 34-kDa subunit of replication protein A (RPA, also known as HSSB and RFA). RPA is a stable complex of three polypeptides of 70, 34, 11 kDa and has been shown to be essential in the early steps of NER as well as in replication and recombination. We also demonstrate here that the RPA complex associates with XPA. These results suggest that RPA may cooperate with XPA in the early steps of the NER processes.
Xeroderma pigmentosum (XP) ()is an autosomal
recessive human disease characterized by hypersensitivity to sunlight
and a high incidence of skin cancer in sun-exposed areas(1) .
Cells from XP patients are hypersensitive to UV irradiation and have a
defect in nucleotide excision repair (NER). Complementation analysis by
cell fusion has identified seven different complementation groups
(A-G) and a variant form in XP(2, 3) . In
addition, there are 11 different complementation groups (groups
1-11) in UV-sensitive rodent mutant cell lines. The XPA (XP group A) and XPC (XP group C) genes have been
directly cloned by transfections of XP cells with mouse genomic DNA and
a human cDNA library, respectively(4, 5) .
Additionally, human genes that can correct the repair deficiency of the
UV-sensitive rodent mutant cell lines have been cloned by the same
transfection cloning strategy and are denoted ERCC (excision
repair cross-complementing rodent repair deficiency) genes. The ERCC3, ERCC2, ERCC5, and ERCC6 genes have shown to be equivalent to XPB (XP group B), XPD (XP group D), XPG (XP group G), and Cockayne
syndrome group B (CSB) genes, respectively (6, 7, 8, 9, 10, 11) .
These genes are involved in the early steps of NER, suggesting that
protein interactions and/or protein complex formations are required for
the early steps of NER processes, including the recognition of DNA
damage, generation of dual incisions on either side of the damage, and
excision of the oligonucleotides containing the damaged
sites(12) . It has been shown that ERCC1 forms a complex with
ERCC4 (XPF) and ERCC11(15, 16) , and that XPC
associates tightly with a human homologue of yeast RAD23(17) ,
although the biological meaning of these interactions are not clear
yet. Moreover, it is known that XPB is a component of
TFIIH(13) , a part of the RNA polymerase II transcription
initiation complex, and that XPD associates with TFIIH (14) ,
indicating a tight connection between NER and the transcription
processes.
We have previously cloned the XPA gene and shown
that it encodes a protein of 273 amino acids containing a C zinc finger motif(4) . The XPA protein (XPA) binds
preferentially to UV- or chemical carcinogen-damaged
DNA(18, 19, 20) , suggesting that XPA is
involved in the recognition of several types of DNA damage in the NER
processes. In light of the observation that the defect in NER in group
A XP cells is particularly severe, we hypothesized that XPA might have
an important DNA repair function other than damage recognition. We
expected that XPA might have domains that interact with other proteins
to coordinate the NER processes. Recently, it has been reported that
XPA and ERCC1 specifically interact(21, 22) . Here, we
have used yeast two-hybrid system to identify other XPA-associated
proteins by screening a HeLa cDNA library and found that the 34-kDa
subunit of replication protein A (RPA, also known as HSSB and RPF)
binds to XPA. RPA is composed of three tightly associated subunits of
70, 34, and 11 kDa (p70, p34, and
p11)(23, 24, 25) . We also demonstrate on
interaction between the RPA complex and XPA. It has been shown that RPA
is involved in NER (26, 27) as well as in replication
and recombination(28) . These results strongly suggest that a
specific interaction between XPA and RPA is required for the early
steps of the NER process.
After cells were lysed by
sonication, the lysates was centrifuged at 15,000 rpm for 20 min
(Sorvall type SS-34 rotor) at 4 °C. The pellet was saved and
resuspended in buffer A (50 mM Tris-HCl, pH 7.5, 6 M guanidine HCl, 0.5 mM PMSF and 1 mM dithiothreitol) in order to denature the inclusion bodies. The
suspension was stirred at 4 °C for 1 h and then centrifuged at
50,000 rpm for 1 h (Hitachi RP 65T rotor) at 4 °C. The supernatant
was loaded onto a Superose CL-6B (Pharmacia) gel filtration column
equilibrated with buffer A. The protein was eluted with buffer A (flow
rate, 0.5 ml/h) and then the GST-XPA peak fraction was dialyzed against
buffer B (20 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 1
mM MgCl, 20 mM zinc acetate, 0.5 mM PMSF, and 1 mM dithiothreitol) at 4 °C. The purity
was checked by SDS-PAGE (11%) and Coomassie staining. The DNA repair
activity of the GST-XPA protein was checked by the restoration of
UV-induced unscheduled DNA synthesis in group A XP cells (GM5509 cells
obtained from NIGMS Human Genetic Mutant Cell Repository, Camden, NJ)
after microinjection of the protein(20) .
Figure 1: Panel A, interaction of XPA protein. Yeast cells (HF7c) were simultaneously transformed with pGBT9-XPA and plasmids expressing the pGAD424 fused to the NER genes ERCC1 (section 1), XPB (section3), XPC (section4), XPD (section5), or to pGAD424 fused to p34 (section2). Panel B, interaction of the 34-kDa subunit of RPA. Yeast cells (HF7c) were simultaneously transformed with pGAD424-p34 and plasmids expressing the pGBT9 fused to the NER genes ERCC1 (section1), XPA (section2), XPB (section3), XPC (section4), and XPD (section5). Cells containing both plasmids were streaked on plates with or without histidine. The ability to grow in the absence of histidine depends on the expression of the HIS3 gene under the control of a Gal1-responsive promoter.
Figure 2:
Interaction of XPA with p34 in
vitro. In vitro translated
[S]methionine-labeled p34 (lane1) was incubated with GST beads (lane2) or GST-XPA beads (lane3). After
washing the beads, the bound proteins were separated by SDS-PAGE and
analyzed by fluorography.
Figure 3:
Interaction of XPA with the RPA complex in vitro. Panel A, HeLa nuclear extract was incubated
with GST beads (lanes1 and 3) or GST-XPA
beads (lanes2 and 4). After washing the
beads, the bound proteins were analyzed by SDS-PAGE and immunoblotting
with either anti-p70 or anti-p34 antibody. Lanes1 and 2, immunoblotting with anti-p34 antibody; lanes3 and 4, immunoblotting with anti-p70 antibody. PanelB, [S]methionine-labeled
HeLa nuclear extracts were incubated with GST (lane 1) or
GST-XPA (lane 2) beads, and the bound proteins were analyzed
by SDS-PAGE and fluorography. The HeLa nuclear extracts were
immunoprecipitated with the corresponding antibodies, directly loaded
onto SDS-PAGE, and analyzed by fluorography (lane 3,
immunoprecipitation with anti-p70 antibody; lane 4,
immunoprecipitation with anti-p34 antibody; lane 5,
immunoprecipitation with control supernatant media). On the one hand,
the proteins bound to GST-XPA beads were eluted with NETN containing 1 M NaCl, immunoprecipitated with the protein G plus/protein
A-agarose beads, which have been bound with the corresponding
monoclonal antibodies (lane 6, anti-p70 antibody; lane
7, anti-p34 antibody; lane 8, control supernatant media). Lane 9, residual GST-XPA beads after the elution. The bound
proteins were separated by SDS-PAGE and analyzed by fluorography. Panel C, purified RPA was incubated with GST beads (lanes2 and 5) or GST-XPA beads (lanes3 and 6). After washing the beads, the bound proteins were
extracted by boiling in SDS sample buffer and separated by SDS-PAGE. As
a control, purified RPA was loaded directly onto an SDS-polyacrylamide
gel (lanes 1 and 4). The 70- and 34-kDa subunits of
RPA were detected by immunoblotting with the corresponding monoclonal
antibodies. Lanes1-3, immunoblotting with
anti-p34 antibody; lanes 4-6, immunoblotting with
anti-p70 antibody.
To examine whether the interaction between RPA and XPA is direct or is mediated through other factors, purified RPA complex was incubated with GST or GST-XPA beads. The GST or GST-XPA bound fractions were analyzed by SDS-PAGE and immunoblotting with anti-p70 or anti-p34 monoclonal antibody. As shown in Fig. 3c, both p34 and p70 bound directly to GST-XPA but not to GST.
Figure 4:
Co-immunoprecipitation of RPA with XPA.
HeLa whole cell extracts were immunoprecipitated with control (lane
2) or anti-XPA (lane 3) antibody. The precipitates were
immunoblotted with anti-p70 and anti-p34 antibodies. Lane1, whole cell extract from 2.5 10
cells.
XPA is a zinc metalloprotein and binds preferentially to UV-,
cisplatin-, or osmium tetroxide-damaged
DNA(18, 19, 20) , indicating that it is
involved in the damage-recognition step of the NER processes. We
recently identified the DNA-binding domain of XPA. The region of XPA
containing a C type zinc finger domain is sufficient for
its preferential binding to damaged DNA. The E-cluster and the
C-terminal regions are not necessary for the DNA binding activity of
XPA. (
)In addition, we have cloned homologues of the human XPA cDNA from mouse, chicken, Xenopus laevis, and Drosophila melanogaster(34) . A comparison of the
amino acid sequences of these homologues revealed that the N-terminal
region is not well conserved except in its nuclear localization signal
and E-cluster region, whereas the C-terminal region is highly
conserved. These results suggest that the E-cluster and the C-terminal
regions may have an important DNA repair function separate from DNA
binding. We speculated that they might be domains for protein-protein
interactions that coordinate the NER processes.
We therefore searched for proteins that bind to XPA using the yeast two-hybrid system, and found that the p34 subunit of RPA bound to XPA. The direct association between XPA and the RPA complex was confirmed by in vitro experiments. Furthermore, the RPA complex was co-immunoprecipitated from HeLa whole cell extracts with XPA by anti-XPA antiserum, suggesting the association of XPA and the RPA complex in vivo.
RPA was originally purified as a factor essential for the in vitro replication of SV40(23, 24, 25) . It is a three-subunit protein complex and binds strongly to single-stranded DNA and weakly to RNA and double-stranded DNA(35, 36) . It is requisite for the helicase action of the virus-encoded T antigen to open the SV40 origin, and it stimulates in vitro DNA synthesis by DNA polymerases on artificial templates(37, 38, 39, 40) . It has been demonstrated that the p70 subunit of RPA, which retains single-stranded DNA binding activity(35, 36, 39) , can interact with the tumor suppressor p53 and the transcription activators GAL4, VP16, and E2, functioning synergistically to enhance replication(41, 42, 43) . SV40 large T antigen also can bind to intact RPA but not to any of the separated subunits of RPA(44) . Less is known about the functions of the p34 and p11 subunits of RPA. It has been speculated that the two acidic regions of p34 are involved in protein-protein interactions with other proteins involved in replication or other functions(45) . Our results indicate that XPA is one of these proteins that bind to the p34 subunit of RPA.
It has been found that RPA is involved in NER (26, 27) as well as in DNA replication and recombination(28) . Neutralizing monoclonal antibodies which recognize the 70- and 34-kDa subunits inhibited DNA repair synthesis in vitro and the addition of an excess amount of RPA into an in vitro NER assay system caused a 2-3-fold stimulation of DNA repair synthesis. Interestingly, when damaged DNA is incised by Uvr ABC excinuclease, human cell extracts can carry out DNA repair synthesis even when RPA has been neutralized with antibodies(27) , suggesting that RPA might be involved in the early step of the NER process. All these results taken together suggest that XPA might cooperate with RPA for the recognition of DNA damages.
It has been shown that XPA forms a complex with the ERCC1/ERCC4 (XPF) heterodimer(22) . The ERCC1/ERCC4 (XPF) heterodimer might be a major component of the incision complex(15, 16) . XPA might play a role in loading the incision complex onto a damaged DNA site. Thus, XPA is a multifunctional protein that coordinates the NER processes. Similarly, RPA also plays multiple roles during NER. RPA might also bind to the single-stranded gap created by the excision of damaged nucleotides. The binding of RPA to the gap protects the single-stranded region and DNA termini from degradation by other nucleases and could facilitate the recycling of the incision protein complex by means of protein-protein interactions(27) . Therefore, the interaction between XPA and RPA is likely to have plural biological significance in the early steps of the NER processes.