(Received for publication, March 6, 1995; and in revised form, July 12, 1995)
From the
Xeroderma pigmentosum (XP) is a human hereditary disease
characterized by a defect in DNA repair after exposure to ultraviolet
light. Among the seven groups of XP, group A (XP-A) patients show the
most severe deficiency in excision repair and a wide variety of
cutaneous and neurological disorders. We have cloned homologs of the
human XPA gene from chicken, Xenopus, and Drosophila, and sequence analysis revealed that these genes
are highly conserved throughout evolution. Here, we report
characterization of the Drosophila homolog of the human XPA gene (Dxpa). The Dxpa gene product shows
DNA repair activities in an in vitro repair system, and Dxpa cDNA has been shown to complement a mutant allele of
human XP-A cells by transfection. Polytene chromosome in situ hybridization mapped Dxpa to 3F6-8 on the X
chromosome, where no mutant defective in excision repair was reported.
Northern blot analysis showed that the gene is continuously expressed
in all stages of fly development. Interestingly, the Dxpa protein is
strongly expressed in the central nervous system and muscles as
revealed by immunohistochemical analysis using anti-Dxpa antibodies,
consistent with the results obtained in transgenic flies expressing a Dxpa--galactosidase fusion gene driven by the Dxpa promoter.
Xeroderma pigmentosum (XP) ()is a human autosomal
recessive disease characterized by hypersensitivity to UV light and a
high incidence of skin cancer because of a defect in nucleotide
excision repair (NER) (Cleaver and Kraemer, 1989). Complementation
analyses by cell fusion between cells from XP patients have revealed
seven complementation groups (A through G) and a variant form,
representing different components that are thought to work together in
NER. Five of the relevant genes (complementing XP-A, -B, -C, -D, and -G
mutations) have been already identified (Tanaka et al., 1990;
Weeda et al., 1990; Legerski and Peterson, 1992; Masutani et al., 1994; Flejter et al., 1992; O'Donovan
and Wood, 1993; Scherly et al., 1993). Some of these have been
shown to correspond to excision repair cross-complementing rodent
repair deficiency (ERCC) genes, which are human genes that
correct NER defects of a set of UV-sensitive rodent cell lines
comprising 11 different complementation groups; the XPB gene
is equivalent to the ERCC3 gene (Weeda et al., 1990), XPD to ERCC2 (Flejter et al., 1992), and XPG to ERCC5 (O'Donovan and Wood, 1993).
Homologs of the XP and ERCC genes have been
identified in many organisms, most notably in Saccharomyces
cerevisiae. A number of yeast radiation-sensitive mutants (rad) have been isolated, and some of the RAD genes
are known to be the homologs; XPA, XPB, XPC, XPD, XPG, and ERCC1 share extensive sequence
homology at the protein level with RAD14 (Bankmann et
al., 1992), RAD25/SSL2 (Park et al., 1992;
Gulyas and Donahue, 1992), RAD4 (Legerski and Peterson, 1992;
Masutani et al., 1994), RAD3 (Weber et al.,
1990), RAD2 (Scherly et al., 1993), and RAD10 (van Duin et al., 1986), respectively. Therefore, the
basic features of the NER mechanism are likely to be conserved among
eukaryotes. Indeed, expression of the XPD gene in S.
cerevisiae was reported to complement the lethality of a mutation
in the RAD3 gene (Sung et al., 1993).
XP patients manifest a wide variety of symptoms, the most characteristic of which is hypersensitivity to UV light and certain chemical mutagens. XP patients have a 2000-fold increased frequency of skin cancer upon UV exposure compared with the general population (Cleaver and Kraemer, 1989). Among the seven complementation groups, XP-A is the most severe form, and these patients exhibit a wide variety of neurologic abnormalities including microcephaly, progressive mental deterioration, ataxia, abnormal reflexes, and sensory deafness (Cleaver and Kraemer, 1989). The human XPA gene encodes a protein of 273 amino acids with a zinc-finger motif (Tanaka et al., 1990). Replacement of each of the 4 cysteine residues of the zinc-finger structure by serine or glycine resulted in loss of repair activity, indicating the functional importance of the motif (Miyamoto et al., 1992). The XPA protein binds to DNA with a preference for UV-irradiated over unirradiated DNA, suggesting that XPA functions as a key component in recognition of DNA damage during repair (Robins et al., 1991; Jones and Wood, 1993; Asahina et al., 1994).
Recently, the haywire gene of Drosophila was found to encode a protein with 66% identity to that encoded by ERCC3, the human gene associated with XP-B (Mounkes et al., 1992; Koken et al., 1992). Flies compromised for haywire function display phenotypes including UV sensitivity, central nervous system (CNS) defects, ataxia, and lethality, suggesting that a Drosophila mutant defective in the function of a human XP homolog could be useful as an animal model of XP. Sophisticated genetic tools available in Drosophila should help in investigating the functions of XP genes.
Previously, we cloned a Drosophila homolog of the human XPA gene (Dxpa) (Shimamoto et al., 1991). The Dxpa cDNA encodes a protein of 296 amino acids with 45% identity to that encoded by the human XPA gene. In this report, we show that the Dxpa protein is involved in NER both in vitro and in vivo. We also reveal that the Dxpa gene is expressed strongly in the CNS of Drosophila and determine the localization of the Dxpa gene to 3F6-8 on the X chromosome, where no mutant defective in NER has yet been reported.
For UV survival experiments, cells were plated at
densities varying from 8 10
to 1.6
10
cells/100-mm Petri dish, depending on the cell line and UV dose
used. Cells were rinsed with PBS and exposed to UV light approximately
1 day after plating. A series of dishes was irradiated for each cell
line, receiving a single UV dose (three dishes/UV dose). Colonies were
fixed and stained with 0.1% crystal violet approximately 10 days after
UV irradiation, and percent colony-forming abilities were determined by
comparing the colony counts of the irradiated plates with those of
unirradiated control plates.
Figure 6: Organization of genomic Dxpa and fusion gene construct. A, sequence of genomic Dxpa. Underlining indicates SalI and HaeII sites. The Dxpa gene has one intron of 65 bp, the boundaries of which are marked by arrowheads. B, restriction map of 3 kb of the Dxpa gene. Restriction sites are shown for SalI (S), PstI (P), PvuII (Pv), HindIII (H), HaeII (Ha), EcoRI (E), and XbaI (X). The open reading frame is represented by the stripedboxes. The openboxes indicate 5`- and 3`-untranslated regions. The asterisk indicates the SalI-HaeII fragment used for the fusion gene construction described in C. C, diagram of the fusion gene, constructed as described under ``Experimental Procedures.''
Germ line transformation was carried out
essentially by the method of Rubin and Spradling(1982). The recipient Drosophila strain was rosy/Kip
P[D2-3,
ry
], and the DNA concentration for
injection was 1 mg/ml of the Carnegie 20 construct. The embryos for
injection were collected, dechorionated, and aligned to the edge of a
coverslip. Following injection, embryos were placed in a moist chamber
and incubated at 20 °C until hatching. Surviving larvae were
transferred to standard food vials and allowed to develop into adults
(G
), which were then backcrossed to rosy
flies. G
progeny with the wild-type eye color were
crossed again to rosy
flies, and the resulting
wild-type G
progeny were mated to each other to establish
homozygous lines. Southern blotting of genomic DNA was performed to
confirm the presence of the inserted DNA in transformed lines.
Immunohistochemical staining was performed
using a Vectastain Elite ABC kit (Vector Laboratories, Inc.). Sections
10 µm thick were fixed with 2% paraformaldehyde in PBS and washed
in PBS. After preincubation with PBSG buffer (PBS, 1% normal goat
serum, 0.01% Triton X-100, and 0.2% bovine serum albumin) at room
temperature for 30 min, they were incubated with affinity-purified
rabbit anti-Dxpa antibodies or control rabbit IgG in PBSG buffer at
room temperature for 1 h and washed with washing buffer (PBS and 0.01%
Triton X-100). The sections were incubated with biotinylated goat
anti-rabbit IgG for 30 min at room temperature and washed, followed by
incubation with ABC complex for 30 min at room temperature. After
washing, the sections were submerged in peroxidase substrate solution
(0.1 M Tris-HCl, pH 7.2, 0.5 mg/ml diaminobenzidine, and 0.02%
HO
) for 2-5 min, washed again, and
mounted. Immunohistochemistry of whole embryos was performed as
described above except for the procedures of fixation and
permeabilization; dechorionated embryos were fixed in 0.1 M PIPES, 2 mM EGTA, 1 mM MgSO
, pH 6.9,
3.7% formaldehyde solution saturated with n-heptane, and the
concentrations of Triton X-100 were raised to 0.1% in PBSG buffer and
0.3% in washing buffer.
Figure 1:
Recombinant and native Dxpa
proteins. A, Coomassie Blue staining of a recombinant Dxpa
protein from E. coli (lane Dxpa) on
SDS-polyacrylamide gel electrophoresis. Lane M, molecular
weight standard. B, immunoprecipitation of
transcription-translation products of pGEM Dxpa and pET-XPAH
using affinity-purified rabbit anti-Dxpa antibodies (lanes 1 and 3) or control rabbit IgG (lanes 2 and 4). Details are described under ``Experimental
Procedures.'' C, effect of UV irradiation on synthesis of
the Dxpa protein. Kc, C10, and mei-9 cells were irradiated
with 6 J/m. Cells were harvested after 0 h (lanes1, 4, and 7), 4 h (lanes2, 5, and 8), and 8 h (lanes3, 6, and 9), and immunoblotting using
affinity-purified rabbit anti-Dxpa antibodies was carried out as
described under ``Experimental
Procedures.''
To investigate whether this system would also work for Drosophila, a whole-cell extract was prepared from growing Kc cells by the method of Manley et al.(1980) (see Wood et al.(1988)). Fig. 2A shows that Drosophila Kc whole-cell extracts supported specific incorporation of deoxyribonucleotides into UV-irradiated SV40 minichromosomes. Little nonspecific incorporation was detected in unirradiated SV40 minichromosomes and pUC19, indicating that this system could detect Drosophila NER in vitro (Fig. 2A, lanes1 and 2). When anti-Dxpa antibodies were added to the reaction mixtures, DNA repair synthesis with UV-irradiated SV40 minichromosomes was inhibited in a dose-dependent manner (Fig. 2A, lanes 5-12), whereas control rabbit IgG had no effect (lanes3 and 4). Addition of the recombinant Dxpa protein to the reactions after mixing the anti-Dxpa antibodies with extracts restored the DNA repair synthesis inhibited by the antibodies in a dose-dependent manner (Fig. 2B). These findings are consistent with results obtained in experiments using the human XPA protein (Masutani et al., 1993), verifying that the Dxpa protein is involved in DNA repair synthesis in Drosophila.
Figure 2: DNA repair synthesis in Kc cell extract. A, DNA repair synthesis and its inhibition by affinity-purified anti-Dxpa antibodies. UV-irradiated (lanes1, 3, 5, 7, 9, and 11) or unirradiated (lanes2, 4, 6, 8, 10, and 12) SV40 minichromosomes were incubated in standard reaction mixtures, which were preincubated in the presence of 1 µg of control rabbit IgG (lanes3 and 4) or 1 ng (lanes5 and 6), 10 ng (lanes7 and 8), 100 ng (lanes9 and 10), and 1000 ng (lanes11 and 12) of anti-Dxpa antibodies for 30 min on ice. Plasmids were linearized and resolved by 1% agarose gel electrophoresis as described under ``Experimental Procedures.'' Upperpanel, autoradiogram; lowerpanel, ethidium bromide staining of the gel. B, restoration by recombinant Dxpa protein of the repair synthesis inhibited by anti-Dxpa antibodies. One ng (lanes9 and 10), 10 ng (lanes11 and 12), 100 ng (lanes13 and 14), or 1000 ng (lanes 5, 6, 15, and 16) of recombinant Dxpa protein was added to the standard reaction mixtures containing 1 µg of control rabbit IgG (lanes 3-6) or 100 ng of anti-Dxpa antibodies (lanes 7-16), and the DNA repair reaction was performed in the mixtures in the presence of UV-irradiated (lanes1, 3, 5, 7, 9, 11, 13, and 15) or unirradiated (lanes2, 4, 6, 8, 10, 12, 14, and 16) SV40 minichromosomes as described under ``Experimental Procedures.'' Upperpanel, autoradiogram; lower panel, ethidium bromide staining of the gel.
Figure 3: Partial correction of the XP12ROSV repair defect by Dxpa cDNA. A, immunoblotting of wild-type human cell line WI38VA13 (lane1), XPA cell line XP12ROSV (lane2), and Dxpa cDNA-transfected cells derived from XP12ROSV, designated XPACDR1-3 (lanes 3-5), using anti-Dxpa antibodies. B, UV survival curves of WI38VA13, XP12ROSV, and XPACDR1-3 (see ``Experimental Procedures'').
Figure 4: Cytologic mapping of the Dxpa gene by in situ hybridization. A digoxygenin-dUTP-labeled Dxpa cDNA was used as a probe to determine the chromosomal location. The hybridization signal is indicated by the arrowhead at the 3F6-8 region on the X chromosome.
Figure 5: The Dxpa cDNA hybridized to a 1.4-kb transcript on Northern blots. Lane1, Kc cells; lane2, embryos; lane3, third instar larvae; lane4, early pupae; lane5, middle pupae; lane6, adult whole bodies; lane7, heads of adults; lane8, thorax and abdomen fractions of adults. The probe for rp49 hybridized to a 0.6-kb transcript.
The expression pattern of the fusion protein in adult flies is shown in Fig. 7(A-C). The fusion protein was strongly expressed in the nuclei of cells in the lamina and retina of the eyes and in the muscles of the whole body. The staining pattern was the same in all three transformants. The nuclear location was attributed to the presence of a putative nuclear localization signal in the first exon of the Dxpa gene (Shimamoto et al., 1991; Miyamoto et al., 1992). The signal is conserved evolutionarily, and the functioning of the signal of the human XPA protein has already been verified (Miyamoto et al., 1992). During embryogenesis, the fusion protein was expressed in all cells (data not shown), consistent with its universal function involved in NER. Fig. 7(D-F) shows immunohistochemical staining of adult flies using affinity-purified anti-Dxpa antibodies. The staining pattern was almost the same as that in the transformants. Strong expression of the Dxpa protein was detected in the nuclei of cells in the CNS and the muscles, although all cells showed different levels of expression. No such staining was seen when control rabbit IgG was used (data not shown), indicating the specificity of the staining by the anti-Dxpa antibodies. Comparison of the staining of transformants using X-gal with that of wild-type flies by the anti-Dxpa antibodies showed that the latter was more sensitive than the former. Therefore, the X-gal staining of the transformants only reflected the strong expression of the Dxpa protein. Interestingly, the ventral nerve cord of embryos was strongly stained with the anti-Dxpa antibodies, but not with control rabbit IgG (Fig. 7G; data not shown).
Figure 7:
Expression of the Dxpa gene in
embryos and adults. Dxpa gene expression was monitored by
localizing -galactosidase activity in transformants harboring a lacZ fusion gene under control of the Dxpa promoter
as described under ``Experimental Procedures'' (A-C), and expression of the Dxpa protein was
immunohistochemically detected in wild-type Drosophila (D-G). A-C, X-gal staining of adult
head sections at lower (A) and higher (B)
magnification and of the adult leg (C) of transformants.
-Galactosidase expression was detected in the nuclei of neurons in
the brain and retina in the head and muscle cells in the leg. Re, retina; R1-R7, nuclear region of distal
photoreceptor cells; R8, nuclear region of proximal
photoreceptor cells; La, lamina; Me, medulla; Lo, lobula and lobula plate. D-G,
immunohistochemical staining of sections of the adult head and thorax (D), the adult head at higher magnification (E), the
adult leg (F), and embryos (G) using
affinity-purified anti-Dxpa antibodies. The Dxpa protein was expressed
strongly in the CNS and muscle cells in adults and in the ventral nerve
cord (VNC) in embryos.
In this report, we have shown that the Dxpa gene product is involved in NER in vitro and in vivo. For the in vitro experiment, we employed a cell-free system for excision repair reactions on UV-irradiated SV40 minichromosomes. DNA repair synthesis was specifically suppressed when the anti-Dxpa antibodies were added to the reaction mixtures containing Kc cell extracts, and the inhibition was restored by adding the recombinant Dxpa protein. These results provided direct evidence for the involvement of the Dxpa protein in NER (Fig. 2). This is the first demonstration that the in vitro system can be used in the analysis of NER in Drosophila. This in vitro system might be an efficient assay to purify novel factors such as Mei-9 and Mus 201 gene products, which have not been cloned but are known to be involved in NER in Drosophila (Yamamoto et al., 1990; Todo and Ryo, 1992).
Furthermore, we demonstrated that the Dxpa gene could
improve UV survival of the human XP-A cell line XP12ROSV (Fig. 3). The functions of the human and Drosophila XPA genes are thus evolutionarily conserved. However, UV resistance of
transfectants conferred by Dxpa expression has not been
restored completely (Fig. 3). This incomplete correction was not
due to the quantity of the expressed Dxpa protein because the Dxpa gene was expressed strongly under control of the -actin
promoter in the transfectants at both mRNA and protein levels compared
with the human XPA gene in WI38VA13 cells (data not shown).
The big difference between primary structures of human XPA and Drosophila Dxpa is the presence or absence of the E-cluster
region. There is no E-cluster region in Dxpa (Shimamoto et
al., 1991), and the mutant human XPA protein shows very weak
repair activities in human XP-A cells (Miyamoto et al., 1992).
There is a possibility that the Dxpa protein having no E-cluster might
show low repair activities in human XP-A cells. Previously, it was
reported that functional cross-complementation was not observed in
human XP-B cells transfected with ERCC3
cDNA,
which is a Drosophila homolog of the human XPB/ERCC3 gene (Koken et al., 1992). ERCC3
is
identical to the haywire gene and more highly conserved during
evolution than the Dxpa gene (Mounkees et al., 1992).
The cause of this discrepancy is unknown, but there are several
possible explanations among the experimental procedures. First, Koken et al.(1992) cotransfected XP-B cells with an ERCC3
expression vector together with a separate selection marker
(neomycin resistance gene)-containing vector, while we used an
expression vector in which a selection marker was already inserted.
Second, they assayed the resulting G418-resistant transfectants without
cloning, which might include those without ERCC3
expression, while we picked up independent transformants and confirmed
the expression of the Dxpa protein in each clone by immunoblotting.
This allowed us to examine UV survival with homogeneous cell
populations expressing the Dxpa protein.
Although the calculated
molecular mass of the Dxpa protein was 34 kDa, it migrated with masses
of 43 and 40 kDa on SDS-polyacrylamide gel electrophoresis (Fig. 1, B and C). Since the recombinant Dxpa
protein synthesized in E. coli was detected as a band of
43 kDa (Fig. 1A) and could restore the DNA repair
synthesis inhibited by the anti-Dxpa antibodies, the products seemed to
be unmodified Dxpa proteins.
Northern hybridization indicated that
the Dxpa gene is expressed continuously throughout fly
development (Fig. 5). This is consistent with the important role
of the gene product in NER. As compared with the internal control rp49,
expression of the Dxpa gene changed during development and was
stronger in the thorax and abdomen than in the head. For detailed
examination of the expression of the Dxpa gene, we made
transgenic flies expressing a marker protein, E. coli -galactosidase, fused to a partial coding region of the Dxpa gene in frame under the control of the Dxpa promoter (Fig. 6). The fusion gene product was expressed
strongly in the CNS in adults (Fig. 7, A and B). Immunohistochemical staining provided similar results and
furthermore clarified the intense expression of the Dxpa protein in the
CNS in embryos (Fig. 7, D, E, and G).
Recently, haywire was identified as a Drosophila mutant defective in the homolog of the human XPB gene,
and this mutant could be used as an animal model of human XP-B disease
(Mounkes et al., 1992). In fact, haywire flies
manifest neurological defects that are thought to mimic the CNS defects
observed in XP-B patients. In a similar way, the Dxpa protein may play
an important role in the CNS. Satoh et al.(1993) reported that
the DNA repair defect of XP prevented removal of a class of oxygen free
radical-induced base lesions. Their data suggested that accumulation of
endogenous oxidative damage in cellular DNA of XP patients contributed
to the neural degeneration occurring in serious cases of the syndrome.
Since the O
consumption in the CNS is known to be
particularly high compared with other organs, it is probable that the
preferential accumulation of oxidative damage occurs in the CNS. The
large amount of Dxpa protein in the CNS of Drosophila might
correspond to the necessity of NER in the CNS and is suggestive of a
mechanism of the variety of neurological symptoms exhibited by XP-A
patients.
mei-9 was originally identified as a mutant defective in meiosis. This mutant also has a defect in NER, and it was possible that its locus was identical to that of the Dxpa gene. However, we verified that they are different genes on the basis of the following data. First, genome mapping demonstrated that their locations were different; we mapped the Dxpa gene to 3F6-8 of the X chromosome (Fig. 4), while mei-9 had been mapped to 4B. Second, we identified the Dxpa protein in mei-9 whole-cell extracts. The size of the protein was the same as that of the wild-type cell lines Kc and C10, and the amount was almost equal to that of C10 (Fig. 1C). Thus, we concluded that the Dxpa gene is different from mei-9.
To date, no mutant defective in the Dxpa gene has yet been identified. Future isolation of such mutants will facilitate further characterization of the function of the Dxpa protein in vivo in addition to providing a potentially useful animal model of human XP-A.
The nucleotide sequences reported in this paper have been submitted to the GSDB, DDBJ, EMBL, and NCBI nucleotide data bases with accession number D31892[GenBank].
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