Department of Pathology and Laboratory Medicine, UMDNJ New Jersey Medical School and the Graduate School of Biomedical Sciences, Newark, NJ 07103, USA
* Author for correspondence (e-mail: mlambert{at}umdnj.edu)
Accepted 26 November 2002
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Summary |
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Key words: Spectrin, FANCA, XPF, DNA interstrand cross-link, DNA repair
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Introduction |
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Cells from patients with the autosomal recessive genetic disorder Fanconi
anemia (FA) provide an excellent experimental model for examination of the
mechanism of repair of DNA interstrand cross-links and the proteins involved.
FA is characterized by progressive bone marrow failure, a marked
predisposition to development of cancer, particularly acute myeloid leukemia,
spontaneous chromosome instability and hypersensitivity to DNA interstrand
cross-linking agents (Glanz and Fraser,
1982; Auerbach,
1995
; Auerbach et al.,
1998
). Correlated with sensitivity to these agents is a defect in
ability to repair DNA interstrand cross-links
(Papadopoulo et al., 1987
;
Averbeck et al., 1988
;
Lambert et al., 1992
;
Zhen et al., 1993
;
Lambert et al., 1997
;
Lambert and Lambert, 1999
).
There are eight complementation groups of FA and, although the genes
responsible for six of the complementation groups have been cloned
(Strathdee et al., 1992
;
Lo Ten Foe et al., 1996
;
Fanconi Anemia/Breast Cancer Consortium, 1996;
de Winter et al., 1998
;
de Winter et al., 2000a
;
de Winter et al., 2000b
;
Timmers et al., 2001
) and the
interactions of their protein products studied
(Kupfer et al., 1997
;
Garcia-Higuera et al., 1999
;
Waisfisz et al., 1999
;
de Winter et al., 2000c
;
Christianson et al., 2000
;
Reuter et al., 2000
;
Medhurst et al., 2001
;
Ahmad et al., 2002
), neither
their role in the etiology of the disorder nor their involvement in the repair
of DNA interstrand cross-links has been elucidated. We have shown that, in all
of the FA complementation groups we have examined (FA-A, FA-B, FA-C, FA-D1,
and FA-G), there is a deficiency in levels of
SpII
* present
(Brois et al., 1999
;
McMahon et al., 1999
). In FA-A
cells, this correlates with a deficiency in ability to produce dual incisions
at sites of interstrand cross-links
(Kumaresan and Lambert, 2000
).
This incision defect is not due to decreased levels of XPF in FA-A cells
because the level of this protein in these cells is similar to that of normal
cells (Brois et al., 1999
). We
have shown that FA-A cells are deficient in a DNA binding protein that
recognizes TMP interstrand cross-links
(Hang et al., 1993
). Because
our studies demonstrate that
SpII
* binds directly to DNA
containing TMP interstrand cross-links
(McMahon et al., 2001
), it is
possible that this is the deficient binding protein in FA-A cells. That FANCA
is involved in this repair process is indicated by our studies, which show
that it also binds to DNA containing interstrand cross-links, although it is
not clear whether this binding is direct or indirect
(McMahon et al., 2001
). It is
possible that
SpII
* acts as a scaffold to aid in the recruitment
of repair proteins to the site of damage and in their alignment at these
sites, thus enhancing the efficiency of the repair process. In FA-A cells,
where levels of
SpII
* are decreased, there would be reduced
recruitment of repair proteins to the sites of damage, which would in turn
lead to reduced levels of DNA repair, as has been observed
(Brois et al., 1999
;
McMahon et al., 1999
). The
same could hold true for the other FA complementation groups in which levels
of
SpII
* have also been shown to be decreased
(McMahon et al., 1999
).
In the present report, the relationship between SpII
* and the
FANCA and XPF proteins was analyzed by examining the localization of
SpII
*, in relation to that of FANCA and XPF, in the nucleus of
human cells damaged with a DNA interstrand cross-linking agent,
8-methoxypsoralen (8-MOP) plus UVA light, and by assessing the interaction of
these proteins by coimmunoprecipitation analysis. For these studies,
damage-induced foci formation of
SpII
*, FANCA and XPF was
examined not only in normal human nuclei, in which
SpII
* is
present, but also in FA-A cell nuclei, in which there is a deficiency in
SpII
*. Here, we show that, in response to 8-MOP plus UVA light,
SpII
*, FANCA and XPF co-localize with each other in discrete
foci in the nucleus and that
SpII
* plays an important role in
modulating the formation of these damage-induced foci. Co-immunoprecipitation
results show that
SpII
*, FANCA and XPF interact with each other.
These results together support the concept of a close functional link between
these proteins and an involvement for them in the repair of DNA interstrand
cross-links. These studies further suggest that an important function of
SpII
* in the nucleus is to act as a scaffold and aid in the
recruitment of repair proteins to sites of DNA damage.
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Materials and Methods |
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Treatment of cells with 8-methoxypsoralen and UVA light
The cells in culture were treated with 3.5 µM 8-methoxypsoralen (8-MOP)
(Sigma-Aldrich, St Louis, MO) in RPMI 1640 media for 20 minutes at room
temperature in the dark. They were then irradiated with UVA light (principally
366 nm) (6 kJ m-2), washed with fresh media and exposed to a second
dose of UVA irradiation (6 kJ m-2), as previously described, so as
to increase the number of DNA interstrand cross-links produced
(Lambert et al., 1988;
Lambert et al., 1992
). For
those experiments in which the effect of dosage of 8-MOP plus UVA irradiation
was examined, the concentration of 8-MOP was kept constant at 3.5 µM and
the dosage of UVA light was increased over a range from 0 kJ m-2 to
10 kJ m-2. At each dosage, an aliquot of cells was counted to
determine the number of surviving cells using a trypan blue exclusion
assay.
Indirect immunofluorescence
Cells were applied to poly-L-lysine coated chamber slides and allowed to
attach for 40 minutes at 37°C. They were then fixed with 4%
paraformaldehyde for 20 minutes, washed with PBS and permeabilized with 0.2%
Triton-X100 in PBS for 10 minutes at room temperature. The cells were then
blocked in 10% goat serum (Gibco, Grand Island, NY), 10% rabbit serum (Jackson
Immuno Research, Westgrove, PA) or 10% donkey serum (Sigma-Aldrich) as
appropriate, for 1-2 hours. The primary antibody was then added and allowed to
bind for 2 hours at 37°C. The primary antibodies used were purified mouse
monoclonal anti-SpII
* antibody, 1:100 dilution
(McMahon et al., 1999
),
purified mouse monoclonal anti-
-spectrin antibody, 1:150 dilution (mAb
1622, Chemicon International, Temecula, CA), affinity-purified rabbit
polyclonal anti-FANCA antibody, against the C-terminal region of FANCA, 1:100
dilution (Bethyl Laboratories, Montgomery, TX)
(McMahon et al., 2001
) and
affinity-purified goat polyclonal anti-XPF antibody, 1:100 dilution (Ab
sc-10161, Santa Cruz Biotech, Santa Cruz, CA). Pre-immune mouse, rabbit and
goat serum (Sigma-Aldrich) was used as a negative control. After five 5-minute
washes with PBS, the appropriate secondary antibody was added: Alexafluor 488
goat anti-mouse IgG conjugate, highly cross-adsorbed, 1:250 dilution (green
fluorescence), when anti-
-spectrin was the primary antibody; Alexafluor
594 goat anti-rabbit IgG conjugate, highly cross-adsorbed, 1:500 dilution (red
fluorescence), when anti-FANCA was the primary antibody; and Alexafluor 594
rabbit anti-goat IgG conjugate, highly cross-adsorbed, 1:500 dilution (red
fluorescence) (Molecular Probes, Eugene, OR), when anti-XPF was the primary
antibody. Incubation with the secondary antibodies was carried out for 20
minutes at room temperature in the dark. Primary and secondary antibodies were
diluted in PBS. In double-labeling experiments, the cells were treated
sequentially with the appropriate blocking agent and then the primary and
secondary antibodies against each of the proteins under investigation. The
secondary antibodies used were as follows: for dual staining with
anti-
-spectrin and anti-FANCA, Alexa 488 goat anti-mouse IgG and Alexa
594 goat anti-rabbit IgG were used, respectively; for dual staining with
anti-
-spectrin and anti-XPF, Alexa 488 goat anti-mouse IgG and Alexa
594 rabbit anti-goat IgG were used; and for dual staining with anti-FANCA and
anti-XPF, Alexa 488 donkey anti-rabbit IgG and Alexa 594 donkey anti-goat IgG
were used, respectively. The slides were then mounted with cover slips using
an aqueous anti-fade mounting agent (Molecular Probes). For those cells which
were examined with a DNA counter stain, after the last antibody labeling step,
the cells were treated with 4'-6' diamidino-2-phenylindole (DAPI)
(Sigma-Aldrich) at 100 ng ml-1.
Stained cells were then viewed using a Leitz DMRB microscope (Leica, Deerfiels, IL) equipped with a 40x oil objective lens. Appropriate filter sets were used to distinguish between red and green emissions. Settings were optimized using positively stained cells. Images were captured using a cooled-head three-color high resolution DEI-750 analog camera (Optronics, Bolton, MA) using the same parameters (brightness/contrast). A fixed exposure time was used for direct comparison of the image intensity. Images were imported into a computerized imaging system, Image Pro-Plus 4.0 (Media Cybernetics, Silverspring, MD) and Adobe Photoshop 5.0 (Adobe Systems, San Jose, CA) and similarly processed for presentation. Quantitation of foci was computerized using Image Pro-Plus. For dual staining experiments, images were merged and co-localization of foci examined. Overlapping foci appeared yellow. 100 cells were counted for each experiment and cells were scored as positive for staining based on comparison with the control pre-immune serum-treated cells.
Immunoprecipitation and western blotting
For immunoprecipitation (IP), chromatin-associated protein extracts from
normal cells were utilized. For this, cell nuclei were isolated and the
chromatin-associated proteins were extracted in a series of steps as described
previously (Lambert et al.,
1992; Hang et al.,
1993
). For anti-
-spectrin IPs, anti-
-spectrin or
mouse IgG1 (Sigma-Aldrich) was bound to protein-G-coated agarose
beads (Sigma-Aldrich) and the binding reactions and IPs were carried out as
previously described (McMahon et al.,
1999
; McMahon et al.,
2001
). For IP, anti-
-spectrin was used because our
anti-
SpII
* is of the IgM class and cannot be used effectively in
IPs (McMahon et al., 1999
).
For anti-FANCA IPs, affinity-purified rabbit polyclonal antisera generated
from the C-terminal region of the FANCA protein, or pre-immune serum, was
bound to protein-A-coated agarose beads (Sigma-Aldrich) and the binding
reactions and IPs carried out as previously described
(McMahon et al., 1999
). For
anti-XPF immunoprecipitation, anti-XPF (an affinity-purified polyclonal
antibody against the XPF protein, a generous gift of Michael Thelen, Lawrence
Livermore National Laboratory) was bound to protein-A-coated agarose beads as
described (McMahon et al.,
1999
). All of the IPs were subjected to SDS-PAGE, transferred to
nitrocellulose and immunoblotted as previously described
(McMahon et al., 1999
;
McMahon et al., 2001
).
Immunoblots were developed using Pierce Ultra chemiluminescent substrate
(Pierce) and then exposed to X-ray film
(McMahon et al., 1999
;
McMahon et al., 2001
). The
primary antibodies used were anti-
SpII
*
(Brois et al., 1999
;
McMahon et al., 1999
),
anti-FANCA (C-terminal) (McMahon et al.,
2001
) and anti-XPF (from M. Thelen). Images were scanned using a
Hewlett-Packard ScanJet 4c/T scanner and analyzed with ImageQuant (Molecular
Dynamics).
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Results |
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|
Co-localization of FANCA and XPF with SpII
* in nuclear
foci in normal cells after DNA damage
In order to determine the localization of FANCA and XPF in the nucleus of
normal human cells and whether they co-localize with SpII
* after
exposure of the cells to 8-MOP plus UVA, immunofluorescence studies were
carried out using a dual-staining technique. In undamaged normal cell nuclei,
staining of the cells with an affinity-purified polyclonal anti-FANCA antibody
showed that FANCA, like
SpII
*, is present in a diffuse and
fairly homogenous pattern in the undamaged nucleus
(Fig. 2A). When the cells were
treated with 8-MOP plus UVA light, FANCA was observed to relocalize to
prominent nuclear foci (Fig.
2A). Merging of the fluorescent signals for
SpII
*
(green) and FANCA (red) showed that there was clear colocalization of most of
these foci (Fig. 2A). The
merged foci appeared yellow. For these studies, staining was carried out using
either purified monoclonal anti-
SpII
* antibody or purified
monoclonal anti-
-spectrin antibody. The results using either antibody
were the same. We have previously shown that these two antibodies both
recognize
SpII
* (McMahon et
al., 1999
). Similarly, staining with an affinity-purified
polyclonal anti-XPF antibody showed that XPF was also present in a diffuse
pattern in the nucleus of undamaged normal cells and that XPF relocalized to
prominent damaged induced foci after exposure of the normal cells to 8-MOP
plus UVA light (Fig. 2B).
Merging of the fluorescent signals for
SpII
* (green) and XPF
(red) showed that there was co-localization of these foci
(Fig. 2B). This same pattern of
relocalization of FANCA and XPF to nuclear foci that was observed after double
staining of the proteins was also observed in cells singly stained for FANCA
and XPF (data not shown). This result supports the above finding that both
FANCA and XPF relocalize to damage-induced foci after treatment with 8-MOP
plus UVA light. Cells stained with preimmune serum showed a slight signal for
SpII
* under these experimental conditions
(Fig. 2A,B). This correlates
with our previous finding that a very low level of
SpII
*
antibodies are present in this preimmune serum
(McMahon et al., 1999
). Little
signal was observed for FANCA and XPF in cell nuclei stained with preimmune
serum (Fig. 2A-C).
|
In order to verify that FANCA and XPF are co-localizing to the same sites
after exposure to 8-MOP plus UVA, experiments were carried out in which dual
staining of both FANCA and XPF was examined. The results show that, in normal
cell nuclei, after treatment with 8-MOP plus UVA light, FANCA foci
co-localized with the XPF foci (Fig.
2C). Collectively, these results indicate that, in response to an
agent that produces DNA interstrand cross-links, FANCA and XPF co-localize to
the same foci as SpII
*.
Dosage of 8-MOP plus UVA light affects levels of nuclear foci
formation
Formation of foci in normal cell nuclei after exposure to 8-MOP plus UVA
light was quantitatively assessed to determine whether there was a
relationship between the number of cells displaying foci and the number of
damage-induced foci per nucleus, and the dosage of 8-MOP plus UVA light used.
For these studies, cells stained singly with either anti--spectrin,
anti-FANCA or anti-XPF antibodies were examined 15 hours after exposure to
8-MOP plus UVA. The dosage of 8-MOP plus UVA was increased by increasing the
levels of UVA light at a constant concentration of 8-MOP. As shown in
Fig. 3, as the dosage of UVA
light was increased from 0 kJ m-2 to 2 kJ m-2, 4 kJ
m-2 and 6 kJ m-2, the percentage of nuclei showing
SpII
* foci increased from 0% to 29%. The number of foci per
nucleus also increased in a UVA-dose-dependent manner, with the greatest
number of foci forming at 6 kJ m-2 (an average of 51 foci per
nucleus). For these experiments, 100 nuclei were counted for each dosage of
UVA light and each experiment was repeated three times. The viability of these
cells was 94% in undamaged cells and 93%, 92% and 91.5% at 2 kJ
m-2, 4 kJ m-2 and 6 kJ m-2, respectively. As
the dose of UVA light was increased to 8 kJ m-2 and 10 kJ
m-2, the number of
SpII
* foci per nucleus and the
number of cells showing nuclear foci decreased
(Fig. 3). However, the
viability of these cells also decreased, to 75.5% and 58%, respectively, at
these higher dosages.
|
Similar results were obtained for the XPF nuclear foci and the FANCA
nuclear foci (data not shown). In both of these instances, as the dosage of
UVA light was increased stepwise from 0 kJ m-2 to 6 kJ
m-2, both the number of nuclei showing foci and the average number
of foci per nucleus increased to levels comparable to those of the
SpII
* foci. At 6 kJ m-2, the percentage of nuclei
showing FANCA and XPF foci was 25% and 27%, respectively, and the average
number of foci per nucleus was 57 for FANCA and 49 for XPF. At 8 kJ
m-2 and 10 kJ m-2, the average number of FANCA and XPF
foci decreased as it did for
SpII
*. These results show that
there is a relationship between the number of
SpII
*, FANCA and
XPF nuclear foci formed per cell and the percentage of cells showing nuclear
foci with the dose of 8-MOP plus UVA light the cells are exposed to.
Time course of the formation of nuclear foci
To further examine the characteristics of nuclear foci formation, the time
course for formation of SpII
*, FANCA and XPF nuclear foci
following exposure of normal cells to 8-MOP plus UVA (6 kJ m-2) was
investigated. For these studies, cells were fixed at various periods of time
after treatment and stained independently for either
SpII
*,
FANCA or XPF. The number of foci per nucleus was counted in 100 cells for each
sample at each time point. Each of these experiments was repeated three times.
As seen in Fig. 4A, nuclei
showing
SpII
* foci were first visible between 8 hours and 10
hours after exposure to 8-MOP plus UVA, and the number of nuclei showing foci
increased with time and peaked at 16 hours. By 24 hours after exposure, nuclei
showing
SpII
* foci were no longer observed and
SpII
* showed a diffuse pattern of staining in the nucleus,
similar to untreated cells. FANCA and XPF nuclear foci also first appeared
8-10 hours after exposure to 8-MOP plus UVA
(Fig. 4A). The number of nuclei
showing FANCA and XPF foci were similar and increased up to 16 hours just as
for
SpII
*. By 24 hours, nuclei containing FANCA and XPF foci
were also no longer visible and a diffuse pattern of staining for these
proteins in the nucleus was observed as it was for
SpII
*. These
results show that
SpII
*, FANCA and XPF foci appear in the
nucleus at the same time after exposure to 8-MOP plus UVA light.
|
The average number of SpII
*, FANCA and XPF foci per nucleus
also increased up to a period between 14 hours and 16 hours after exposure to
8-MOP plus UVA (Fig. 4B). These
numbers were similar for
SpII
*, FANCA and XPF. After 16 hours,
the number of foci per nucleus decreased for each of these proteins. No foci
were observed by 24 hours.
Failure of localization of SpII
*, FANCA and XPF to
nuclear foci in FA-A cells after damage
In FA-A cells, there is a deficiency in levels of SpII
* that
correlates with a defect in ability to repair DNA interstrand cross-links
produced by either TMP or 8-MOP plus UVA light
(Brois et al., 1999
;
Lambert and Lambert, 1999
;
McMahon et al., 1999
;
Kumaresan and Lambert, 2000
).
This makes these cells an excellent source for studying the influence of
SpII
* on formation of damage-induced nuclear foci, particularly
because levels of the DNA repair protein XPF are normal in these cells
(Brois et al., 1999
). Studies
were undertaken to determine the localization of
SpII
* in the
nuclei of undamaged FA-A cells and FA-A cells exposed to 8-MOP plus UVA. In
addition, the localization of XPF and FANCA was examined in these cells. It
was found that, in undamaged FA-A cells, a diffuse pattern of staining was
observed with
SpII
*, but that this staining was much fainter
than in normal cells (Fig. 5A).
This correlates with the reduced levels of
SpII
* in FA-A cells
(Brois et al., 1999
;
McMahon et al., 1999
). When
the FA-A cells were damaged with 8-MOP plus UVA,
SpII
* was shown
to relocalize to a few foci in the nucleus
(Fig. 5A), but there were far
fewer foci than in damaged normal cell nuclei. The mean number of
SpII
* foci per nucleus 15 hours after treatment was 12 (20% of
normal). No staining for FANCA was observed above background levels in the
nuclei of FA-A cells, either undamaged or damaged with 8-MOP plus UVA
(Fig. 5A). This correlates with
reports of lack of detectable levels of this protein in this FA-A cell line
(HSC 72) owing to the mutation in the FANCA gene
(Kupfer et al., 1997
;
de Winter et al., 2000c
).
|
Examination of the XPF protein in FA-A cell nuclei showed that a diffuse
staining pattern of XPF was present at levels similar to normal
(Fig. 5B), which is consistent
with our finding that levels of XPF are similar in FA-A cells and normal cells
(Brois et al., 1999). However,
in FA-A cells damaged with 8-MOP plus UVA, XPF only relocalized to a few foci
in the nucleus and mainly showed a more diffuse pattern of staining
(Fig. 5B). The average number
of foci per nucleus 15 hours after treatment was 11 (18% of normal). Merging
of the fluorescent signals for
SpII
* and XPF showed
co-localization of only a few foci (Fig.
5B). To verify this deficiency in localization of XPF to
damage-induced foci after treatment with 8-MOP plus UVA, experiments were
carried out in which dual staining for XPF and FANCA was examined. The results
show that XPF again relocalized to only a few damage-induced nuclear foci
(Fig. 5C). No staining for
FANCA was observed above background levels. Thus in these FA-A cells, in which
there is a deficiency in
SpII
*, XPF was not able to relocalize
to many discrete nuclear foci after damage with a DNA interstrand
cross-linking agent.
SpII
*, FANCA and XPF co-localize to nuclear foci in
corrected FA-A cells after DNA cross-link damage
When FA-A cells express the FANCA cDNA, the DNA repair defect and
the deficiency in levels of SpII
* and FANCA are corrected; in
our corrected FA-A cells, we have also found that these levels are actually
slightly greater than normal (Brois et al.,
1999
). Studies were therefore undertaken to determine whether, in
the corrected FA-A cells, the defect in ability of
SpII
*, FANCA
and XPF to localize to nuclear foci after DNA damage had been corrected. In
the nuclei of corrected undamaged FA-A cells (HSC72) transduced with a
retroviral vector expressing the FANCA cDNA, a diffuse staining
pattern was observed for
SpII
* and FANCA just as in normal cell
nuclei (Fig. 6A). In corrected
FA-A cells exposed to 8-MOP plus UVA,
SpII
* relocalized to
discrete nuclear foci as in normal cells
(Fig. 6A). The average number
of
SpII
* foci per nucleus at 15 hours after treatment with 8-MOP
plus UVA light was 66 (108% of normal). Similarly, FANCA relocalized to foci
in the nucleus after damage (Fig.
6A). At 15 hours after damage, the average number of FANCA foci
per nucleus was 68 (111% of normal). Merging the fluorescent signals for
SpII
* and FANCA showed that these proteins localized to the same
discrete foci in the nucleus of corrected FA-A cells exposed to 8-MOP plus UVA
light (Fig. 6A).
|
Similar results were obtained for XPF in the corrected FA-A cells. In the
nuclei of untreated FA-A cells, XPF was present in a diffuse pattern
(Fig. 6B), as it was in the
uncorrected FA-A cells. However, in the corrected FA-A cells, XPF relocalized
to discrete nuclear foci after treatment with 8-MOP plus UVA
(Fig. 6B), as it did in damaged
normal cells. At 15 hours after damage, the average number of XPF foci per
nucleus was 62 (105% of normal). XPF also co-localized with
SpII
* to the same nuclear foci
(Fig. 6B). Dual-staining
experiments for both XPF and FANCA showed that these two proteins co-localized
to the same damage-induced nuclear foci
(Fig. 6C). These results
indicate that correcting the deficiency in levels of
SpII
* in
FA-A cells restores the ability of XPF to relocalize to nuclear foci after the
cells are damaged with 8-MOP plus UVA light.
SpII
*, FANCA and XPF bind to each other
The above immunofluorescence studies show that SpII
*, FANCA
and XPF localize to the same nuclear foci in normal cells after exposure to
8-MOP plus UVA, and suggest that these proteins interact with each other in
the nucleus. Studies were undertaken to provide additional evidence for the
existence of this interaction using immunoprecipitation. A series of IPs were
carried out using chromatin-associated-protein extracts from normal human
lymphoblastoid cells. Anti-
-spectrin IP and immunoblotting with
anti-
SpII
*, anti-FANCA or anti-XPF demonstrated that FANCA and
XPF co-immunoprecipitated with
SpII
*
(Fig. 7A). IP using anti-FANCA
and immunoblotting with anti-
SpII
*, anti-FANCA or anti-XPF
showed that XPF and
SpII
* co-immunoprecipitated with FANCA
(Fig. 7B). Anti-XPF
immunoprecipitation and immunoblotting with anti-
SpII
*,
anti-FANCA or anti-XPF demonstrated that FANCA and
SpII
*
co-immunoprecipitated with XPF from the normal extracts
(Fig. 7C). These IP studies
thus further confirm that
SpII
*, FANCA and XPF interact with
each other in the nucleus, although whether this interaction is direct or
indirect is not yet clear.
|
![]() |
Discussion |
---|
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---|
There is evidence that repair of DNA interstrand cross-links in mammalian
cells involves elements of both nucleotide excision repair (NER) and
recombination (Calsou et al.,
1996; Thompson,
1996
; Li et al.,
1999
; De Silva et al.,
2000
; Kuraoka at al.,
2000
; Wang et al.,
2001
; Dronkert and Kanaar,
2001
; Kumaresan and Lambert,
2000
; Kumaresan et al.,
2002
) and that double-strand breaks might form as intermediates in
this repair process (De Silva et al.,
2000
; Zhang et al.,
2002
). There might be more than one pathway for the repair of DNA
interstrand cross-links (Bessho et al.,
1997
; Li et al.,
1999
; De Silva et al.,
2000
; Wang et al.,
2001
; Kumaresan and Lambert,
2000
; Dronkert and Kanaar,
2001
). Our studies have previously shown that
SpII
*,
FANCA and XPF are involved in the initial damage recognition and incision
steps of the repair process (McMahon et
al., 1999
; Kumaresan and
Lambert, 2000
; McMahon et al.,
2001
; Kumaresan et al.,
2002
), though this does not preclude their involvement in
subsequent steps as well. The evidence for this is that
spectrin binds
directly to DNA containing a TMP interstrand cross-link and FANCA also binds
to this cross-linked DNA, although whether this binding is direct or indirect
is not clear (McMahon et al.,
2001
). A mAb against
SpII
*
(McMahon et al., 2001
) and a
polyclonal antibody against FANCA (M.W.L., L.W.M. and K. Kumaresan,
unpublished) inhibit the dual incisions we observe at sites of a TMP
interstrand cross-link. A mAb against XPF also specifically inhibits the
5' and 3' incisions we observe at the site of a cross-link
(Kumaresan and Lambert, 2000
),
and XPF cells, deficient in the XPF protein, are defective in ability to
produce dual incisions at sites of cross-links
(Kumaresan and Lambert, 2000
).
In addition, Kuraoka et al. (Kuraoka et
al., 2000
) have also shown that XPF-ERCC1 is involved in
production of the 5' and 3' incisions at the sites of DNA
interstrand cross-links. However, although our studies indicate that these
three proteins play a role in repair of DNA interstrand cross-links, the exact
relationship between them has not yet been elucidated.
Two different approaches were used in the present study to examine the
relationship between SpII
*, FANCA and XPF. One was to
investigate whether any of these proteins co-localize in the nucleus after
damage with a DNA interstrand cross-linking agent; the other was to determine
whether any of these proteins interact with each other as ascertained by
immunoprecipitation. Immunofluorescence studies using dual-staining techniques
showed that, after normal cells were treated with 8-MOP plus UVA,
SpII
*, FANCA and XPF changed their localization in the nucleus
and co-localized to the same discrete nuclear foci. Time course experiments on
foci formation showed that the appearance of FANCA and XPF foci coincide with
that of
SpII
* foci. The co-localization of these three proteins
to the same foci after damaging the DNA with 8-MOP plus UVA indicates that
they might act in concert and play a role together in the repair of DNA
interstrand cross-links. The average number of
SpII
*, FANCA and
XPF foci per nucleus as well as the number of nuclei showing foci increased
with increasing dosage of 8-MOP plus UVA light, thus indicating that the foci
assembled in response to DNA damage and that the number of foci depended on
the levels of DNA damage. Presumably, these foci are forming at sites of
damage. Other studies have shown that proteins involved in DNA repair and
checkpoint signaling pathways are relocalized to nuclear foci after DNA
damage. These include XPG, RPA, FANCD2, BRCA1, Rad51, H2AX, 53BP1, BLM, hMLH1
and the Mre11-Rad50-Nbs1 complex (Park et
al., 1996
; Scully et al.,
1997
; Wang et al.,
2000
; Paull et al.,
2000
; Cantor et al.,
2001
; Anderson et al.,
2001
; Pedrazzi et al.,
2001
; Garcia-Higuera et al.,
2001
; Choudhary and Li,
2002
). The increased local concentration of these proteins has
been proposed to facilitate their various enzymatic activities and their
functioning in processes such as signal transduction
(Anderson et al., 2001
). In the
present study, the number of foci formed peaked between 14-16 hours, then
started to decrease and was back to background levels at 24 hours. Presumably,
this reflected the repair of cross-links and dispersal of the proteins in the
foci at the completion of the repair process.
Immunoprecipitation studies showed that SpII
*, FANCA and XPF
have binding affinity for each other, although whether this binding is direct
or indirect is not yet clear. We have previously reported, in separate
experiments, that FANCA co-immunoprecipitates with
SpII
*
(McMahon et al., 1999
) and
that XPF co-immunoprecipitates with
SpII
*
(McMahon et al., 2001
). In the
present study, co-immunoprecipitation of all three proteins in the same
experiment was examined. When immunoprecipitation was carried out using
anti-
-spectrin, FANCA and XPF were shown to co-immunoprecipitate with
SpII
* from normal chromatin-associated proteins. The binding of
these proteins to each other was confirmed by anti-FANCA and anti-XPF
immunoprecipitation. This demonstrated association between these three
proteins again indicates that they might be involved in a common biochemical
pathway such as repair of DNA interstrand cross-links. Whether they form one
complex at the site of damage or are part of at least two interacting
complexes is not yet clear.
The use of FA cells in the present study has enabled us to get a much
better understanding of the importance of SpII
* in the repair of
DNA interstrand cross-links and its relationship to other proteins involved in
the repair process. Because the deletion of
spectrin from a cell has
been shown to be lethal (e.g. in Drosophila melanogaster and
Caenorhabditis elegans) (Lee et
al., 1993
; Lee et al.,
1997
; Norman and Moerman,
2002
), the FA-A cell line examined in the present study provides
an excellent model for examination of the effects on the repair process of
decreased levels of
SpII
* (reduced to 30-35% of normal) in the
nucleus. When FA-A cells were treated with 8-MOP plus UVA light, the reduction
in number (but not elimination) of
SpII
* nuclear foci correlated
with decreased levels of
SpII
* in the FA-A nuclei, which was
quantitatively assessed by examination of the
SpII
* band on an
SDS gel that was electroblotted onto a nitrocellulose membrane and stained
with colloidal gold (Brois et al.,
1999
; McMahon et al.,
1999
). This decrease in
SpII
* levels in FA-A cells
correlated in turn with a decreased number of endonucleolytic incisions
produced at the site of a TMP interstrand cross-link by
chromatin-associated-protein extracts from FA-A cells and with the observed
reduction of DNA repair levels, measured as unscheduled DNA synthesis, in
these cells (Brois et al.,
1999
; Kumaresan and Lambert,
2000
). All these values were approximately 25-35% of those of
normal cells (Brois et al.,
1999
; Kumaresan and Lambert,
2000
). Preliminary studies using a FA-C cell line (HSC 536)
indicate that there is a similar correlation between decreased levels of
SpII
*, a decreased number of
SpII
* nuclear foci
after damage with 8-MOP plus UVA light and decreased repair of interstrand
cross-links in these cells. In addition, the present demonstration that, in
FA-A cells, there is a markedly reduced recruitment of XPF to nuclear foci
after damage with 8-MOP plus UVA light, compared with normal cell nuclei,
further supports our model that
SpII
* is involved in targeting
repair proteins to sites of damage and that, when levels of
SpII
* are significantly reduced, so is the recruitment of repair
proteins to these damage sites. Because the FANCA protein is absent in this
FA-A cell line, it is also possible that it is needed for formation of XPF
foci. However, the present study shows that, in the absence of FANCA, there is
still a low level of
SpII
* and XPF focus formation and that the
number of foci per nucleus correlates with the levels of
SpII
*
present in FA-A cell nuclei (McMahon et
al., 1999
). If FANCA were essential for the formation of these
foci, no foci should be observed in this FA-A cell line.
The present studies also show that a functional FANCA gene is
essential for the re-localization of SpII
*, FANCA and XPF to
damage-induced nuclear foci. In corrected FA-A cells, which express the
FANCA cDNA,
SpII
*, FANCA and XPF again co-localize to
nuclear foci and both the number of foci per nucleus and the number of nuclei
showing foci are restored to normal. This correlates with return to levels of
SpII
* and FANCA in the nucleus that are slightly greater than
normal and of levels of DNA repair (unscheduled DNA synthesis) that are
slightly higher than normal (McMahon et
al., 1999
; Brois et al.,
1999
). It is possible that the FANCA protein is needed for
stability of
SpII
* in the nucleus or that the FANCA
gene is involved in regulating the expression of
SpII
*. Our
studies show that levels of
SpII
* mRNA are the same in
FA-A cells as in normal cells, which would indicate that reduced levels of
SpII
* in the FA-A nucleus are not due to decreased expression of
SpII
* (J. Lefferts and M.W.L., unpublished). It is possible that
FANCA and other FANC proteins are involved in the stability of
SpII
*. Studies indicate that FANCA, FANCC, FANCE, FANCF and
FANCG form a complex in the nucleus; they further suggest that the presence of
each of these proteins is important for the stability of the complex and that,
in the absence of any of these proteins, this complex is disrupted
(Kupfer et al., 1997
;
Yamashita et al., 1998
;
Garcia-Higuera et al., 1999
;
Waisfisz et al., 1999
;
de Winter et al., 2000c
;
Reuter et al., 2000
;
Garcia-Higuera et al., 2000
;
Medhurst et al., 2001
;
Siddique et al., 2001
;
Pace et al., 2002
;
Taniguchi and D'Andrea, 2002
).
In the FA-A cells used in the present work, in addition to undetectable levels
of FANCA, there are reduced levels of this protein complex
(Yamashita et al., 1998
;
Garcia-Higuera et al., 1999
;
Garcia-Higuera et al., 2000
).
In the present work, the reduced levels of
SpII
* observed in the
FA-A cells could thus possibly be due not only to the reduced levels of FANCA
in these cells but also to the reduced levels of this FA protein complex.
These studies show that there is localization of at least three different
proteins in the nucleus of normal human cells to common sites after damage
with a DNA interstrand cross-linking agent: one is a structural protein,
SpII
*, which we hypothesize acts as a scaffolding protein; one
an FA protein, FANCA; and one a DNA repair protein, XPF, which has been shown
to be involved in repair of DNA interstrand cross-links
(de Laat et al., 1998
;
Kumaresan and Lambert, 2000
;
Kuraoka et al., 2000
;
Kumaresan et al., 2002
). The
use of FA-A cells, which contain normal levels of XPF, in these studies
enabled us to demonstrate that
SpII
* is needed for the
localization of XPF to these nuclear foci after damage with a DNA interstrand
cross-linking agent and emphasizes the importance of
SpII
* in
the recruitment of repair proteins to sites of DNA damage. These results,
combined with the present demonstration that these three proteins interact
with each other and with our previous studies on the involvement of these
proteins in the initial damage-recognition and incision steps of the repair
process, greatly strengthen the concept that
SpII
*, FANCA and
XPF play an important role in the repair of DNA interstrand cross-links. In
addition, they emphasize the importance of a structural protein,
SpII
*, in aiding in the targeting and interaction of specific
proteins in the nucleus, a role that could extend beyond an involvement in DNA
repair to an involvement in other processes as well.
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Acknowledgments |
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