1159
Journal of Cell Science 110, 1159-1168 (1997)
Printed in Great Britain © The Company of Biologists Limited 1997
JCS4372
Translocation of a UV-damaged DNA binding protein into a tight association
with chromatin after treatment of mammalian cells with UV light
Vesna Rapić Otrin*, Mary McLenigan*, Masashi Takao†, Arthur S. Levine‡ and Miroslava Protić §
Section on DNA Replication, Repair and Mutagenesis, National Institute of Child Health and Human Development, National
Institutes of Health, Bethesda, MD 20892, USA
*These two authors contributed equally to this work
†Present address: Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-77, Japan
‡Author for correspondence (e-mail: [email protected])
§Present address: Center for Mechanistic Biology and Biotechnology, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL 60439, USA
SUMMARY
A UV-damaged DNA binding protein (UV-DDB) is the
major source of UV-damaged DNA binding activity in
mammalian cell extracts. This activity is defective in at
least some xeroderma pigmentosum group E (XP-E)
patients; microinjection of the UV-DDB protein into their
fibroblasts corrects nucleotide excision repair (NER). In an
in vitro reconstituted NER system, small amounts of UVDDB stimulate repair synthesis a few fold. After exposure
to UV, mammalian cells show an early dose-dependent inhibition of the extractable UV-DDB activity; this inhibition
may reflect a tight association of the binding protein with
UV-damaged genomic DNA. To investigate the dynamics
and location of UV-DDB with respect to damaged
chromatin in vivo, we utilized nuclear fractionation and
specific antibodies and detected translocation of the p127
component of UV-DDB from a loose to a tight association
with chromatinized DNA immediately after UV treatment.
A similar redistribution was found for other NER proteins,
i.e. XPA, RP-A and PCNA, suggesting their tighter association with genomic DNA after UV. These studies revealed
a specific protein-protein interaction between UVDDB/p127 and RP-A that appears to enhance binding of
both proteins to UV-damaged DNA in vitro, providing
evidence for the involvement of UV-DDB in the damagerecognition step of NER. Moreover, the kinetics of the reappearance of extractable UV-DDB activity after UV
treatment of human cells with differing repair capacities
positively correlate with the cell’s capacity to repair 6-4
pyrimidine dimers (6-4 PD) in the whole genome, a result
consistent with an in vivo role for UV-DDB in recognizing
this type of UV lesion.
INTRODUCTION
long. In subsequent steps, the resulting DNA gap is filled in by
DNA polymerase and ligase in a PCNA (proliferating cellnuclear antigen)-dependent reaction (Ma et al., 1995; Wood,
1996, and references therein).
It has been suggested that in addition to XPA, yet another
protein, UV-DDB, has a role in the damage-recognition step of
NER because of its high binding affinity for 6-4 PD in vitro
(Hirschfeld et al., 1990; Treiber et al., 1992; Reardon et al.,
1993; Protic and Levine, 1993). However, the exact role of UVDDB in NER is still unknown despite the availability of its
purified protein components (Abramić et al., 1991; Hwang and
Chu, 1993; Keeney et al., 1993) and their corresponding cloned
genes (Takao et al., 1993; Lee et al., 1995; Dualan et al., 1995;
Hwang et al., 1996). UV-DDB has been purified from primate
cells as a homodimer of 127 kDa subunits, and as a heterodimer
of the 127 kDa protein and a 48 kDa protein (Abramić et al.,
1991; Keeney et al., 1993). p127 appears to be a DNA-binding
protein, while the function of p48 in the formation of the
binding complex is still unresolved (Reardon et al., 1993). The
DNA binding activity of the complex (UV-DDB activity) is
absent in cells from only a subset of XP group E patients,
The major DNA repair process that removes structurally
unrelated lesions from DNA in both prokaryotic and eukaryotic cells is the NER pathway. Genetic and biochemical studies
with mammalian cells implicate more than 20 proteins in the
complete reaction, and defects in many of these proteins are
associated with various inherited human diseases characterized
by defective DNA repair, such as xeroderma pigmentosum
(XP), Cockayne’s syndrome (CS), and trichothiodystrophy.
The best characterized proteins correspond to genetic complementation groups of XP (A to G), and are involved in the early
steps of NER (Friedberg et al., 1995). Protein-protein interaction studies and in vitro reconstitution experiments using
damaged DNA have shown that XPA, a damage recognition
protein, first forms a complex with RP-A (a major human
single-stranded DNA-binding protein); other proteins are then
recruited to the lesion, including TFIIH (a transcription factor
containing DNA helicases XPB and XPD), XPC, and two DNA
endonucleases, XPG and XPF/ERCC1. The result of this
process is the removal of a damaged oligomer ~30 nucleotides
Key words: Xeroderma pigmentosum, UV light, DNA binding
protein, DNA repair, Chromatin
1160 V. Rapić Otrin and others
although cells from all XP-E patients tested thus far show a 4060% reduction in unscheduled DNA synthesis (UDS) and
moderate sun-sensitivity (Chu and Chang, 1988; Hirschfeld et
al., 1990; Kataoka and Fujiwara 1991; Keeney et al., 1992).
Microinjection of the purified UV-DDB protein into XP-E
fibroblasts that lack the binding activity restored UDS to the
levels found in normal human cells. However, this biochemical complementation had no effect on UDS in cells from XPE patients that demonstrate binding activity nor in cells from
patients in other XP groups (Keeney et al., 1994). Although
single-base mutations have been identified in the gene
encoding the p48 subunit in cells from three XP-E patients who
lack UV-DDB binding activity (Nichols et al., 1996), this result
could simply reflect polymorphism and the molecular basis of
XP group E requires further definition. Moreover, while UVDDB is not essential for in vitro reconstituted NER (the core
reaction) (Mu et al., 1995; Guzder et al., 1995), the protein can
stimulate repair synthesis two-fold when added to the reaction
in small amounts (Abussekhra et al., 1995). However, larger
amounts can have an inhibitory effect on repair (Wood, 1996).
We reported previously that mammalian cells show an early
dose-dependent inhibition of the extractable UV-DDB activity
after treatment with UV light (Protić et al., 1989; Hirschfeld et
al., 1990). Such a UV response appears to be common to UVdamaged DNA binding activities in a diversity of vertebrates
because a similar inhibition of extractable binding activities
that recognize either cyclobutane pyrimidine dimers (CPD) or
non-CPD lesions has been detected in cells from fish to
primates (McLenigan et al., 1993). We also suggested that this
inhibition is probably due to translocation of the UV-DDB
from a loose to tight chromatin/UV-DDB/UV-damaged DNA
structure; this tight structure would prevent release of UVDDB, with the consequence that it could not be extracted under
standard conditions for 0.3 M salt nuclear extract preparation
(Hirschfeld et al., 1990; McLenigan et al., 1993). Since our
previous data concerned only UV-DDB activity, and not the
protein harboring the activity, we designed the present study to
address both the activity and the protein. To test the hypothesis of UV-DDB binding to damaged DNA directly, and to learn
more about the location and dynamics of UV-DDB after UV
treatment in vivo, we have carried out an extensive nuclear
fractionation analysis and are now able to demonstrate translocation of the p127 component of UV-DDB, as well as of the
DNA repair and replication proteins XPA, RP-A and PCNA,
from low-salt (loose association) to high-salt (tight association)
chromatin following UV-irradiation of primate cells. Furthermore, we detected a specific interaction between UVDDB/p127 and RP-A in vitro and in vivo, and this interaction
appears to enhance binding of both proteins to UV-damaged
DNA in vitro. Finally, we studied extractable UV-DDB activity
over the course of 48 hours after UV treatment in a variety of
normal and repair-deficient human cell lines, and found a
positive correlation between the kinetics of reappearance of
extractable UV-DDB activity after UV treatment and the
capacity of these cell lines to repair 6-4 PD.
MATERIALS AND METHODS
Cells and cell treatments
Monolayer cultures of TC-7 cells, a clone of the African green
monkey kidney cell line CV-1, were grown at 37°C and 10% CO2 in
Dulbecco’s modified Eagle’s medium (MEM) supplemented with
10% fetal bovine serum and 20 mM-glutamine. For UV-irradiation,
cells were washed twice with phosphate-buffered saline (PBS),
precooled for 15 minutes at 4°C, and exposed to 254 nm light for 8
seconds (total energy of 27.2 J/m2). This dose of UV was chosen
because, in our preliminary experiments, it caused complete inhibition of UV-DDB activity in 0.3 M-salt nuclear extracts from monkey
cells immediately after UV treatment.
For kinetic studies, normal (GM00011, GM01652, GM05757,
GM00037 and its SV40-transformed cell line, GM00637), CS-A
(CS3BE), XP-A (XP12BE), and XP-C (XP9BE) human fibroblasts
were obtained from the Human Genetic Mutant Repository, Camden,
NJ. The XP-A cell line XP12R0 and its revertant XP129 were
generous gifts from K. Valerie (MCV-VCU, Virginia) and J. Cleaver
(UCSF, California), respectively. Cells were grown and UV-irradiated
as above, except that medium was supplemented with 20% fetal
bovine serum and 2× MEM amino acids solution, and the following
UV doses were used: 12 J/m2 (normal human fibroblasts and CS3BE),
6 J/m2 (XP12BE, XP9BE and GM00637), and 3 J/m2 (XP12R0 and
XP129).
Preparation of nuclei and nuclear fractionation
TC-7 cells were harvested by trypsinization immediately after irradiation (UV 0-hours) or mock treatment. Nuclei were prepared from 57×108 cells as described previously (Tamiya-Koizumi et al., 1989).
Briefly, cells were swollen in 2 volumes of hypotonic solution containing 5 mM MgCl2, 1 mM NaHCO3, 1 mM phenylmethylsulfonyl
fluoride (PMSF), and disrupted in a Dounce homogenizer with 8-10
strokes of pestle A. The homogenate was immediately adjusted with
2 M sucrose to yield 0.25 M. Crude nuclei were collected by centrifugation at 800 g for 10 minutes. Following one wash with isotonic
buffer, the nuclear pellet was resuspended in 50 volumes of 1.7 M
sucrose, 5 mM MgCl2, and sedimented through a 1.7 M sucrose layer
at 52,000 g for 60 minutes. The purified nuclei were washed twice
with 0.25 M sucrose, 5 mM MgCl2. Nuclei obtained in this way were
further fractionated according to the modified procedure of Smith and
Berezney (1982). Nuclei were resuspended to 1.6 mg/ml DNA in 25
mM Tris-HCl, pH 7.4, 0.25 M sucrose, 5 mM MgCl2, 1 mM PMSF,
and digested with 50 units/ml bovine pancreatic DNAse I (USB,
Cleveland, OH) at 4°C for 16 hours. The nuclei were spun for 15
minutes at 1,000 g, the supernatant was saved, and the pellet extracted
3 times with low-salt buffer (10 mM Tris-HCl, pH 7.4, 0.2 mM
MgCl2, 1 mM PMSF) to yield soluble bulk chromatin and an insoluble
low-salt pellet. The pellet was consecutively extracted 3 times with
the same buffer as above, but with increasing concentrations of NaCl.
The resulting supernatants are referred to as 0.3 M, 0.5 M and 2.0 M,
respectively. To obtain the nuclear matrix, the high-salt pellet was
finally extracted with low-salt buffer, 1% Triton X-100 and washed
twice with buffer only. Supernatants from 3 extractions were
combined into a single sample. Extractions were done for 15 minutes
at 4°C by dropping magnetic stirrers into Eppendorf tubes containing
the samples. Centrifugations were for 15 minutes at 1,000 g after the
low-salt and 0.3 M-salt extractions, at 1,500 g after the 0.5 M-salt
extraction, and at 6,000 g after all subsequent extractions. The nuclear
matrix was dispersed by sonication using a Hert System sonicator
(Misonix Inc., Farmingdale, NY), at the maximum setting for 3 times
at 20 second intervals in low-salt buffer supplemented with 1 mM
EDTA, 1 mM DTT.
Whole-cell and nuclear extract preparation
For kinetic studies, human cells were harvested 0, 1, 3, 6, 24, and 48
hours after irradiation or mock treatment. Cells were washed with icecold PBS, scraped from the culture plates, and collected by centrifugation. Cell pellets were kept frozen at −80°C until preparation of
whole-cell extracts. They were then resuspended in an equal volume
of lysis buffer: 20 mM Hepes, pH 7.9, 25% glycerol, 420 mM NaCl,
Translocation of UV-DDB after UV 1161
1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT and a protease inhibitors
cocktail, freeze-thawed (dry ice/37°C water bath) 5 times, and
incubated on wet ice for 60 minutes with occasional mixing. The
samples were spun at 4°C for 10 minutes and the supernatant was used
as a whole cell extract. Protein concentration was determined by the
Bio-Rad microassay (Bio-Rad Laboratories, Hercules, CA) using
bovine serum albumin (BSA) as the standard.
For immunoprecipitation studies, the nuclear extract from mock or
UV 0-hour TC-7 cells was prepared as follows: cells were washed
once with solution A (10 mM Hepes, pH 7.9, 1.5 mM MgCl2, 10 mM
KCl, 1 mM DTT, 0.3 mM PMSF, 40 ng/ml leupeptin, 40 ng/ml
pepstatin, 40 ng/ml chymostatin), and homogenized in 2 volumes of
the same solution. Nuclei, recovered by centrifugation, were
incubated in lysis buffer (100 mM Tris-HCl, pH 8, 100 mM NaCl, 1%
Nonident P-40, 5 mM MgCl2, 0.3 mM PMSF, 40 ng/ml leupeptin, 40
ng/ml pepstatin, 40 ng/ml chymostatin) at 4°C for 15 minutes and
digested with 100 units/ml DNAse I at 37°C for 1 hour. The lysate of
1.2×107 nuclei/ml was adjusted to a final concentration of 1 mM
EDTA and clarified by centrifugation at 3,000 rpm at 4°C. The
resulting supernatant is referred to as nuclear lysate.
Immunoprecipitation and western analysis
Nuclear lysates and purified UV-DDB fractions (Takao et al., 1993),
solubilized in NENT (100 mM Tris-HCl, pH 8, 100 mM NaCl, 1%
Nonident P-40, 1 mM EDTA, 0.3 mM PMSF, 40 ng/ml leupeptin, 40
ng/ml pepstatin, 40 ng/ml chymostatin), were incubated with antip127 or anti-RP-A antibodies and immune complexes were absorbed
to Protein A Sepharose (Pharmacia Biotech Inc., Piscataway, NJ), and
Protein G agarose (Life Technologies Inc., Gaithersburg, MD),
respectively. The precipitates were washed four times with NENT,
resuspended in electrophoresis sample buffer, and analyzed by SDSPAGE.
Western analysis was done using chemiluminescent detection as
recommended by the manufacturer (Tropix, Bedford, MA). The
following antibodies and dilution factors were used: polyclonal antibodies anti-p127 (1:250) and anti-XPA (1:5,000; kindly provided by
R. Legerski, UT, Texas); monoclonal antibodies anti-PCNA (1:100;
clone PC10), anti-lamin B (1:100; clone 101-B7) and anti-actin
(1:200; clone JLA20), all from Oncogene Science Inc., Cambridge,
MA; anti-RP-A 34A and 70C (0.5 mg/ml, kindly provided by R.
Wood, ICRF, England; Kenny et al., 1990); goat anti-rabbit IgG (H+L)
AP-conjugate (1:20,000; Bio-Rad Laboratories, Hercules, CA); goat
anti-mouse AP-conjugate (1:10,000; Novagen, Madison, WI); and
goat anti-mouse IgM (1:15,000; Boehringer Mannheim Corp., Indianapolis, IN).
Polyclonal anti-p127 antibodies were affinity-purified by absorption to a PVDF strip containing the pure 40 kDa C-terminal domain
of the recombinant p127 protein (Takao et al., 1993), and elution with
100 mM glycine, pH 2.5, 150 mM NaCl, followed by neutralization
with 1 M Tris-HCl, pH 8.0.
DNA-binding assay
The band-shift assay of UV-DDB activity was carried out with a UVirradiated double-stranded 60mer DNA oligonucleotide (‘60/54’), and
protein/DNA complexes were separated on a 5% nondenaturing polyacrylamide gel as described previously (Hirschfeld et al., 1990;
Protić and Levine, 1993).
Quantitation
Photographic images of bands generated by the band-shift assay and
immunoblotting were measured by densitometry using the Image
program, version 1.51 (National Technical Information Service,
Springfield, VA) on an Apple Macintosh Quadra 800 computer, and
normalized per total amount of protein per fraction.
RESULTS
UV-DDB activity in fractionated nuclei after UVtreatment
To gain insight into the nature of the early inhibition of
extractable UV-DDB activity after UV treatment, we fractionated nuclei from mock- and UV-treated TC-7 cells into seven
components differing in salt and detergent solubility (Fig. 1A)
and assayed the extractable binding activity of each fraction.
The majority (~70%) of the UV-DDB activity in mock-treated
cells is present in low-salt and 0.3 M-salt supernatants (Fig.
1B, Unirradiated; lanes b and c). The rest of the activity is in
less soluble complexes and could be traced all the way to the
nuclear matrix which contains ~1% of the total binding activity
(lanes d to g). In contrast, the majority of the detectable UVDDB activity in nuclei from UV-irradiated cells was present in
the high-salt chromatin (Fig. 1B, UV-irradiated; lanes d and e),
while ~30% of the activity was in the low-salt fractions (lanes
A
Isolated nuclei
DNase I
(a)
Low salt
(b)
0.3 M salt
(c)
0.5 M salt
(d)
2.0 M salt
(e)
1% Triton X-100
(f)
Nuclear Matrix
(g)
Fig. 1. UV-DDB activity in fractionated nuclei after UV-treatment. (A) The procedure for fractionation of nuclei from unirradiated or UVirradiated TC-7 cells. (B) The band-shift assay of UV-DDB activity with unirradiated and UV-irradiated DNA probe and 3 µg of nuclear
protein; 1 ng of UV-DDB purified protein complex was run on the gel as a positive control.
1162 V. Rapić Otrin and others
20
UV-treated
0
60
fractions
UV-treated
Matrix
Triton
2.0M salt
mock
0.5M salt
0.3M salt
Low salt
UV-treated
DN-aze sup
Matrix
mock
Triton
20
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ase
I
Low
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0.3
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0.5
Ms
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2.0
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FRACTIONS
ACTIN (%/fraction)
Triton
Matrix
mock
2.0M salt
0.5M salt
0.3M salt
Low salt
DN-ase sup.
Matrix
Triton
2.0M salt
0.5M salt
Low salt
UV-treated
XPA (%/fraction)
60
0
60
40
20
0
LAMIN B (%/fraction)
40
mock
2.0M salt
60
UV-treated
40
0.5M salt
0
0.3M salt
20
40
0.3M salt
60
DN-ase sup.
(%/fraction)
0
mock
UV-treated
20
Low salt
20
mock
40
DN-ase sup
40
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p127 (%/fraction)
60
DN
Fig. 2. Translocation of repair and replication proteins into high-salt
chromatin after UV. (A) Fluorogram of UV-DDB (p127), lamin B
and actin after separation of proteins (40 µg) from each nuclear
fraction of unirradiated and UV-irradiated TC-7 cells on an 8% SDSPAGE, transfer to a PVDF membrane and probing with specific
antibodies. For immunodetection of PCNA (40 µg), RP-A /p34 (40
µg), and XPA (60 µg) proteins, the same nuclear fractions were
analyzed by 8%, 10% and 12% SDS-PAGE, respectively (B). For
identification of fractions, refer to Fig. 1A. (C) Fluorogram of each
protein was measured by densitometry as described in Materials and
Methods. Each bar represents a percentage of total nuclear protein
per fraction.
C
RP-A
The p127 component of UV-DDB translocates into
high-salt chromatin after UV
To test for UV-induced translocation of UV-DDB, we carried
out western analyses of the same fractions that had been tested
in the binding assay using anti-p127 antibodies (Fig. 2A). In
mock-treated cells, the fractions most abundant in p127 were
the low-salt and 0.3 M-salt supernatants, containing 76% of the
total protein (Fig. 2C). The Triton X-100 supernatant and the
nuclear matrix had <1% of the total protein (which was visible
only after longer film exposure; not shown). Such a distribution of p127 reflects the binding activity of the UV-DDB in
these nuclear fractions. In UV-treated cells, the p127 was again
found in all nuclear fractions despite the fact that UV-DDB
activity was primarily detected in the 2 M fraction (Fig. 1B).
However, the 2 M fraction from UV-treated cells (Fig. 2A, UVirradiated, lane e) appears to be enriched in p127 because it has
3- to 4-fold more p127 (~8% of the total nuclear p127; Fig.
2C) than the corresponding fraction from unirradiated cells.
Although only a small portion of the total nuclear p127 was
translocated into the 2 M-salt extractable structure, this result
was reproducible (three experiments) for different nuclear
PCNA (%/fraction)
a-c). Because the total DNA binding activity of UV-DDB in
UV-irradiated cells was only ~20% of that found in mocktreated cells, the in vitro band-shift assay could not reveal the
true redistribution of the UV-DDB protein within nuclei after
UV.
This loss of UV-DDB activity was not due to leakage into
the hypotonic cell extract, as we have seen with 5-10% of the
total nuclear activity from mock-treated cells, because examination of the cytosolic fraction from UV-irradiated cells did not
uncover any detectable UV-DDB activity (gels not shown). To
eliminate the possibility that this inhibition was primarily the
result of UV-DDB binding to lesions on genomic DNA present
in the extracted fractions, we tested the extracts in a modified
band shift assay performed at 37°C (which allows both association and dissociation of the protein complex with UVdamaged DNA). Under such conditions, UV-DDB activity in
irradiated cells slightly increased, but was still inhibited about
70% (gel not shown). Moreover, the total amount of genomic
DNA in each fraction does not positively correlate with the
extent of inhibition in the same fraction. In contrast, the 2 M
salt extract of irradiated cells contained most of the binding
activity (Fig. 1B, UV-irradiated; lane e) and also contained the
highest amount of genomic DNA (not shown). Several other
approaches, including replacement of DNase I treatment of
nuclei with sonication, and mixing of cytoplasmic and nuclear
extracts from untreated and UV-treated cells, did not affect the
inhibition of extractable UV-DDB activity by UV.
In DNaseI and Triton X-100 supernatants, from UV-irradiated or unirradiated cells, we detected two novel fast-migrating
binding activities specific for UV-damaged DNA, but these
activities have not been analyzed further (Fig. 1B, lanes a and
f).
Translocation of UV-DDB after UV 1163
preparations, and the shift in p127 distribution was observed
only when a gradual increase of salt was applied in the
procedure for fractionation of nuclei. In the same fractions, the
distribution of two control proteins, a cytoskeletal protein
(actin) and lamin B, did not change after UV-treatment (Fig.
2A and C). (Lamin B is a marker protein for the nuclear matrix,
and our immunoblotting detection reveals 55%, 30%, and 10%
of total cellular protein in the nuclear matrix, detergent soluble
and 2 M salt fractions, respectively; Fig. 2C.)
SDS-PAGE; the same membrane was cut into three strips, and
each strip was probed immunologically for a specific protein.
As shown in Fig. 3B, p127 was coimmunoprecipitated with
both p34 and p70 subunits of RP-A. Similarly, both RP-A
subunits were detected as coimmunoprecipitates of p127.
The presence of a specific interaction between UV-DDB and
RP-A was further confirmed by coimmunoprecipitation of
p127 from the nuclear lysate using anti-RP-A antibodies (Fig.
Translocation of nucleotide excision repair and
replication proteins into high-salt chromatin after UV
To examine whether the other members of the NER pathway
respond to UV as does UV-DDB (p127), we analyzed in
parallel the same nuclear fractions with antibodies against
XPA, RP-A (its two subunits, p34 and p70) and PCNA. We
chose to test for RP-A and XPA as marker proteins for the
recognition/incision step of NER (Robins et al., 1991;
Coverley et al., 1991; He et al., 1995; Li et al., 1995), and
PCNA for the repair synthesis step (Miura et al., 1992; Nichols
and Sancar, 1992; Shivji et al., 1992). All three proteins
showed a redistribution after UV (Fig. 2B and C). While the
fraction most enriched in PCNA was the 0.5 M-salt supernatant, translocated RP-A was mainly detected in the 2 M-salt
supernatant. Longer film exposure also revealed traces of RPA in the detergent-soluble fraction and the nuclear matrix, but
no difference in RP-A amounts was observed between the two
treatments (not shown). Similarly to p127, RP-A and PCNA
were enriched in the high-salt chromatin about 3-fold (Fig. 2B,
lanes d and e), which corresponds to 14-20% of their total
nuclear content (Fig. 2C).
In contrast, the distribution of XPA, a protein exclusively
localized in nuclei (Miyamoto et al., 1992), changed dramatically after UV. While in mock-treated cells, the DNase I supernatant contained all of the XPA, nuclear fractions from UVirradiated cells had a more uniform distribution of the protein
with ~50% of the total XPA present in a 0.5 and 2 M salt
extractable structure (Fig. 2B and C). However, the total
amount of XPA protein in nuclei of UV-treated cells was ~30%
higher when compared to the amounts found in unirradiated
cells (Fig. 2C). This difference may be due to leakage of the
protein since a faint XPA signal was detected in the corresponding cytosol from unirradiated cells (not shown).
UV-DDB interacts with RP-A in vitro and in vivo
The similar pattern of redistribution after UV treatment found
for p127 and RP-A (Fig. 2A,B and C) prompted us to examine
whether these two proteins interact specifically under our
experimental conditions. First, we did a western analysis of our
UV-DDB fractions that had been eluted from UV-irradiated
DNA cellulose during the process of UV-DDB purification
(Takao et al., 1993) and found that they contain the p70 and
p34 subunits of RP-A (Fig. 3A). (The presence of p11, the third
RP-A subunit (Kenny et al., 1992), has not been tested here.)
To eliminate the possibility that these proteins copurified only
as a result of binding to the affinity column used for purification, we tested for a specific interaction between p127 and RPA within the same fractions by coimmunoprecipitation
analysis. Purified UV-DDB fractions were first incubated with
affinity purified anti-p127, anti-p70 or anti-p34 antibodies;
bound proteins in immunoprecipitates were then analyzed by
Fig. 3. Interaction of UV-DDB/p127 with RPA in vitro and in vivo.
UV-DDB purified fractions from UV-irradiated TC-7 cells (lane 1),
mock-treated HeLa cells (lane 2) and purified RP-A protein from
HeLa cells (lane 3) were separated on a 4-12% SDS-PAGE with
Rainbow protein markers (Amersham Life Sci. Inc., Arlington
Heights, IL) and silver stained or immunoblotted with anti-p127,
anti-p34 RPA and anti-p70 RPA antibodies (A). The same UV-DDB
purified fractions were immunoprecipitated (IP) with control (Protein
A and Protein G), anti-p127, anti-p70, and anti-p34 antibodies,
resolved by SDS-PAGE and analyzed by fluorography (B). Nuclear
lysates from 1×107 TC-7 cells were immunoprecipitated with control
(Protein G), anti-p70 and anti-p34 antibodies. The precipitates were
immunoblotted (IB) with anti-p127, anti-p70, and anti-p34
antibodies. Nonspecific bands shown at the bottom of membranes are
the chains of immunoglobulin G (C).
1164 V. Rapić Otrin and others
3C). The amount of p127 coimmunoprecipitated with anti-p34
or anti-p70 antibodies was very low compared with the starting
material, and did not increase even after cells received a high
dose (100 J/m2) of UV (not shown), suggesting a constitutive
interaction of the two proteins in the cell. When a similar
analysis was performed using an anti-PCNA antibody, we did
not detect either p127 in immunoprecipitates or PCNA in our
UV-DDB purified fractions (not shown). Nonspecific trapping
of immunoblotted proteins was not detected in any of the
control lanes with Protein A or G only (Fig. 3B,C).
The interaction of UV-DDB and RP-A appears to
enhance binding of both proteins to UV-damaged
DNA
To test whether the specific interaction between UV-DDB and
RP-A affects binding of UV-DDB to UV-damaged DNA,
increasing amounts of purified RP-A were added to an RP-Aimmunodepleted UV-DDB fraction and binding to UVdamaged DNA was assessed by the band-shift assay. Purified
RP-A from HeLa cells bound to the UV-damaged DNA probe,
and the complex migrated to about the same position on the
gel as the UV-DDB/UV-DNA complex under our experimental conditions (Fig. 4, lanes 1 and 3). Addition of both proteins
to the binding mixture enhanced the signal of the protein/UVDNA complex about 5-fold and 3-fold when compared to UVDDB or RP-A binding alone, respectively (Fig. 4, lanes 1-3).
The enhanced binding is specific for UV-damaged DNA (lanes
14-16), and can be partly supershifted with anti-p70 antibodies
Fig. 4. A specific UV-DDB and RPA interaction appears to enhance
binding of both proteins to UV-damaged DNA. An RP-Aimmunodepleted UV-DDB fraction (0.1 ng) and RP-A (5 ng), alone
or in combination, were tested for their effect on binding activities to
a UV-damaged DNA probe in the absence (0) or presence (+UV, −UV)
of a 50-fold molar excess of unlabeled 60/54 DNA competitor, using
the band-shift assay. Under the same conditions, the effect of SSB
(0.05 ng) and BSA (1 ng) on UV-DDB binding was tested. For
supershifting, 100 ng of anti-p70 antibody was added to the binding
reaction.
(Fig. 4, lanes 5 and 6). Since the supershifted signal of the RPA binding was about 2-fold greater when UV-DDB was present
in the binding mixture, it appears that UV-DDB stimulates
binding of RP-A to UV-damaged DNA. The residual (nonsupershifted) binding was also enhanced (about 2.7-fold),
suggesting coordinate stimulation of UV-DDB binding by RPA. However, this interpretation is not certain because our antip127 antibodies do not supershift the UV-DDB (not shown)
and therefore the enhanced DNA binding cannot be assigned
to p127 alone.
In contrast to the above findings, the addition of E. coli
single-stranded DNA binding protein (SSB) (USB, Cleveland,
OH) or bovine serum albumin (BSA) (Sigma Chemical Co., St
Louis, MO) to the binding mixture did not affect the binding
of UV-DDB to UV-damaged DNA (Fig. 4, lanes 7-10), further
suggesting the specificity of the UV-DDB/RP-A interaction in
enhancing the binding of both proteins to UV-damaged DNA.
Recovery of UV-DDB activity after UV is delayed in
repair-deficient human cell lines
To further explore the dynamics of UV-DDB in NER, we
studied the kinetics of its binding activity within 48 hours
following UV exposure, in a variety of repair-deficient cell
lines. Four normal human fibroblast lines (Fig. 5A) demonstrated a kinetic profile which correlates well with our previous
results using monkey CV-1 cells (Hirschfeld et al., 1990); 6080% of extractable UV-DDB activity was lost upon UV
treatment but recovered to normal levels within 3 to 6 hours.
A similarly normal profile of recovery of the activity was
obtained with extracts from CS-A fibroblasts, a cell type that
is defective in preferential repair of a subset of DNA lesions
in transcriptionally active genes (van Hoffen et al., 1993;
Henning et al., 1995); UV-DDB activity was inhibited 50%
immediately after UV, but almost complete recovery occurred
by 6 hours (Fig. 5B).
UV radiation is highly cytotoxic to cells from most XP
groups due to low levels of their NER (Friedberg et al., 1995;
Ma et al., 1995); therefore, instead of the 12 J/m2 dose that was
used for normal and CS-A fibroblasts, here we used a 2- to 4fold lower fluence of UV. At these lower doses, normal cells
showed slight or no inhibition of UV-DDB activity (not
shown). XP-A cells, which have severe defects in repair either
in the bulk DNA or in active genes (Miura et al., 1992; Evans
et al., 1993; Wood, 1996), had 50% UV-DDB activity at UV
0-hours that declined to 30% by 6 hours post-treatment (Fig.
5B). With longer culturing times of XP12BE cells, we either
saw no further change in this extractable activity or a very slow
but incomplete recovery. The UV-DDB activity profile for XPC fibroblasts, a cell type that is defective in repair of bulk DNA
but proficient in repair of transcriptionally active genes
(Venema et al., 1991; Evans et al., 1993; van Hoffen et al.,
1995), was similar to that found for XP-A cells: UV-DDB
activity was at near normal levels (~80%) early after UV
treatment, but then dropped dramatically to ~10% by 6 hours.
Further recovery of the activity was slow and incomplete by
48 hours (Fig. 5B).
We also tested XP129, a UV-resistant revertant of the SV40immortalized XP-A cell line XP12RO (Cleaver et al., 1987).
The XP12RO cells contain a termination codon, resulting in no
detectable level of XPA protein, and no evidence of removal
of 6-4 PD and CPD, although a very low level of repair
Translocation of UV-DDB after UV 1165
delayed by 48 hours (Fig. 5B,C), about 60% of the activity was
recovered in XP12RO cells (in contrast to 30% in XP12BE).
We do not have an explanation for this difference between the
two XP-A lines; although different mutations have been identified in the XPA gene in these two cell lines, neither line
expresses XPA mRNA, and they both have negligible residual
repair (States and Myrand, 1996; McDowell et al., 1993).
A
% Activity
100
Normal
GM01652
GM05757
GM00037
10
DISCUSSION
50
40
30
20
10
0
GM00011
B
% Activity
100
XP12BE (XP-A)
10
XP9BE (XP-C)
50
40
30
20
10
0
CS3BE (CS-A)
C
% Activity
100
XP12RO (XP-A)
10
50
40
30
20
10
0
XP129 (XP-A
rev.)
GM00637 (normal)
Hours
Fig. 5. Recovery of UV-DDB activity after UV in normal and repairdeficient human cell lines. Whole cell extract proteins (3 µg) from
human fibroblasts: normal (A), XP-A, XP-C and CS-A (B), and XPA revertant XP129, its parental cells XP12RO, and a normal SV40transformed cell line GM00637 (C), were tested for the presence of
extractable UV-DDB activity by the band-shift assay within the
course of 48 hours after UV treatment. Activity curves were plotted
as the log of percentage activity of treated cells compared to mocktreated cells vs hours post-treatment.
synthesis was detected. The reversion of the parental mutation
in XP129 allows the cells to make a reduced amount of full
length XPA protein, and might also contribute to its altered
substrate specificity; the revertant shows normal repair of 6-4
PD and normal repair synthesis (Jones et al., 1992; McDowell
et al., 1993). In contrast to the delayed recovery of UV-DDB
activity in the parental line, which has also been seen with
XP12BE cell lines, the revertant’s activity profile is similar to
that of the corresponding SV40-transformed normal cell line
GM00637 (Fig. 5C). Even though recovery of UV-DDB
activity in both XP-A cell lines, XP12RO and XP12BE, was
UV-induced translocation of UV-DDB and other
proteins involved in NER in the nuclei of mammalian
cells
We have studied the nuclear dynamics of UV-DDB and several
other NER and DNA replication proteins before and immediately after UV treatment of mammalian cells. Following irradiation, the p127 component of UV-DDB, as well as XPA, RPA and PCNA, were translocated to different extents within the
nuclei from low- to high-salt chromatin, suggesting that these
proteins move to a tight chromatin association, presumably
with UV-damaged DNA. In contrast to the dramatic translocation of XPA, which involved more than 50% of the total nuclear
protein, only a small fraction of p127 was translocated to the
high-salt chromatin (Fig. 2A,B,C), a result that could possibly
be attributed to the different extent of involvement of these
proteins in NER. XPA has binding affinity for a broad spectrum
of DNA lesions (Jones and Wood, 1993), and is a key protein
for assembling the NER complex (Matsuda et al., 1995; He et
al., 1995; Li et al., 1995). So far, a role in assembly is the only
known biological function of this protein. Therefore, it is to be
expected that virtually all of this protein will be recruited to
chromatin after UV damage, as is demonstrated in this study
(Fig. 2). UV-DDB is an abundant cellular protein complex with
high affinity for 6-4 PD in vitro (Abramić et al., 1991; Reardon
et al., 1993), but the role of UV-DDB in NER has yet to be
demonstrated clearly, even though the data here and elsewhere
suggest that such a role is likely. At our UV doses, 6-4 PD
represent only about 25% of the total UV-induced DNA lesions
(Friedberg et al., 1995) and hence the requirement for the
protein would be less in NER. The amount of p127 that we
have observed to translocate into high-salt chromatin after UV
(Fig. 2A) correlates with the results of Keeney et al. (1994),
who restored the normal level of UDS in XP-E cells that lack
UV-DDB activity by microinjecting less than 10% of the
normal cellular content of UV-DDB. Park et al. (1996) reported
that by using indirect immunofluorescence detection, the core
NER protein XPG also shows dynamic regulation of its
intranuclear distribution within 2 hours after a moderate UVC dose (10 J/m2).
RP-A and PCNA, besides their roles in NER (Coverley et
al., 1991; Shivji et al., 1992 and 1995), are involved in other
cell processes (Kenny et al., 1990; Hurwitz et al., 1990); thus,
only a fraction of these proteins (14-20%) is moved into a
tightly bound compartment after UV (Fig. 2). This result is
consistent with previous reports that an increased association
of PCNA with nuclei occurs after UV irradiation of nonreplicating DNA in normal human fibroblasts; only a small proportion of PCNA is tightly bound to the nucleus (Toschi and
Bravo, 1988). The immunostaining of PCNA in various XP cell
lines after UV irradiation correlates with their capacity for
1166 V. Rapić Otrin and others
incision of damaged DNA (Miura et al., 1992; Aboussekhra
and Wood, 1995). Taken together, these findings support the
notion that only a fraction of PCNA and RP-A, as well as UVDDB proteins, tightly associated with chromatin after UV, are
involved in NER. The similar shift of p127 to that of RP-A and
PCNA could be attributed to UV-DDB having an accessory
role in NER (Aboussekhra et al., 1995), but another role(s) in
other cell processes.
The increased levels of UV-DDB/p127, XPA, RP-A and
PCNA associated with high-salt chromatin after UV treatment
were due to redistribution of these proteins within the nuclei
because the total amount of each nuclear protein, with the
exception of XPA, was similar whether extracted before or
after irradiation (Fig. 2C). Much data, especially from studies
of in vitro interactions of eukaryotic NER proteins, suggest that
a partial (He et al., 1995; Nagai et al., 1995) or complete
‘repairosome’ (the protein complex essential for NER) exists
constitutively in the absence of damaged DNA (Svejstrup et
al., 1995). Consistent with this evidence, our findings with the
subset of proteins that likely participate in the formation of a
repairosome in vivo suggest that upon irradiation of
mammalian cells, such a repairosome becomes translocated
from a loose to tight association with genomic DNA.
The distribution of UV-DDB activity within nuclear
fractions (Fig. 1B) correlated with our initial observation
(Protić et al., 1989; Hirschfeld et al., 1990) that only a trace of
UV-DDB activity could be detected in low- and 0.3 M salt
extracts after UV-treatment. Although our immunoblotting
data confirmed that we recovered all p127 from the nuclei of
irradiated cells (Fig. 2), 80% of control UV-DDB activity
remained inhibited (Fig. 1B). At present, we cannot exclude a
role for the p48 subunit of UV-DDB (Keeney et al., 1993, 1994;
Nichols et al., 1996), nor a role for RP-A, since we found that
UV-DDB and RP-A specifically interact in vivo (Fig. 3C).
Further studies are needed to elucidate the mechanism of inhibition of UV-DDB activity, specifically related to UV, and its
importance for NER. However, ionizing radiation that does not
create high-affinity binding sites for UV-DDB had no effect on
UV-DDB binding activity (V. Rapić Otrin and M. Protić,
unpublished data).
UV-DDB interaction with RP-A
Here we have uncovered for the first time a specific interaction
between the p127 protein component of UV-DDB and the p70
and p34 subunits of RP-A. These interactions were detected,
in vitro and in vivo, by coimmunoprecipitation from UV-DDB
purified fractions and nuclear lysates, respectively (Fig. 3B and
C). The protein-protein interactions occur constitutively in
vivo, and are not stimulated by UV treatment (Fig. 3C), which
may account for the unenhanced level of copurified RP-A in
the UV-DDB purified fraction from UV-irradiated cells,
compared to mock treated cells (Fig. 3A). This is also consistent with the notion that NER may proceed by interactions of
intermediate subassemblies. Most likely only a fraction of the
nuclear p127 and RP-A (8 and 20%) that is translocated into a
tight complex with chromatin after UV (Fig. 2), presumably
involved in NER, is also involved in this protein-protein interaction. The very low level of coprecipitation of p127 with RPA in vivo (Fig. 3C), and the fact that RP-A-immunodepletion
does not immunoprecipitate all p127 from the UV-DDB
purified fraction (this fraction still exhibits UV-DDB binding
activity, Fig. 4, lane 1), support this notion. In fact, we cannot
eliminate the possibility that the interaction between UV-DDB
and RP-A is mediated by the p48 component, especially since
the UV-DDB fractions used contain other protein bands (Fig.
3A).
RP-A is primarily a single-stranded DNA-binding protein
(Kenny et al., 1990), but it also binds to bulky lesions like
cisplatin- (Clugston et al., 1992), AAAF- (He et al., 1995), and
UV-damaged DNA (Burns et al., 1996). We also detected preferential binding of RP-A to UV-damaged versus undamaged
DNA (Fig. 4, lanes 17-19). However, human RP-A purified
from HeLa cells, which we used, has higher binding activity
than an RP-A purified from E. coli; we obtained saturation of
binding to a UV-damaged-DNA probe with 10 ng of RP-A (not
shown), while 70-100 ng of an RP-A fusion protein were
required (Burns et al., 1996). Although UV-DDB itself has
high affinity for 6-4 PD, our results suggest that the combination of the two interacting proteins enhanced the binding to
UV-damaged DNA shown by either protein alone (Fig. 4). At
this point, we can only speculate that the RP-A/UV-DDB interaction leads to some conformational change in one or both
proteins that results in its/their enhanced binding affinity for
damaged DNA, which would be consistent with our earlier
finding that the UV-DDB monomer is an inactive form of UVDDB protein with respect to complexing with damaged DNA
(Abramić et al., 1991; Takao et al., 1993). Substitution of
human RP-A for SSB protein does not affect the binding of
UV-DDB to UV-damaged DNA, further supporting the significance of the UV-DDB/RP-A interaction for enhancing the
binding to UV-damaged DNA. Additional evidence favoring
the role of this interaction in NER awaits a more detailed cellfree reconstruction analysis.
Several groups have shown that XPA binds specifically to
the p70 and p34 subunits of RP-A, both in vitro and in vivo
(Matsuda et al., 1995; He et al., 1995), although only the interaction with p70 seems to be essential for NER (Li et al., 1995).
The interaction enhances the otherwise low affinity of XPA for
damaged DNA (Jones and Wood, 1993), and provides a mechanistic basis for the role of RP-A in the initial step of NER.
This similarity of UV-DDB/RP-A to XPA/RP-A interactions
supports our earlier hypothesis that UV-DDB has a role in the
early damage-recognition step of NER.
Kazantsev et al. (1996), reported that a high concentration
of RP-A can correct the in vitro repair defect of XP-E cell
extracts, but found no RP-A mutations in an XP-E cell line that
lacked UV-DDB binding activity. The authors propose that the
XPE gene product acts as a molecular chaperone to aid in the
assembly of RP-A with the other components of the DNAbound repairosome; given a high enough level of RP-A, XPE
would be unnecessary. The specific UV-DDB/RP-A interaction
which we reported in this paper does not exclude this notion,
but the fact that RP-A itself binds to 6-4 PD in vitro (Burns et
al., 1996), could account for the observed complementation.
UV-DDB activity in normal and repair-deficient
human cell lines
We have found previously that the kinetics of recovery of UVDDB activity measured in cell extracts in vitro positively correlates with the kinetics of NER in vivo (Hirschfeld et al., 1990;
McLenigan et al., 1993). Now we have extended this analysis
in order to determine whether a correlation can be found
Translocation of UV-DDB after UV 1167
between the kinetics of UV-DDB recovery and a specific type
of NER. As seen before with monkey cells (Hirschfeld et al.,
1990), normal human fibroblasts recover UV-DDB activity 36 hours after UV treatment, the time needed for repair of 6-4
PD in primate cells (Friedberg et al., 1995). In contrast, cells
deficient in some aspect of NER showed two patterns of
recovery of UV-DDB activity: XP-A and XP-C cells had
delayed and incomplete recovery of the binding activity even
48 hours post-irradiation, while CS-A cells and an XPA
revertant line, XP129, expressed a pattern similar to that of
normal human fibroblasts.
The absence of a functional XPA protein alters the recovery
of UV-DDB activity after UV (Fig. 5B and C), suggesting that
UV-DDB might cooperate with XPA in recognizing 6-4 PD.
Additional evidence may be found in the XP-A revertant
(XP129) cells, which have acquired the ability to repair 6-4 PD
in the genome overall and CPD to a certain extent, but only in
the transcribed strand of active genes (Cleaver et al., 1987;
Jones et al., 1992; Lommel and Hanawalt, 1993), and have
normal recovery of UV-DDB activity (Fig. 5C). Furthermore,
when XP-A is the limiting factor, as in XP129 cells, the
presence of UV-DDB might promote the repair of 6-4 PD in
preference to other photoproducts. The cooperative efforts of
XPA and UV-DDB proteins may explain the greater efficiency
at which 6-4 PD are repaired when both proteins are present
in an in vitro reconstituted NER assay (Aboussekhra et al.,
1995). As with XP-A cell lines, the fact that the absence of a
functional XPC protein, which is involved in the repair of bulk
genomic DNA and in the repair of the nontranscribed strand of
active genes (Evans et al., 1993; Venema et al., 1991; van
Hoffen et al., 1995), affects the normal profile of the UV-DDB
binding recovery (Fig. 5B), offers indirect evidence that UVDDB may be involved in repair of bulk DNA. Further evidence
in support of UV-DDB involvement in efficient removal of 64 PD from the genome overall is the normal pattern of recovery
of UV-DDB activity in CS-A cells, which is most likely a
reflection of their normal repair of 6-4 lesions in the bulk
genome (Parris and Kraemer, 1993).
We thank Dr Richard Wood for providing us with purified RP-A
and for useful discussions throughout this work.
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(Received 6 November 1996 – Accepted 11 March 1997)