BRCA1 contributes to transcription-coupled repair of DNA damage through polyubiquitination and degradation of Cockayne syndrome B protein.
ABSTRACT BRCA1 is an important gene involved in susceptibility to breast and ovarian cancer and its product regulates the cellular response to DNA double-strand breaks. Here, we present evidence that BRCA1 also contributes to the transcription-coupled repair (TCR) of ultraviolet (UV) light-induced DNA damage. BRCA1 immediately accumulates at the sites of UV irradiation-mediated damage in cell nuclei in a manner that is fully dependent on both Cockayne syndrome B (CSB) protein and active transcription. Suppression of BRCA1 expression inhibits the TCR of UV lesions and increases the UV sensitivity of cells proficient in TCR. BRCA1 physically interacts with CSB protein. BRCA1 polyubiquitinates CSB and this polyubiquitination and subsequent degradation of CSB occur following UV irradiation, even in the absence of Cockayne syndrome A (CSA) protein. The depletion of BRCA1 expression increases the UV sensitivity of CSA-deficient cells. These results indicate that BRCA1 is involved in TCR and that a BRCA1-dependent polyubiquitination pathway for CSB exists alongside the CSA-dependent pathway to yield more efficient excision repair of lesions on the transcribed DNA strand.
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ABSTRACT: Nucleotide excision repair (NER) removes a wide variety of helix distorting DNA lesions. NER comprises two damage recognition sub-pathways: GG-NER operates genome wide, whereas TC-NER specifically removes transcription-blocking lesions from the transcribed strand of actively transcribed genes. NER is a multistep process, which requires the concerted action of 30 proteins that need to be tightly controlled at the right time and place for efficient repair. Post translational protein modifications (PTMs) are common regulators of complex protein networks. Several NER factors were shown to be modified by ubiquitin, whereas others are actively involved in the ubiquitin-proteasome system itself. PTMs by ubiquitylation can be swiftly induced in a reversible manner and have the ability to regulate protein function, localization or stability. This makes the regulation by ubiquitin highly suitable for the coordination of the complex NER reaction. Accumulating evidence showed, including proteome wide quantitative proteomics approaches that especially NER factors involved in the damage recognition are regulated by ubiquitin, emphasizing the high level of regulation during the initiation of the NER reaction. In this review we will therefore focus on the different functions of ubiquitylation during the DNA damage recognition steps of NER.Experimental cell research. 07/2014;
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ABSTRACT: A quarter of a century has elapsed since the discovery of transcription-coupled repair (TCR), and yet our fascination with this process has not diminished. Nucleotide excision repair (NER) is a versatile pathway that removes helix-distorting DNA lesions from the genomes of organisms across the evolutionary scale, from bacteria to humans. TCR, defined as a subpathway of NER, is dedicated to the repair of lesions that, by virtue of their location on the transcribed strands of active genes, encumber elongation by RNA polymerases. In this review, we will report on newly identified proteins, protein modifications, and protein complexes that participate in TCR in Escherichia coli and in human cells. We will discuss general models for the biochemical pathways and how and when cells might choose to utilize TCR or other pathways for repair or bypass of transcription-blocking DNA alterations.DNA repair 04/2014; · 4.20 Impact Factor
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ABSTRACT: The Javanese medaka, Oryzias javanicus, is a fish highly adaptable to various environmental salinities. Here, we investigated the effects of the environmental pollutant bisphenol A (BPA; an endocrine disrupting chemical) on gene expression levels in this species acclimated to different salinities. Using cDNA microarrays, we detected the induction of differential expression of genes by BPA, and compared the transcriptional changes caused by chemical exposure at different salinities. There were marked transcriptional changes induced by BPA between treatments. While 533 genes were induced by a factor of more than two when O. javanicus was exposed to BPA in seawater, only 215 genes were induced in freshwater. Among those genes, only 78 were shared and changed significantly their expression in both seawater and freshwater. Those genes were mainly involved in cellular processes and signaling pathway. We then categorized by functional group genes specifically induced by BPA exposure in seawater or freshwater. Gene expression changes were further confirmed in O. javanicus exposed to various concentrations of BPA, using quantitative real-time reverse transcription PCR based on primer sets for 28 selected genes.Marine Drugs 01/2014; 12(2):983-98. · 3.98 Impact Factor
BRCA1 contributes to transcription-coupled repair of
DNA damage through polyubiquitination and
degradation of Cockayne syndrome B protein
Leizhen Wei,1,2,8Li Lan,3Akira Yasui,3,4Kiyoji Tanaka,5Masafumi Saijo,5Ayako Matsuzawa,1Risa Kashiwagi,1
Emiko Maseki,1Yiheng Hu,6Jeffrey D. Parvin,6Chikashi Ishioka2and Natsuko Chiba1,7
Departments of1Molecular Immunology,2Clinical Oncology and3Molecular Genetics, and4Division of Dynamic Proteome in Aging and Cancer, Institute of
Development, Aging and Cancer, Tohoku University, Sendai;5Human Cell Biology Group, Graduate School of Frontier Biosciences, Osaka University, Osaka;
6Department of Biomedical Informatics and the Ohio State University Comprehensive Cancer Center, Ohio State University, Columbus, Ohio, USA
(Received April 27, 2011 ⁄ Revised July 7, 2011 ⁄ Accepted July 9, 2011 ⁄ Accepted manuscript online July 14, 2011 ⁄ Article first published online August 18, 2011)
BRCA1 is an important gene involved in susceptibility to breast
and ovarian cancer and its product regulates the cellular response
to DNA double-strand breaks. Here, we present evidence that
BRCA1 also contributes to the transcription-coupled repair (TCR) of
ultraviolet (UV) light-induced DNA damage. BRCA1 immediately
accumulates at the sites of UV irradiation-mediated damage in cell
nuclei in a manner that is fully dependent on both Cockayne syn-
drome B (CSB) protein and active transcription. Suppression of
BRCA1 expression inhibits the TCR of UV lesions and increases the
UV sensitivity of cells proficient in TCR. BRCA1 physically interacts
with CSB protein. BRCA1 polyubiquitinates CSB and this polyubiq-
uitination and subsequent degradation of CSB occur following UV
irradiation, even in the absence of Cockayne syndrome A (CSA)
protein. The depletion of BRCA1 expression increases the UV sensi-
tivity of CSA-deficient cells. These results indicate that BRCA1 is
involved in TCR and that a BRCA1-dependent polyubiquitination
pathway for CSB exists alongside the CSA-dependent pathway to
yield more efficient excision repair of lesions on the transcribed
DNA strand. (Cancer Sci 2011; 102: 1840–1847)
breast and ovarian cancers(3,4)and its expression in these can-
cers is often reduced,(5)suggesting that BRCA1 plays a role in
both hereditary and sporadic carcinogenesis. BRCA1 contains a
RING domain at the amino (N)-terminus and two BRCA1 car-
boxy-terminal (BRCT) domains at the carboxy (C)-terminus.
RING domain is an essential component of many ubiquitine E3
ligase. BRCA1 associates with BARD1, which also has a RING
domain,(6)and the BRCA1⁄BARD1 heterodimer has ubiquitin
BRCA1 has been implicated in a variety of biological pro-
cesses, including DNA repair, transcription, chromatin remodel-
ing and centrosome duplication.(10)BRCA1 localizes to nuclear
foci during S-phase of the cell cycle.(11)Various mediators of
DNA damage such as ultraviolet (UV) irradiation disperse the
BRCA1 foci, followed by the reappearance of BRCA1 foci.(12)
BRCA1 is phosphorylated in response to UV-induced dam-
age.(13)BRCA1 associates with RNA polymerase II (RNA-
PII)(14)and mediates the ubiquitination of RNAPII following
The main type of DNA damage induced by UV irradiation is
the formation of cyclobutane pyrimidine dimers (CPD) and (6-4)
photoproduct adducts. These lesions are removed by nucleotide
excision repair (NER). The NER operates via two pathways:
transcription-coupled repair (TCR) and global genome repair
(GGR). The TCR efficiently removes DNA lesions on the tran-
scribed strands of transcriptionally active genes, whereas the
RCA1 is an important breast and ovarian cancer suscepti-
bility gene.(1,2)BRCA1 mutations are rare in sporadic
GGR repairs DNA lesions throughout the genome. A stalled
RNAPII is presumed to trigger the initiation of TCR in harmony
with Cockayne syndrome (CS) proteins. Xeroderma pigmento-
sum (XP) and CS are rare genetic disorders. Xeroderma pigmen-
tosum is characterized by a high incidence of skin cancer and CS
is characterized by photosensitivity and neurodevelopmental
abnormalities.(18,19)There are seven genes (XPA–G) involved in
XP and two genes (CSA and CSB) involved in CS.(18,20)Muta-
tions in XPA–G result in defects in both GGR and TCR, with the
exception of XPC and XPE, which are defective in GGR alone.
Patients with CS have defects in TCR, but have functional GGR.
Although BRCA1 is known to function in the repair of DNA
double-strand breaks (DSB),(21)there have been several reports
suggesting roles for BRCA1 in the excision repair of DNA dam-
age. BRCA1 has been reported to function in NER of oxidative
DNA damage(22)and in TCR.(23,24)BRCA1-deficient cells are
defective with respect to the preferential removal of oxidative
base damage from the transcribed DNA strand.(25)These suggest
that BRCA1 participates in the TCR pathway. BRCA1 mutations
or reduced expression of BRCA1 might result in the deficiency
of TCR, in addition to DSB repair, and cause an increase in
cancer risk and contribute to carcinogenesis.
The aim of the present study was to gain insight into the
mechanisms involved in the BRCA1-mediated regulation of
TCR. Small, restricted areas of cell nuclei were exposed to UV
irradiation using an isopore membrane filter and BRCA1 locali-
zation was analyzed. The results showed the immediate, Cocka-
yne syndrome B (CSB)-dependent accumulation of BRCA1 at
the UV-irradiated sites. A suggested mechanism for BRCA1
function in TCR is also presented.
Materials and Methods
Plasmid construction. pCMV-Myc-ubiquitin and pcDNA3-
HA-BRCA1 have been described previously.(15,26)pcDNA3-
HA-BRCA1-I26A was generated by site-directed mutagenesis.
Cell lines and transfections. Saos-2, HEK-293T, XP3BRSV,
XP12ROSV, XP4PASV, CS3BESV, UVs1KOSV and HA-CSB⁄
UVs1KOSV cells were grown in Dulbecco’s modified Eagle’s
medium supplemented with 10% fetal bovine serum. HEK-293T
cells were transfected with the vectors using Fugene-6 (Roche,
Localized UVirradiation. Localized UV irradiation was
delivered as previously described.(27)Cells were cultured as
7To whom correspondence should be addressed.
8Present address: Genome Structure and Stability Group, Beijing Institute of
Genomics (BIG), No. 7, Beitucheng West Road, Chaoyang District, Beijing 100029,
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ª ª 2011 Japanese Cancer Association
monolayers in 35-mm glass-bottomed dishes (Matsunami Glass,
Osaka, Japan), covered with a polycarbonate isopore membrane
filter containing pores 3 lm in diameter (Millipore, Billerica,
MA, USA), and exposed to 254-nm UV irradiation at a dose of
out as previously described.(28)An anti-CPD antibody (Medical
& Biological Laboratories, Nagoya, Japan) and an anti-BARD1
antibody (H-300; Santa-Cruz Biotechnology, Santa Cruz, CA,
USA), a polyclonal anti-BRCA1 antibody specific for residues
397–1080 of BRCA1, or an anti-BRCA1 antibody (C-20; Santa-
Cruz Biotechnology) were used.
Small interfering RNA (siRNA). A siRNA targeting BRCA1
was synthesized using a Silencer siRNA construction Kit
(Ambion, Austin, TX, USA). The siRNA sequence was
5¢-AAGGUUUCAAAGCGCCAGUCA-3¢.(29)The Silencer neg-
ative control siRNA (Ambion) was used as a negative control.
Cells were transfected with siRNA using Lipofectamine RNAi-
MAX (Invitrogen, Carlsbad, CA, USA).
Immunoprecipitation and western blot. Immunoprecipitation
(IP) was carried out as previously described.(28)Total cell lysates
were prepared from CS3BESV cells in 1 · SDS sample buffer
(2% SDS, 0.67 M 2-Mercaptoethanol, 50 mM Tris–HCl pH 6.8,
12% glycerol, 1% Bromphenol Blue), sonicated and incubated at
95?C for 10 min. Samples were subjected to electrophoresis in
SDS-polyacrylamide gels and immunoblotted using anti-
BRCA1, anti-CSB (Santa-Cruz Biotechnology), anti-BARD1 or
anti-b-actin (Sigma-Aldrich, St. Louis, MO, USA) as indicated.
Colony formation assay. Cells were transfected with control
or BRCA1 siRNA. Forty-eight hours after transfection, the cells
were replated. Eight hours later, the cells were exposed to UV
irradiation and incubated for 10 days. Colonies were stained
with 0.3% crystal violet, and the number of colonies was
counted and expressed as a percentage of the non-irradiated
colonies as a measure of survival.
Analysis of strand-specific DNA repair. The repair of CPD was
examined in the 17.9-kb KpnI fragment within the dihydrofolate
reductase (DHFR) gene in XP4PASV cells transfected with con-
trol or BRCA1 siRNA and irradiated with 8 J⁄m2using a method
previously described.(30)Briefly, DNA was extracted, digested
with KpnI and treated with T4 endonuclease V, which generates
single-strand breaks at CPD sites. The samples were separated
by electrophoresis in 0.65% alkaline agarose gels, transferred
onto Hybond N+membranes (Amersham Biosciences, Little
Chalfont, Bucks, UK), and hybridized with strand-specific
digoxigenin (DIG)-labeled DNA probes. The strand-specific
probes were generated by linear PCR in the presence of DIG-11-
dUTP using a PCR DIG Probe Synthesis Kit (Roche). Hybrid-
ization with DIG-labeled strand-specific probes was detected
using a DIG Detection Kit (Roche).
In vitro ubiquitination assay. Reaction mixtures contained
10 mM HEPES (pH 7.6), 0.5 mM EDTA, 5 mM MgCl2, 2 mM
NaF, 2 mM ATP, 60 mM KCl, 14 lM ubiquitin (Sigma),
36 nM E1, 12 lM UbcH5c-His, 10 or 20 nM BRCA1-FLAG⁄
BARD1 and 24 nM CSB. The preparation of E1, UbcH5c-His
and BRCA1-FLAG⁄BARD1 has been described previously.(31)
CSB was prepared as described previously.(32)After incubation
at 37?C for 1 h, CSB modifications were analyzed using western
BRCA1 accumulates at UV-irradiated sites. To analyze the
response of BRCA1 to UV irradiation, cells covered with an iso-
pore membrane filter were irradiated to generate localized UV
damage to the cell nuclei.(27,33)Saos-2 cells were exposed to
localized UV irradiation and analyzed by co-immunostaining
with antibodies against CPD and BRCA1. Five and 30 min after
UV irradiation, BRCA1 was distributed as fine nuclear dots that
co-localized with CPD (Fig. 1a).
BRCA1 localizes to nuclear foci within a few hours after UV
irradiation, which was explained by the response of BRCA1 to
the DSB formed at the sites of stalled replication forks in
S-phase.(12)However, in asynchronous cells, BRCA1 accumula-
tion at UV-irradiated sites was observed in almost all cells. To
exclude the possibility that we were observing BRCA1 accumu-
lation at UV-induced DSB in S-phase, cells were synchronized
in G0⁄G1. BRCA1 clearly accumulated at the UV-irradiated
sites in cells at G0⁄G1 (Fig. S1a,b). In addition, we analyzed
whether phosphorylated H2AX (cH2AX), which is rapidly phos-
phorylated at DSB, was observed at irradiated sites. Even at a
higher dose of 100 J⁄m2, no cH2AX was detected at UV-irradi-
ated sites 10 min after irradiation (Fig. S1c). These indicate that
DSB are not induced immediately after UV irradiation under
these experimental conditions. Therefore, it can be concluded
that DSB do not induce immediate BRCA1 accumulation after
local UV irradiation.
BRCA1 accumulation at UV-irradiated sites is dependent on
CSB. To determine whether BRCA1 accumulation at UV-irradi-
ated sites depends on NER factors, the response of BRCA1 to
UV irradiation in several NER-deficient cell lines was exam-
ined. The cell lines used were: patient-derived XPG-deficient
XP3BRSV cells, XPA-deficient XP12ROSV cells, XPC-deficient
XP4PASV cells, CSA-deficient CS3BESV cells and CSB-defi-
cient UVs1KOSV cells. XPC is involved in the damage recogni-
tion step of GGR, whereas CSA and CSB function only in TCR.
XPA and XPG are required for both GGR and TCR, and func-
tion downstream of XPC, CSA and CSB (Fig. 1b). BRCA1
accumulated at UV-irradiated sites in almost all XPG-, XPA-,
XPC- and CSA-deficient cells as observed in Saos-2 cells, but
not in CSB-deficient cells (Fig. 1c). Accumulation of BRCA1 at
UV-irradiated sites was observed in a stable transfectant of
UVs1KOSV cells expressing full-length hemagglutinin (HA)-
tagged CSB (HA-CSB⁄UVs1KOSV).(34)The localization of
BRCA1 following irradiation was also examined in mouse
embryonic fibroblasts (MEF). BRCA1 accumulated at UV-irra-
diated sites in CSA-deficient (CSA)⁄)) 6L1030 cells, but not in
CSB-deficient (XPA+⁄) CSB)⁄)) cells (Fig. S2). This suggests
that BRCA1 accumulation at UV-irradiated sites is dependent
Inhibition of transcription abolishes BRCA1 accumulation at
UV-irradiated sites. Cockayne syndrome B plays an important
role in the initiation step of TCR through recognition of a stalled
RNAPII.(35)To investigate whether the response of BRCA1 is
dependent on active transcription, Saos-2 cells were treated with
actinomycin D or a-amanitin prior to UV irradiation. Treatment
with either chemical completely abolished the accumulation of
BRCA1 at the sites of irradiation (Fig. 1d). Although the expres-
sion of BRCA1 was slightly decreased by the treatment with
actinomycin D, expression of BRCA1 was clearly detected in
cells treated with these transcription inhibitors by western blot-
ting (Fig. S3). Thus, the accumulation of BRCA1 at the UV-
irradiated sites is dependent on transcription.
Depletion of BRCA1 impairs TCR but not GGR. To determine
whether the loss of BRCA1 expression affects TCR, the UV sen-
sitivity of GGR- and TCR-deficient cells transfected with
BRCA1 siRNA was examined. In XPC-deficient XP4PASV
cells, DNA lesions are repaired by TCR, but not by GGR,
whereas in CSB-deficient UVs1KOSV cells only the GGR path-
way is functional. BRCA1 siRNA efficiently suppressed the
expression of BRCA1 in these cells (Fig. 2a). Cells were irradi-
ated with varying doses of UV and their ability to form colonies
was assessed (Fig. 2b). When BRCA1 expression was reduced,
XP4PASV cells were more sensitive to UV irradiation. By
contrast, BRCA1 knockdown did not appear to affect the UV
sensitivity of UVs1KOSV cells.
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Strand-specific DNA probes were also used to examine the
removal of CPD from the transcribed and non-transcribed
strands of the active DHFR gene in XP4PASV cells transfected
with control or BRCA1 siRNA and irradiated. Knockdown of
BRCA1 expression reduced the efficiency of CPD removal from
the transcribed strand 6 h after UV irradiation (Fig. 2c). This
suggests that BRCA1 is important for efficient TCR and medi-
ates resistance to the UV lesion.
Association between BRCA1 and CSB is accompanied by
polyubiquitination. To obtain molecular insights into the role of
BRCA1 in TCR, the association of BRCA1 with CSB was
assessed. HEK-293T cells were UV irradiated and cell extracts
were prepared 1 h after exposure. The extracts were then
immunoprecipitated with a control IgG or anti-CSB antibody.
BRCA1 co-precipitated with CSB (Fig. 3a). In the anti-CSB
immune complexes, both BRCA1 and CSB were present as dif-
fuse, slowly migrating bands. Although BARD1 was also
detected in anti-CSB immune complexes, it did not show a dif-
fuse pattern. BARD1 accumulated at the UV-irradiated sites
indicated time points after localized UV irradiation and stained with anti-cyclobutane pyrimidine dimers (CPD) and anti-BRCA1 antibodies. (b)
Schematic of nucleotide excision repair (NER). (c) XP3BRSV XPG)⁄), XP12ROSV XPA)⁄), XP4PASV XPC)⁄), CS3BESV CSA)⁄), UVs1KOSV CSB)⁄)
and HA-CSB⁄UVs1KOSV cells were fixed 30 min after UV irradiation and then stained. (d) Saos-2 cells were treated with actinomycin D (Act D)
(10 lg⁄mL) or a-amanitin (100 lg⁄mL) for 1 h and then exposed to UV irradiation. Scale bars, 10 lm. CSA, Cockayne syndrome A; GGR, global
genome repair; TCR, transcription-coupled repair.
BRCA1 accumulation at ultraviolet (UV)-irradiated sites is dependent on Cockayne syndrome B (CSB). (a) Saos-2 cells were fixed at the
ª ª 2011 Japanese Cancer Association
Polyubiquitinated proteins show a diffuse pattern on western
blots, similar to the behavior observed for BRCA1 and CSB.
BRCA1 is ubiquitinated both in vitro and in vivo, whereas
CSB is ubiquitinated in vitro only.(36)To determine whether
CSB was polyubiquitinated in vivo, HEK-293T cells were
transfected with a vector expressing a Myc-tagged ubiquitin
(Myc-ubiquitin)(15)and then exposed to UV irradiation. Cell
lysates were immunoprecipitated with control IgG or anti-Myc
antibodies. The slowly migrating form of CSB was clearly pre-
cipitated by the anti-Myc antibody (Fig. 3b). Although these
slowly migrating bands were observed in non-irradiated cells,
the intensity of the bands was enhanced by UV irradiation
(Fig. 3c). These indicate that the slowly migrating form of CSB
is polyubiquitinated and that UV irradiation increases the poly-
ubiquitination of BRCA1 and CSB.
degradation after UV irradiation. Polyubiquitination is a signal
for proteasomal degradation. To determine whether the poly-
ubiquitinated BRCA1 and CSB were targeted for proteasomal
degradation following UV irradiation, HEK-293T cells were
incubated in the presence or absence of the proteasome inhibi-
tor, MG132, after UV irradiation (Fig. 3d). Treatment with
MG132 markedly increased the polyubiquitination of CSB, but
not BRCA1. This suggests that CSB is polyubiquitinated
and targeted for degradation after UV irradiation, whereas
ubiquitination of BRCA1 is unlikely to be coupled to degrad-
BRCA1 polyubiquitinates CSB and is involved in CSA protein-
independent resistance to UV irradiation. Cockayne syndrome
B is polyubiquitinated in vitro by an E3 ubiquitin ligase complex
containing CSA and degraded by a proteasomal pathway in a
CSA-dependent manner after UV irradiation.(36,37)In contrast, it
is reported that CSB expression is downregulated after UV irra-
diation even in CSA-deficient cells.(38)There might be another
pathway for the polyubiquitination and degradation of CSB.
Consistent with this expectation, polyubiquitination of CSB was
observed in CSA-deficient cells (Fig. 4a). Polyubiquitination of
CSB in BRCA1-knockdown cells was significantly lower than
that in cells transfected with the control siRNA (Fig. 4b). To test
whether the ubiquitin ligase activity of BRCA1 is involved in
the polyubiquitination of CSB, HEK-293T cells were transfected
with expression vectors for wild-type BRCA1 (HA-BRCA1) or
a BRCA1 mutant in which the ubiquitin ligase activity is abol-
ished (HA-BRCA1-I26A).(39,40)In BRCA1-I26A-transfected
cells, the polyubiquitination of CSB was markedly lower than
that in cells transfected with wild-type BRCA1 (Fig. 4c). These
suggest that the ubiquitin ligase activity of BRCA1 is involved
in the polyubiquitination of CSB.
Next, ubiquitination assays were performed to determine
whether BRCA1 directly ubiquitinates CSB in vitro. Purified
recombinant CSB protein was incubated with ATP, ubiquitin,
E1, UbcH5c and the BRCA1⁄BARD1 heterodimer and analyzed
using western blotting (Fig. 4d). In the complete reaction, poly-
ubiquitinated CSB was observed as slowly migrating diffuse
bands and the amount of polyubiquitinated CSB was propor-
tional to the amount of BRCA1⁄BARD1. This suggests that
CSB is a substrate for BRCA1⁄BARD1.
Next, to assess whether CSB is degraded following UV
irradiation in CSA-deficient cells, the amount of CSB was
examined in CSA-deficient cells following UV irradiation in the
presence of cycloheximide (CHX) (Fig. S5a). The amount of
CSB protein decreased after UV irradiation in the presence of
CHX in CSA-deficient cells. In contrast, CSB protein was not
downregulated after UV irradiation in the absence of CHX, as
previously reported.(36)The amount of CSB did not alter signif-
icantly after treatment with CHX alone (Fig. S5b). Treatment
with the proteasome inhibitor together with CHX prevented
downregulation of CSB after UV irradiation (Fig. S5c). To
examine whether BRCA1 was involved in the downregulation
of CSB, CSA-deficient cells were transfected with control or
BRCA1 siRNA and the amount of CSB was analyzed after UV
irradiation in the presence of CHX. Knockdown of BRCA1
suppressed the downregulation of CSB after UV irradiation in
the presence of CHX (Fig. 4e). The amount of BRCA1 in cells
transfected with control siRNA decreased following UV irradi-
ation in the presence of CHX, consistent with the report by
Hammond-Martel et al.(41)These suggest that CSB polyubi-
quitinated by BRCA1 is degraded via a proteasomal pathway
independent of CSA.
Finally, the effect of BRCA1 depletion on the UV sensitivity
of CSA-deficient cells was analyzed. BRCA1 knockdown
T4 endo V
UV dose (J/m2)
0.6 0.9 1.20.6
UV dose (J/m2)
repair (TCR). (a) XP4PASV and UVs1KOSV cells were transfected with
control or BRCA1 siRNA. Cell lysates analyzed using western blot with
anti-BRCA1 and anti-b-actin antibodies. (b) Colony formation assay for
XP4PASV and UVs1KOSV cells. XP4PASV and UVs1KOSV cells were
mean ± standarddeviationsof
**P < 0.01 versus the corresponding value for control siRNA. (c)
Removal of ultraviolet (UV)-induced CPD from the dihydrofolate
reductase (DHFR) fragments in XP4PASV cells immediately or 6 h after
UV irradiation. The DHFR fragments were analyzed using strand-
specific probes recognizing the transcribed (TS) or non-transcribed
siRNA knockdown of BRCA1 impairs transcription-coupled
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ª ª 2011 Japanese Cancer Association
increased the UV sensitivity of CSA-deficient cells, supporting a
role for BRCA1 in the TCR of UV lesions (Fig. 4f).
In the present study we showed that BRCA1 immediately
accumulated at locally UV-irradiated sites in a manner that is
dependent on CSB and transcription (Fig. 1). Although these are
highly suggestive of a role for BRCA1 in TCR, BRCA1 enhances
GGR through the transcriptional induction of XPC and DDB2.(42)
Therefore, we demonstrated that loss of BRCA1 affects the sensi-
tivity of TCR-proficient and GGR-deficient XPC-deficient cells
to UV irradiation, but not of GGR-proficient and TCR-deficient
CSB-deficient cells. Furthermore, the removal of CPD from tran-
scribed strands was suppressed by depletion of BRCAl. Thus, we
concluded that BRCA1 is involved in TCR following UV irradia-
tion and increases the survival of cells harboring UV damage.
GFP-tagged CSB protein accumulates in sub-nuclear areas at
sites of local UV damage.(43)The amount of CSB protein
increases in the chromatin fraction after UV irradiation.(44,45)
CSB was responsible for BRCA1 accumulation at UV-irradiated
sites. To examine whether BRCA1 moves to chromatin follow-
ing UV irradiation in a CSB-dependent manner, we fractionated
cell lysates from XP4PASV and UVs1KOSV cells into soluble
and chromatin-containing fractions (Fig. S6). The amount of
CSB protein within the chromatin-containing fractions from
XP4PASV cells, increased after UV irradiation. Consistent with
Figure 1c, BRCA1 was identified in the chromatin-containing
fraction from XP4PASV cells following UV irradiation, but not
in that from UVs1KOSV cells.
The CSB-dependent accumulation of BRCA1 at the UV
lesions is similar to that seen for other NER factors. Cockayne
syndrome A is translocated to the nuclear matrix in an UV- and
CSB-dependent manner,(46)and other NER proteins are recruited
to TCR sites in a CSB-dependent manner.(44)These suggest that
BRCA1 accumulates at UV-irradiated sites and functions in
TCR together with other NER factors, and that CSB is an inte-
gral factor for recruiting DNA repair factors to TCR sites.
The polyubiquitination of both BRCA1 and CSB was
enhanced after UV irradiation. Auto-ubiquitination of BRCA1
enhances its DNA-binding activity.(47)Enhanced polyubiquitina-
tion of BRCA1 following UV irradiation might be involved in its
function as a DNA repair molecule. However, CSB was poly-
ubiquitinated and processed for proteasomal degradation after
UV irradiation. This is consistent with a report that CSB is
degraded following UV irradiation.(37)As described above, the
amount of CSB increases in the chromatin fraction after UV irra-
diation. Lake et al.(45)reported that the amount of CSB
re-appearing in the soluble fraction 7 h after UV irradiation
decreases compared with the amount of CSB present before UV
irradiation. Cockayne syndrome B might be recruited to the chro-
matin and then polyubiquitinated for proteasomal degradation
after UV irradiation. Since BRCA1 was also recruited to the chro-
matin fraction after UV irradiation, BRCA1 might ubiquitinate
CSB at the chromatin. Phosphorylated RNAPII is also polyubiq-
uitinated by BRCA1 and targeted for proteasomal degradation
following UV irradiation.(15,16)BRCA1 might function in TCR
through the regulation of protein stability at sites of UV damage.
Depletion of BRCA1 increased UV sensitivity in CSA-defi-
cient cells (Fig. 4f), but not in CSB-deficient cells (Fig. 2b).
irradiation. (a) HEK-293T cells were treated with UV irradiation at a dose of 20 J⁄m2. One hour after exposure, cell lysates were subjected to
immunoprecipitation (IP) with control IgG or anti-CSB antibodies followed by western blotting with anti-BRCA1, anti-CSB or anti-BARD1
antibodies. (b) HEK-293T cells were transfected with Myc-ubiquitin and then UV irradiated. Lysates were subjected to IP using control IgG or
anti-Myc antibodies 1 h after UV irradiation. (c) Polyubiquitination of BRCA1 and CSB is enhanced by UV irradiation. (d) CSB polyubiquitination
is associated with proteasomal degradation after UV irradiation. HEK-293T cells were treated with or without 50 lM of MG132 after UV
Cockayne syndrome B (CSB) is associated with BRCA1 and polyubiquitinated for proteasomal degradation after ultraviolet (UV)
ª ª 2011 Japanese Cancer Association
This suggests that BRCA1 is involved in TCR for UV damage
in a CSB-dependent manner, but independent of CSA. Polyubiq-
uitination of CSB was observed in CSA-deficient cells. Cocka-
yne syndrome B is polyubiquitinated in a BRCA1-dependent
manner. Furthermore, CSB was polyubiquitinated by BRCA1⁄
BARD1 in vitro, similar to the effect of the CSA complex.
Although CSA and BRCA1 might play similar redundant roles
in the ubiquitination of CSB, the presence of two independent
pathways of CSB polyubiquitination might reflect different roles
for CSA and BRCA1 in TCR.
Clinical differences between CSA-deficient and CSB-deficient
patients have not been observed. However, CSA and CSB
have different functions in the response to oxidative damage.
CSB)⁄) MEF and keratinocytes are hypersensitive to oxidative
damage, but CSA-deficient cells are not(48). Cockayne syndrome
A functions in the response to oxidative damage, and CSA-defi-
cient cell extracts show normal oxidative damage cleavage
activity while CSB-deficient cell extracts do not.(49)This sug-
gests that downstream pathways that involve CSB exist, one of
which might be independent of CSA. Although we identified a
function for BRCA1 in TCR of UV lesions in the present study,
BRCA1 is also involved in the TCR of oxidative damage(25)and
DNA damage induced by ionizing irradiation(23,24). Therefore,
BRCA1 might also be involved in TCR of these DNA damage
independent of CSA. Additional studies are needed to gain
further understanding of the role played by BRCA1 in TCR
This study was supported by grants-in-aid from the Ministry of Educa-
tion, Science, Sports and Culture (N. C. and C. I.), The Naito Foundation
ultraviolet (UV)-irradiation. (a) CS3BESV cells were treated with UV irradiation. One hour after exposure, cell lysates were subjected to IP. (b)
HEK-293T cells were transfected with control or BRCA1 siRNA. One hour after exposure, cell lysates were subjected to immunoprecipitation (IP).
(c) HEK-293T cells were transfected with wild-type HA-BRCA1 or HA-BRCA1-I26A. One hour after exposure, cell lysates were subjected to IP. (d)
BRCA1 polyubiquitinates CSB in vitro. Long and short exposures of the same blot are presented to show that polyubiquitination of CSB is
dependent on the amount of BRCA1⁄BARD1 (10 or 20 nM). (e) CS3BESV cells were transfected with control or BRCA1 siRNA. Cells were pre-
treated with cycloheximide (CHX) for 1 h and UV irradiated. Cells were incubated with CHX and total cell lysates were prepared at the indicated
times for western blot with anti-CSB antibody. (f) Colony formation assay for CS3BESV cells transfected with control or BRCA1 siRNA. The cells
were UV irradiatied as indicated. Data represent the mean ± standard deviations of four independent experiments.
BRCA1 polyubiquitinates Cockayne syndrome B (CSB) and is involved in the Cockayne syndrome A (CSA)-independent resistance to
Wei et al.Cancer Sci|
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ª ª 2011 Japanese Cancer Association
(N. C.), The Ichiro Kanahara Foundation (N. C.), Suzuken Memorial
Foundation (N. C.), Osaka Cancer Research Foundation (N. C.) and a
genome network project grant from the Ministry of Education, Science,
Sports and Culture of Japan (A. Y.).
The authors have no conflict of interest.
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is inhibitedby BRCA1-dependent
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Additional Supporting Information may be found in the online version of this article:
Fig. S1. Immediate BRCA1 accumulation after local ultraviolet (UV) irradiation is not induced by double-strand breaks (DSB).
Fig. S2. Accumulation of BRCA1 at ultraviolet (UV) irradiated sites is dependent on Cockayne syndrome B (CSB).
Fig. S3. Expression of BRCA1 in Saos-2 cells treated with actinomycin D or a-amanitin.
Fig. S4. BARD1 accumulates at sites of ultraviolet (UV) irradiation.
Fig. S5. Cockayne syndrome B (CSB) protein is downregulated after ultraviolet (UV) irradiation in the presence of cycloheximide in Cockayne
syndrome A (CSA)-deficient cells.
Fig. S6. Ultraviolet (UV)-irradiation recruits Cockayne syndrome B (CSB) and BRCA1 proteins to the chromatin fraction.
Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries
(other than missing material) should be directed to the corresponding author for the article.
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