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Oncogene (2006) 25, 5864–5874
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REVIEW
The roles of BRCA1 and BRCA2 and associated proteins in the
maintenance of genomic stability
K Gudmundsdottir and A Ashworth
The Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, Fulham Road, London, UK
The BRCA1 and BRCA2 proteins are important in
maintaining genomic stability by promoting efficient and
precise repair of double-strand breaks. The main role of
BRCA2 appears to involve regulating the function of
RAD51 in the repair by homologous recombination.
BRCA1 has a broader role upstream of BRCA2,
participating in various cellular processes in response to
DNA damage. The DNA repair defect associated with
mutations in BRCA1 or BRCA2 could be exploited to
develop new targeted therapeutic approaches for cancer
occurring in mutation carriers.
Oncogene (2006) 25, 5864–5874. doi:10.1038/sj.onc.1209874
Keywords: BRCA1; BRCA2; repair; genomic stability;
DSS1
Pathways for the repair of DNA damage
DNA single-strand lesions
The accurate repair of damaged DNA is essential for the
maintenance of genomic integrity. The eukaryotic cell has
evolved several ways of repairing the multiple different
types of DNA damage that occur (Hoeijmakers,
2001). There are pathways that deal with the repair of
individual damaged bases and single-strand breaks
(SSBs), and distinct mechanisms that cope with the
potentially more severe consequences of double-strand
breaks (DSBs). Lesions affecting only one of the DNA
strands, where the intact complementary strand can be
used as a template for repair, are repaired by the baseexcision repair (BER), nucleotide-excision repair (NER)
or mismatch repair (MMR) pathways. Although partially overlapping, these pathways show preference for
specific DNA lesions.
DNA double-strand breaks
DNA DSBs are more problematic than SSBs since the
complementary strand is not available as a template for
repair. DSBs may arise as a result of either exogenous
Correspondence: Professor A Ashworth, The Breakthrough Breast
Cancer Research Centre, The Institute of Cancer Research, Fulham
Road, London SW3 6JB, UK.
E-mail: [email protected]
insults, such as exposure to ionizing radiation (IR), or
endogenous events, such as the collapse of replication
forks upon encountering a SSB. Three DSB repair
pathways have been identified within eukaryotic cells:
nonhomologous end-joining (NHEJ), gene conversion
(GC) and single-strand annealing (SSA). Both GC and
SSA rely on sequence homology for repair while NHEJ
utilizes no, or little, homology (van Gent et al., 2001;
Shin et al., 2004).
In NHEJ, the initial event involves the binding of the
Ku70/Ku80 heterodimer to broken DNA ends (Lieber
et al., 2003; Hefferin and Tomkinson, 2005). Two
heterodimers then come together to bridge matching
ends and recruit the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). The kinase activity of
DNA-PKcs is required for NHEJ but exactly why is not
clear, although the autophosphorylation of DNA-PKcs
appears to be important. A substrate of DNA-PKcs is
Artemis, a nuclease that forms a complex with DNAPKcs. Together they act as an endonuclease, which can
process the DNA ends preparing them for ligation by
XRCC4-Ligase IV. NHEJ is the predominant mechanism for the repair of DSBs during G0, G1 and early
S phases of the cell cycle, although it seems to be active
in all phases of the cell cycle (Takata et al., 1998;
Rothkamm et al., 2003). Traditionally, NHEJ has been
thought of as an error-prone mechanism of repair,
which frequently results in minor changes in the DNA
sequence at the break site and occasionally in the joining
of previously unlinked DNA molecules, resulting in
gross chromosomal rearrangements. However, in recent
years, it has become apparent that a precise subpathway
of NHEJ exists in eukaryotic cells alongside the more
error-prone pathway. The precise repair pathway is
thought to be Ku/DNA-PKcs dependent and is associated with minimal sequence modification at the break
site. The error-prone pathway of NHEJ, however, uses
microhomology flanking the break site to anneal and religate the broken ends and is thought to be dependent on
the MRE11-RAD50-NBS1 (MRN) complex (Ma et al.,
2003; Zhang et al., 2004).
GC uses a homologous sequence, preferably the sister
chromatid, as a template to resynthesize the sequence
surrounding the DSB and this pathway is therefore
generally thought to result in the accurate repair of the
break. Furthermore, crossover events are suppressed in
mammalian cells as these can result in genome alterations (Johnson and Jasin, 2000). Repair by GC is
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dependent upon the recombinase function of RAD51
and the assistance of various other proteins. The MRN
complex is thought to play a role in bringing the two
broken DNA ends together and the complex has also
been suggested to be involved in the nucleolytic
resection of the ends of the DSB. The resulting 30
overhangs become coated by RPA, which prevents the
formation of, and removes, secondary structures.
RAD51 forms a nucleoprotein filament on the ssDNA
and catalyses the invasion of the homologous sequence
on the sister chromatid, which is then used as a template
for accurately repairing the broken ends by DNA
synthesis. Recombination intermediates are then resolved, either with or without sequence exchange. Other
proteins implicated in assisting this process include
RAD54, the RAD51 paralogues and BRCA2 (van Gent
et al., 2001; West, 2003). Although RAD52 is essential
for GC in yeast, this does not seem to be the case in
mammalian cells (Stark et al., 2004). This pathway is
mainly active during the S and G2 phases of the cell
cycle following DNA replication, when a sister chromatid is available for use as a template for repair (Takata
et al., 1998; Rothkamm et al., 2003).
SSA also involves the use of homologous sequences
for the repair of DSBs. However, unlike GC, which
involves strand invasion, SSA is RAD51 independent
and involves the annealing of DNA strands formed after
resection at the DSB. The proteins and mechanisms
involved in SSA are still somewhat unclear (Valerie and
Povirk, 2003; Stark et al., 2004). Initially, DNA ends are
resected by an exonuclease, most likely the MRN
complex, to yield long single-strand overhangs that
RPA and RAD52 may bind to. Once homology is
exposed in the overhangs, they are annealed and the
protruding ends are trimmed by the ERCC1/XPF
nuclease and the gap is filled by DNA polymerase. This
pathway is error-prone as it results in the retention of
only one of the homologous sequences and deletion of
the intervening sequence. SSA is potentially an important pathway of mutagenesis as a large fraction of the
genome consists of repetitive elements (Lander et al.,
2001; Elliott et al., 2005). Both GC and SSA require
resection of DNA ends as an initial step in the repair
process and this strand resection has been shown to be
cell cycle regulated in yeast, being much reduced in G1
cells (Ira et al., 2004). Therefore SSA, like GC, is
thought to be active in S–G2 phases of the cell cycle
(Elliott et al., 2005).
BRCA1 and DSB repair
One of the earliest indications that BRCA1 was involved
in DNA repair was the observation that BRCA1
associates and colocalizes with RAD51 in nuclear foci
in mitotic cells (Scully et al., 1997b). These foci were also
observed to contain BRCA2 and the BRCA1-binding
protein BARD1, both before and after DNA damage
(Jin et al., 1997; Scully et al., 1997a; Chen et al., 1998).
Further evidence came from the observation that
BRCA1-deficient cells were highly sensitive to IR and
displayed chromosomal instability, with both numerical
and structural chromosome aberrations, which may be a
direct consequence of unrepaired DNA damage (Shen
et al., 1998; Xu et al., 1999). The similar genomic
instability in BRCA1- and BRCA2-deficient cells and
the interaction of both BRCA1 and BRCA2 with
RAD51 suggested a functional link between the three
proteins in the RAD51-mediated DNA damage repair
process. However, while BRCA2 is directly involved in
RAD51-mediated repair, affecting the choice between
GC and SSA (see below), BRCA1 acts upstream of these
pathways (Stark et al., 2004). Brca1 deficiency in mouse
embryonic stem cells leads to impaired repair of DSBs
by GC induced by the site-specific I-SceI endonuclease
(Moynahan et al., 1999, 2001a). The involvement of
BRCA1 in the repair of DSBs by GC is consistent with
its association and colocalization with RAD51 in
nuclear foci, and, accordingly, BRCA1 is required for
their formation (Bhattacharyya et al., 2000). DSB repair
by SSA is also reduced in BRCA1-deficient cells, placing
BRCA1 before the branch point of GC and SSA (Stark
et al., 2004). However, NHEJ has been reported to be
unaffected in BRCA1-deficient cells, although data
concerning the role of BRCA1 within this repair
pathway has been conflicting (Moynahan et al., 1999;
Baldeyron et al., 2002; Zhong et al., 2002; Stark et al.,
2004). This may, at least in part, be a reflection of
different roles of BRCA1 in the subpathways of NHEJ.
In support of this notion, BRCA1 has been shown
recently to be important in promoting precise NHEJ,
while inhibiting more error-prone microhomologymediated NHEJ (Wang et al., 2006; Zhuang et al.,
2006). Therefore, in addition to promoting error-free
repair of DSBs by HR, BRCA1 also reduces the
mutagenic potential of NHEJ, thereby contributing to
genomic stability.
BRCA1 is associated with other DNA repair functions
and complexes
BRCA1 has been associated with a variety of proteins
that are involved in the response to, or the repair of,
DNA damage. BRCA2 and RAD51 have been observed
to coexist in a complex with BRCA1 termed BRCC
(BRCA1-BRCA2-Containing Complex) that also contains BARD1 and other components (Dong et al., 2003).
This complex displays an E3 ubiquitin ligase activity,
which has been implicated in the regulation of factors
involved in DNA repair. However, BRCA2 and RAD51
do not appear to be present in another BRCA1containing complex termed BASC (BRCA1-Associated
Genome Surveillance Complex) (Wang et al., 2000).
This complex includes tumour suppressors, DNA
damage sensors and signal transducers, including the
MRN complex, the mismatch repair proteins MSH2,
MSH6 and MLH1, the Bloom syndrome helicase BLM,
the ATM kinase, DNA replication factor C (RFC) and
PCNA. Many of the proteins in this complex can bind
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Roles of BRCA1 and BRCA2 and associated proteins
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abnormal DNA structures and, therefore, have the
potential to act as sensors of DNA damage. Many also
function directly in DNA replication or DNA-replication associated repair. This suggests a role for BRCA1
in coordinating various functions of DNA replication
that are important for maintaining genomic integrity in
the cell. The identification of the MRN complex in
BASC provides further support for a role of BRCA1 in
the repair of DSBs. The MRN complex is thought to be
involved in the end-processing of DSBs in GC, SSA and
microhomology-mediated NHEJ. BRCA1 colocalizes
with the MRN complex in foci upon DNA damage and
it also inhibits the nucleolytic activity of MRE11 in vitro,
thereby potentially influencing the choice of repair
pathway after a DSB (Zhong et al., 1999; Wang et al.,
2000; Paull et al., 2001). The association of BRCA1 with
MSH2 and MSH6 in the BASC complex also links
BRCA1 to a subpathway of NER that preferentially
repairs base lesions from the transcribed strand, as these
two MSH proteins are known to be involved in this
process (Wang et al., 2000). Furthermore, BRCA1 is
required for the repair of oxidative 8-oxoguanine lesions
by transcription-coupled DNA repair, although the role
of BRCA1 in the repair of oxidative damage has been
subject to re-evaluation following the retraction of a
paper implicating BRCA1 within this pathway (Gowen
et al., 1998; Le Page et al., 2000). BRCA1 has also been
reported to enhance the global genomic repair (GGR)
subpathway of NER by inducing the expression of the
NER genes XPC, DDB2 and GADD45 (Harkin et al.,
1999; Hartman and Ford, 2002). An association of
BRCA1 with a complex that contains the proteins SW1
and SNF links BRCA1 to chromatin remodelling, which
is important to facilitate access of proteins involved in
DNA processing, such as transcription and repair, to
DNA (Bochar et al., 2000).
BRCA1 functions in signalling the response to DNA
damage
The initial step in the response to DNA damage involves
sensing and recognizing the damaged DNA and
transducing a signal onwards to downstream effectors,
resulting in cell cycle arrest and DNA repair. The
protein kinases ATM and ATR are central to the DNA
damage response, relaying the signal onwards by
phosphorylating downstream proteins, including
BRCA1. The role of BRCA1 is to respond to these
signals by participating in various cellular pathways,
including cell cycle regulation and DNA repair (Figure 1).
ATM and ATR phosphorylate BRCA1 in response to
different stimuli and they appear to have both distinct
and overlapping phosphorylation sites, only some of
which have been characterized. ATM phosphorylates
BRCA1 on several different residues in response to IR
(Cortez et al., 1999; Gatei et al., 2000). BRCA1 lacking
two of those phosphorylation sites, Ser1423 and
Ser1524, fails to rescue the radiation hypersensitivity
of a BRCA1-deficient cell line, demonstrating that
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Figure 1 BRCA1 in DNA damage signalling. DNA damage
activates the kinases ATM, ATR and CHK2, which in turn activate
BRCA1 by phosphorylation. In response to IR-induced damage,
BRCA1 is phosphorylated by ATM on several different residues,
including serines 1387, 1423 and 1524 (Cortez et al., 1999; Gatei
et al., 2000) and by CHK2 on serine 988 (Lee et al., 2000). ATR
phosphorylates BRCA1 on serine 1423 in response to UV damage
or HU-induced replication arrest (Tibbetts et al., 2000). The
phosphorylation of serine 1387 is important for S phase arrest,
while phosphorylation on serine 1423 is important for G2–M arrest
(Xu et al., 2001a, 2002). The serine 988 phosphorylation affects the
DNA repair response to DSBs (Zhang et al., 2004; Zhuang et al.,
2006; Wang et al., 2006). BRCA1 participates in the G1–S
checkpoint response indirectly through several ways, including by
affecting CHK2 and p53 phosphorylation (Foray et al., 2003).
phosphorylation of BRCA1 by ATM is critical for the
appropriate response to DSBs (Cortez et al., 1999).
Phosphorylation of the Ser1423 residue appears to be
important for the IR-induced G2–M checkpoint but not
for the IR-induced intra-S phase arrest, which requires
phosphorylation of Ser1387 (Xu et al., 2001a, 2002).
ATR, which is activated by UV-damage and hydroxyurea-induced replication arrest, also phosphorylates
BRCA1 on several residues, including Ser1423 during
the G2–M phase (Tibbetts et al., 2000; Gatei et al., 2001;
Okada and Ouchi, 2003). Moreover, ATR colocalizes
with BRCA1 in foci in cells synchronized in S phase and
after exposure to DNA damaging agents or DNA
replication inhibitors, associating BRCA1 and ATR
with the response to stalled replication forks (Tibbetts
et al., 2000; Gatei et al., 2001). In response to IR, ATM
phosphorylates and activates another checkpoint kinase,
CHK2, which in turn can phosphorylate BRCA1 on
Ser988 (Lee et al., 2000). CHK2 and BRCA1 interact
and colocalize within discrete nuclear foci that disperse
after damage by IR. Phosphorylation on Ser988
has been shown to be required for this dispersion and
the separation of the two proteins, as well as for
BRCA1 to mediate survival after DNA damage in a
BRCA1-mutated cell line. Recently, CHK2 phosphorylation of the Ser988 of BRCA1 was also shown
to be important for the role of BRCA1 in the repair of
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DSBs by promoting error-free HR and by inhibiting the
error-prone microhomology-mediated subpathway of
NHEJ (Zhang et al., 2004; Zhuang et al., 2006; Wang
et al., 2006). The DNA damage response kinases ATM,
ATR and CHK2, therefore, modulate the function of
BRCA1 through phosphorylation, affecting cell cycle
regulation and fidelity of DNA repair.
BRCA1 is essential for activating the CHK1 kinase and
it also induces the expression of the 14-3-3 proteins and
the WEE1 kinase, which is another inhibitor of CDC2
activity (Yarden et al., 2002). In addition, phosphorylation of BRCA1 on Ser1423 by ATM is required to
activate this checkpoint and BRCA1 has been demonstrated to induce the expression of GADD45, which
activates the G2–M checkpoint by inhibiting the activity
of the CDC2–CyclinB complex (Mullan et al., 2001; Xu
et al., 2001a).
BRCA1 and checkpoint control
In response to DNA damage, mammalian cells have to
arrest the cell cycle to initiate repair. Failure of cell cycle
checkpoints can lead to the acquisition and accumulation of genetic alterations and chromosomal abnormalities. The role of BRCA1 in cell cycle checkpoints has
recently been reviewed (Kennedy et al., 2004; Deng,
2006). Mouse embryonic fibroblasts (MEFs) derived
from embryos homozygous for a targeted deletion of
exon 11 of the Brca1 gene, maintain an intact G1–S
checkpoint but are defective in an IR-induced G2–M
checkpoint (Xu et al., 1999). Additionally, these cells
exhibit centrosome amplification, resulting in abnormal
chromosome segregation and aneuploidy. Having a
functional G1–S checkpoint suggests a functional p53
pathway in Brca1 mutant cells, which is consistent with
the finding that removal of p53 partially rescues Brca1
deficiency (Hakem et al., 1997; Ludwig et al., 1997; Shen
et al., 1998). Moreover, MEFs containing a Brca1 exon
11 deletion in a p53 heterozygous or null background
showed partial or complete loss of the G1–S checkpoint,
respectively (Xu et al., 2001b). However, BRCA1 has
been reported to stimulate the transcription of the p21
gene, which results in cell cycle arrest at the G1–S phase
boundary, and is a coactivator for p53, indicating a
more complex mechanism of control (Somasundaram
et al., 1997; Ouchi et al., 1998; Zhang et al., 1998).
BRCA1 has also been shown to be required for the
ATM/ATR-mediated phosphorylation of several proteins following DNA damage, including CHK2 and p53
at Ser15, which is necessary for G1–S arrest via
transcriptional induction of p21 (Foray et al., 2003;
Fabbro et al., 2004). The activation of the intra-S phase
checkpoint by stalled replication forks may also require
BRCA1 and this response may depend on ATR
phosphorylation (Tibbetts et al., 2000). After damage
by IR the activation of the intra-S phase checkpoint
requires phosphorylation of BRCA1 on Ser1387 by
ATM, and, additionally, BRCA1 has been reported to
stimulate the transcription of p27, which induces intraS phase arrest (Williamson et al., 2002; Xu et al., 2002).
BRCA1 regulates the G2–M phase checkpoint at multiple levels. Establishment of the G2–M checkpoint
requires phosphorylation of the CDC2 kinase, while
removal of this phosphorylation by CDC25C activates
the CDC2/CyclinB complex to initiate mitosis. DNA
damage leads to inhibition of CDC25C activity through
phosphorylation by the CHK1 kinase and nuclear
exclusion of CDC25C through binding to 14-3-3s,
leading to cell cycle arrest at the G2–M checkpoint.
The BRCA1-binding protein BARD1 and DNA repair
BRCA1 exists as a heterodimeric complex with BARD1,
a structurally related protein that, like BRCA1, contains
a N-terminal RING-finger domain and two C-terminal
BRCT motifs (Wu et al., 1996). The association between
these two proteins is mediated through their respective
RING domains, and together they exhibit E3 ubiquitin
ligase activity, which can be disrupted by cancerpredisposing mutations within the RING domain of
BRCA1 (Hashizume et al., 2001; Ruffner et al., 2001).
Furthermore, these mutations affect the ability of cells
to repair damaged DNA, as measured by sensitivity to
IR and to arrest at the G2–M phase of the cell cycle.
This suggests that the ubiquitin ligase activity is
important for BRCA1 to execute its role within the
DNA damage response pathway (Ruffner et al., 2001).
The interaction between BRCA1 and BARD1 is
important for the stability of BRCA1 and, accordingly,
the phenotype of Bard1-null mice is strikingly similar to
that of Brca1-null mice, including chromosomal instability and early embryonic lethality due to severe
impairment of cell proliferation, which can be partially
rescued in a p53-null background (Hashizume et al.,
2001; Joukov et al., 2001; McCarthy et al., 2003; Evers
and Jonkers, 2006). A role for BARD1 in the DNA
damage response was suggested by the observation that
BARD1 colocalizes with BRCA1 in S phase cells and in
cells following DNA damage (Jin et al., 1997; Scully
et al., 1997a). BARD1 has also been shown to
participate with BRCA1 in the homology-directed
repair of DSBs (Westermark et al., 2003; Fabbro
et al., 2004). A role for BARD1 in DSB repair was
demonstrated using a dominant-negative truncated
BARD1 peptide, which contained the N-terminal
BRCA1-interacting RING domain but was missing the
C-terminal BRCT repeats (Westermark et al., 2003).
The peptide was, therefore, predicted to compete
with endogenous full length BARD1 for binding to
endogenous BRCA1, disrupting the endogenous
BRCA1–BARD1 interaction. Expression of the
dominant-negative BARD1 peptide resulted in a
significant decrease in the homology-directed repair
of a chromosome DSB, as well as in mitomycin
C (MMC) hypersensitivity, without affecting the
subcellular location of BRCA1. Recently, the BRCA1–
BARD1 complex has been demonstrated to ubiquitinate
RNA polymerase II following DNA damage resulting in
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its degradation and subsequent inhibition of transcription and RNA processing (Kleiman et al., 2005; Starita
et al., 2005). This process could help in eliminating
prematurely terminated transcripts that could produce
truncated proteins and also to clear the damaged
DNA region, creating access and possibly acting as a
recruiting factor for DNA repair proteins.
The function of BRCA2 in regulating RAD51-mediated
recombination
BRCA2 plays an important role in the repair of DSBs
by HR, a pathway which is dependent upon the
recombinase function of RAD51 (Moynahan et al.,
2001b; Tutt et al., 2001). The direct interaction between
BRCA2 and RAD51 and their colocalization in nuclear
foci after DNA damage was the first evidence for a role
for BRCA2 within this DNA repair pathway (Sharan
et al., 1997; Chen et al., 1998). BRCA2 appears to
regulate the function of RAD51 in HR and in the last
few years, a great deal of data has been published on the
interplay between these two proteins and how it affects
the repair of DSBs.
BRCA2 deficiency is associated with a defect in GC
Cells defective in BRCA2 function show a high degree
of chromosome instability, including chromosome
breaks and radial chromosomes (Patel et al., 1998; Tutt
et al., 2001; Kraakman-van der Zwet et al., 2002). These
aberrations have been shown to accumulate spontaneously during passage of cells in culture and can also be
induced by DNA damaging agents that induce DSBs,
such as IR and DNA cross-linking agents. BRCA2 is
thought to promote genomic stability through its role in
the error-free repair of DSBs by GC via its association
with RAD51. How BRCA2 deficiency affects DSB
repair has been investigated in several BRCA2 mutant
cell lines using recombination repair substrates. Mouse
embryonic stem cell lines carrying targeted mutations in
Brca2 exhibit a reduction in the accurate repair of DSBs
by GC, while deletion events are elevated (Moynahan
et al., 2001b; Tutt et al., 2001; Stark et al., 2004). These
deletions are thought to arise predominantly through
the use of the SSA pathway. Deficiency in GC has also
been demonstrated in the human tumour cell line
CAPAN-1 and in the Chinese hamster cell mutant
VC8, both of which are defective in BRCA2 function
(Moynahan et al., 2001b; Larminat et al., 2002). NHEJ,
however, is apparently unaffected in BRCA2-deficient
cells (Patel et al., 1998; Yu et al., 2000). Loss of BRCA2,
therefore, results in the repair of DSBs by a more errorprone mechanism possibly explaining the apparent
chromosome instability associated with BRCA2 deficiency. In addition to this, BRCA2 has been implicated
in the response to stalled replication and in preserving
the stability of stalled replication forks, perhaps
explaining the apparent instability that spontaneously
accumulates in BRCA2-deficient cells (Lomonosov
et al., 2003).
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BRCA2 brings RAD51 to sites of DSBs
RAD51 was the first BRCA2-binding protein to be
identified (Sharan et al., 1997) and the interaction of these
proteins has since been extensively studied. BRCA2
binds directly to RAD51 through its C-terminus
and the BRC repeats located in the middle of the
protein. These repeats are highly conserved across
species, while most of the intervening sequences are
not, suggesting that the BRC repeats are important for
BRCA2 function (Bignell et al., 1997; Takata et al.,
2002; Warren et al., 2002). Of the eight BRC repeats in
the BRCA2 protein, RAD51 has been shown to bind to
BRC1-4, BRC7 and BRC8, which are the more highly
conserved repeats (Bignell et al., 1997; Wong et al.,
1997). These repeats have been shown to bind distinct
regions of RAD51, confirming nonequivalent interactions between the different BRC repeats and RAD51
(Galkin et al., 2005).
The physical interaction between BRCA2 and
RAD51 is essential for error-free HR to take place in
response to DSBs. BRCA2 is thought to be required for
the transport of RAD51 into the nucleus and to sites of
DNA damage, where RAD51 would be released to form
the nucleoprotein filament required for recombination
to take place. While BRCA2 has nuclear localization
signals (NLSs), these have not been identified in
RAD51, prompting the idea that BRCA2 facilitates
the transport of RAD51 into the nucleus. In support
of this, truncated BRCA2, lacking its NLSs at the
C-terminus has been shown to be cytoplasmic, along with
the majority of the RAD51 protein (Spain et al., 1999;
Davies et al., 2001). Accordingly, BRCA2-deficient cells
do not form RAD51 foci in response to DNA damage
(Yuan et al., 1999). RAD51 foci are thought to represent
sites of repair that presumably contain nucleoprotein
filaments of RAD51, and their formation is dependent
on the function of BRCA2 upon DNA damage.
However, there is some evidence that the formation of
RAD51 foci in undamaged S phase cells may be BRCA2
independent (Tarsounas et al., 2003). These spontaneous foci occur in smaller numbers than damage
induced foci and are thought to represent sites at which
stalled or broken replication forks undergo repair. Only
around 20% of RAD51 has been suggested to reside in a
relatively immobile fraction that is bound to BRCA2,
with the remaining 80% of RAD51 existing either as
an immobile oligomerized fraction or as a relatively
mobile fraction. The immobile RAD51 fraction complexed with BRCA2 is then mobilized upon replication
arrest by hydroxyurea (Yu et al., 2003). A model has
been proposed that BRCA2 is involved in both
sequestering and mobilizing RAD51 and holds
RAD51 in a state of readiness until DNA damage or
replication arrest, when the complex becomes localized
to sites of DSBs.
RAD51 nucleoprotein filament formation and
recombination
For HR to take place, RAD51 must be released from
BRCA2 to form a nucleoprotein filament on ssDNA,
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which then invades and pairs with a homologous DNA
duplex, initiating strand exchange between the paired
DNA molecules. To understand how the BRCA2RAD51 interaction affects RAD51 function, the
DNA-binding properties of RAD51 have been analysed
in the presence of synthetic BRCA2 BRC-repeat
peptides (Yuan et al., 1999; Davies et al., 2001; Stark
et al., 2004). Overexpression of a single BRC repeat
prevents RAD51 from forming nucleoprotein filaments
on DNA. Furthermore, the addition of BRC peptides to
preassembled nucleoprotein filaments causes them to
dissociate. This was suggested to be a consequence of a
direct interaction between a RAD51 monomer and a
single BRC repeat, therefore preventing the RAD51
monomers from self-associating (Davies et al., 2001).
Consequently, overexpressing a BRC repeat renders
cells with wild-type BRCA2 hypersensitive to DNA
damaging agents and results in compromised RAD51
foci formation, with an associated defect in GC and
upregulation of SSA (Yuan et al., 1999; Stark et al.,
2004). How this was mediated at the molecular level was
clarified in a paper describing the crystallographic
structure of a RAD51-BRC4 complex (Pellegrini et al.,
2002). The BRC structure mimics the structure of the
interaction domain of the adjacent RAD51 monomer,
thereby impairing the ability of one RAD51 monomer
to interact with another to form nucleoprotein filaments.
This could be a way to prevent RAD51 from interacting
with inappropriate DNA substrates within the nucleus.
However, the inhibition of RAD51 oligomerization by a
BRC repeat contrasts with the essential role of the
BRCA2 protein in the repair of DSBs by HR. Recently,
the BRC3 and BRC4 repeats were shown to be able to
bind and form stable complexes with RAD51–DNA
nucleoprotein filaments. However, only at high concentrations of the BRC repeats were filaments disrupted
(Galkin et al., 2005). These results, therefore, suggest
direct interactions between BRCA2 and RAD51 filaments, rather than with RAD51 monomers. However, in
these experiments individual BRC repeats were utilized,
which may not be representative of their function in the
context of full-length BRCA2. This is supported by
experiments investigating the importance of the BRC
repeats, along with the C-terminal DBD (DNA/DSS1binding domain), in the repair of DNA damage by
BRCA2 using a fusion protein (Saeki et al., 2006). The
DBD of BRCA2 has five domains, including three
oligonucleotide/oligosaccharide-binding (OB) fold domains (OB1, OB2 and OB3) (Yang et al., 2002). OB
folds are found in ssDNA-binding proteins, such as
RPA, to which the three OB folds of BRCA2 are most
similar. The fusion protein consisted of a single (BRC3
or BRC4) or multiple BRC repeats fused to a ssDNAbinding domain from RPA (Saeki et al., 2006).
Expression of any of these fusion proteins in BRCA2
mutant cells rescued BRCA2 deficiency by increasing
GC while suppressing SSA, restoring RAD51 focus
formation and cellular survival in response to IR and
MMC, as well as reducing spontaneous chromosomal
aberrations (Figure 2). This suggests that much of
BRCA2 is dispensable for DSB repair and in maintain-
ing genomic integrity, implying that the key role of
BRCA2 is to deliver RAD51 to sites of DNA damage. A
role for BRCA2 in promoting recombination was
demonstrated in the yeast-like fungus Ustilago maydis.
In this model, BRCA2 was shown to facilitate the
nucleation of the RAD51 filament by recruiting RAD51
to DNA and displacing RPA at the nucleation site
(Yang et al., 2005). This study also showed that the
BRCA2 orthologue acted preferentially at a junction
between dsDNA and ssDNA, stimulating filament
formation only at junctions having 30 overhang ssDNA.
The DBD domain was also found to stimulate the
homologous pairing and strand-exchange activities of
RAD51, indicating that BRCA2 might facilitate
RAD51-mediated recombination and might have a role
at the dsDNA–ssDNA junction of the resected DSB
(Yang et al., 2002). The DBD-binding protein, DSS1,
has also been shown in Ustilago to be important for
properly controlled recombination (Kojic et al., 2005)
and this is discussed in more detail below.
BRCA2 regulation by phosphorylation
In addition to its interaction with the BRC repeats,
RAD51 can also interact with the C-terminal region of
BRCA2. Recently, this region was shown to be
phosphorylated at residue 3291 by cyclin-dependent
kinases (CDKs) in a cell cycle-dependent manner
(Esashi et al., 2005). This phosphorylation was observed
to be low in S phase but to increase as the cell progressed
from G2 to M phase, thereby preventing interaction of
the C-terminus of BRCA2 with RAD51. However,
treatment with IR reduced this phosphorylation by
CDKs and restored the interaction between BRCA2 and
RAD51. These results show that the phosphorylation
status of S3291 is crucial for modulating interactions
between the C-terminus of BRCA2 and RAD51 and
possibly provides a mechanism for the regulation of
recombinational repair. Exactly how the two RAD51binding regions of BRCA2, the BRC repeats and the
C-terminus, work together to control RAD51 function
is still not clear.
BRCA2 and the regulation of cell cycle progression
Several studies have linked BRCA2 with progression
through mitosis (Lee et al., 1999; Futamura et al., 2000;
Marmorstein et al., 2001; Daniels et al., 2004). BRCA2
associates with the DNA-binding protein BRAF35
(BRCA2-associated factor 35) through a region contained within BRC6, 7 and 8 of BRCA2 (Marmorstein
et al., 2001). BRAF35 was observed to bind to branched
DNA structures, such as those formed during recombinational repair, implicating BRAF35 in the DNA
damage response. BRAF35 and BRCA2 were also
found to colocalize on mitotic chromosomes and
injection of antibodies into cells to block either BRCA2
or BRAF35 impeded entry into mitosis. Further
evidence to support a role for BRCA2 in the M-phase
checkpoint is through interaction with BUBR1
Oncogene
Roles of BRCA1 and BRCA2 and associated proteins
K Gudmundsdottir and A Ashworth
5870
Figure 2 Functionally distinct effects of BRC repeat expression on RAD51 function. The BRCA2 BRC repeats bind RAD51 and are
essential for the function of both proteins. Overexpressing a single BRC repeat in mammalian cells disrupts RAD51 filament formation
and dissolves preassembled filaments by structurally mimicking the RAD51-RAD51 oligomer interface creating a BRC-RAD51
complex (Yuan et al., 1999; Davies et al., 2001; Stark et al., 2004). This inhibits RAD51 function, creating a BRCA2-deficient
phenotype. A BRC repeat fused to a ssDNA-binding domain, however, restores RAD51 function in BRCA2-deficient cells, indicating
that a BRC repeat and the DBD domain together are the minimal functional units of BRCA2 and that the main role of BRCA2 is to
deliver RAD51 to the DNA break site (Saeki et al., 2006).
(Futamura et al., 2000), which is important for the
correct attachment of chromosomes to the mitotic
spindle. BUBR1 was found to interact with and
phosphorylate the C-terminus of BRCA2, but the significance of this remains to be elucidated. Additionally,
inactivation of the mitotic checkpoint, through mutation of BUBR1 or BUB1 in BRCA2-deficient
cells was sufficient to overcome growth arrest and
initiate neoplastic transformation (Lee et al., 1999).
A role for BRCA2 in cytokinesis has also been proposed
as the cell cycle is extended from anaphase onset to the
completion of cell division in BRCA2-deficient cells
compared to wild-type cells (Daniels et al., 2004). The
absence of BRCA2 has been associated with centrosome
amplification and the formation of micronuclei, which
are abnormal DNA-containing bodies (Tutt et al.,
1999). These abnormalities could, to some extent,
account for the aneuploidy and chromosome instability
seen in BRCA2-deficient cells. Furthermore, BRCA2
has been reported to be phosphorylated in a cell cycle
dependent manner by Polo-like kinase 1 (Plk1) (Lin
et al., 2003; Lee et al., 2004) but how these phosphorylation events affect BRCA2 function still remains to be
clarified.
Oncogene
The BRCA2-binding protein DSS1 and DNA repair
The BRCA2-binding protein DSS1 has, in recent years,
been demonstrated to be essential for BRCA2 function
(Kojic et al., 2003; Gudmundsdottir et al., 2004; Li
et al., 2006). DSS1 is a highly conserved 70 amino-acid
protein that interacts with the C-terminal DBD domain
of BRCA2 (Crackower et al., 1996; Marston et al.,
1999). It binds to BRCA2 in an extended conformation,
interacting with numerous residues within the helical
domain, OB1 and OB2 of BRCA2, most of which are
highly conserved (Yang et al., 2002). Most of the 25
BRCA2 interacting residues in the DSS1 protein are
also highly conserved. These residues reside in two
regions, referred to as BRCA2 interacting region 1
(residues 7–25) and BRCA2 interacting region 2
(residues 37–63), linked by a 11-residue nonconserved
region.
A functional role for DSS1 in DNA repair was
suggested from studies using the fungus Ustilago maydis
(Kojic et al., 2003). Ustilago contains, unlike the fission
and budding yeast, a clear orthologue of BRCA2, as
well as DSS1 and RAD51 orthologues. The phenotype
of BRCA2 and RAD51 mutants parallel those seen in
Roles of BRCA1 and BRCA2 and associated proteins
K Gudmundsdottir and A Ashworth
5871
mammalian cells, making Ustilago a useful system to
study this pathway. Ustilago null DSS1 mutants exhibit
a phenotype virtually identical to that seen in BRCA2
and RAD51 mutants, including extreme sensitivity to
UV and IR, as well as deficiency in both mitotic and
meiotic recombination. Both spontaneous and UVinduced allelic recombination was abrogated to the
same extent as that observed in BRCA2 and RAD51
mutants and double-strand DNA break repair was
similarly reduced. The defects in meiosis were associated
with sterility, most likely due to failed recombinational
repair of DSBs. DSS1 was also observed to be important
for maintaining genomic stability, as mutants exhibited
elevated spontaneous mutator activity of approximately
40-fold compared to wild-type and frequently harbored
aberrant chromosomes as measured by pulse-field gel
electrophoresis.
Further work in Ustilago implicated DSS1 in the
control of BRCA2 recombinational repair (Kojic et al.,
2005). BRCA2 mutants lacking the C-terminal DSS1binding region were able to rescue the phenotype
associated with DSS1 deficiency. This N-terminal
BRCA2 fragment was still capable of binding RAD51
as the BRC region was intact and it also contained a
NLS enabling BRCA2 to enter the nucleus. The BRCcontaining region is therefore capable of contributing to
DNA repair in the absence of the DBD domain,
demonstrating that DSS1 is not required for delivering
RAD51 to sites of DNA damage or RAD51 nucleoprotein filament formation. Furthermore, fusing the BRCA2
N-terminus to the DNA-binding domain of the Ustilago
orthologue of the RPA70 subunit exaggerated this
phenotype even further. The DNA-binding domain of
RPA70 has a similar structure to the BRCA2 DBD but is
devoid of corresponding DSS1-interacting residues.
DSS1-deficient cells transfected with the BRCA2RPA70 hybrid showed elevated levels of RAD51 foci
formation, both spontaneous and damage-induced, as
well as elevated recombination levels. This exaggerated
phenotype highlights the importance of DSS1 in
controlling BRCA2-dependent recombination.
The importance of DSS1 in BRCA2 function has also
been established in mammalian cells (Gudmundsdottir
et al., 2004; Li et al., 2006). Silencing of DSS1 using
RNA interference (RNAi) in both mouse and human
cells compromised the ability of these cells to form
colonies and importantly DSS1 was shown to be required
for the formation of DNA damage induced RAD51
foci, suggesting a role for DSS1 in BRCA2- and RAD51dependent repair by HR. Consistent with this, DSS1 was
shown to be important for the maintenance of genomic
stability in the presence of MMC (Gudmundsdottir
et al., 2004). The mechanism by which DSS1 affects
BRCA2 function is not yet clear. However, DSS1 has
been shown to be dispensable for the interaction
between BRCA2 and RAD51, but conflicting views
exist as to whether DSS1 is required for the stability
of BRCA2. In contrast to two previous reports
(Gudmundsdottir et al., 2004; Kojic et al., 2005), a
recent paper by Li et al. (2006) suggests that silencing of
DSS1 leads to a considerable reduction in BRCA2
protein levels as measured by western blotting. The halflife of the protein appeared to be affected, although
calpain-, caspase- and proteasome inhibitors were
unable to inhibit its degradation. The stability of a
BRCA2 mutant devoid of the DBD region, however,
was unaffected by DSS1 silencing, suggesting it is the
DBD region that causes BRCA2 to be degraded. These
results complement those mentioned previously, where
removal of the DBD region in Ustilago rescues the
DSS1/BRCA2 deficient phenotype (Kojic et al. 2005).
Additionally, BRCA2 has proven to be largely insoluble
in the absence of DSS1, potentially implicating DSS1 in
maintaining the correct conformation of BRCA2 (Yang
et al. 2002; Kojic et al. 2003). However, as the
mechanism of how DSS1 depletion induces degradation
of BRCA2 remains to be elucidated, further research
into BRCA2 stability is warranted.
DSS1 as a link between the proteasome and DNA repair
A possible role for the proteasome in the repair of DSBs
has emerged with the discovery that DSS1 is involved
both in DSB repair and is a subunit of the proteasome.
DSS1 was recently identified as a component of the lid
subcomplex of the 19S regulatory particle in yeast
(Funakoshi et al., 2004; Sone et al., 2004; Josse et al.,
2006). The 19S regulatory complex serves to recognize
ubiquitinated proteins and is implicated in their unfolding and translocation into the interior of the 20S
proteolytic core complex, where they are degraded into
oligopeptides (Baumeister et al., 1998). A functional link
to the proteasome is provided by the demonstration that
DSS1 is required for efficient ubiquitin-dependent
protein degradation (Funakoshi et al., 2004; Sone
et al., 2004; Josse et al., 2006). To examine whether
the proteasome might be involved in repairing DNA
DSBs, the sensitivity of yeast strains containing a
deletion in one or more proteasome subunits to various
DNA damaging agents have been investigated (Funakoshi
et al. 2004; Krogan et al. 2004). Single mutants
displayed little additional sensitivity to UV, HU,
camptothecin and bleomycin but double mutants
combining DSS1 deletion with another proteasome
mutation exhibited marked hypersensitivity. Furthermore, combining a proteasome mutant with deletion of
a gene involved in HR caused a synthetic growth defect
along with hypersensitivity to HU and MMS, implicating the proteasome in a pathway other than or including
HR. Using ChIP (chromatin immunoprecipitation)
analysis, DSS1 was shown to bind to DNA at the site
of a specific DSB along with components of the 19S and
the 20S proteasome complexes, linking proteolysis to the
repair of DSBs. Recruitment of DSS1 was reduced when
either Rad52, a HR protein, or Pol4, a polymerase
linked to NHEJ, were deleted, suggesting that DSS1 is
recruited by both HR and NHEJ pathways to sites of
DSBs. In support of that, DSS1 was shown to be
important for the repair of DSBs by both HR and
NHEJ (Krogan et al. 2004).
Oncogene
Roles of BRCA1 and BRCA2 and associated proteins
K Gudmundsdottir and A Ashworth
5872
Therapeutic exploitation
BRCA1 and BRCA2 are important for the repair of
DSBs by the potentially error-free pathway of HR by
GC. Cells that lack either BRCA1 or BRCA2 repair
these lesions by alternative more error-prone mechanisms. In the case of BRCA2 deficiency, repair by GC is
impaired while the use of the alternative more errorprone homology-directed repair pathway SSA is upregulated (Moynahan et al., 2001b; Tutt et al., 2001; Stark
et al., 2004). In BRCA1-deficient cells, the use of both
GC and SSA is decreased, along with a precise
subpathway of NHEJ, leaving DSBs to be repaired by
an error-prone microhomology-mediated subpathway
of NHEJ (Moynahan et al., 1999; Stark et al., 2004;
Wang et al., 2006; Zhuang et al., 2006). The use of less
accurate pathways for the repair of DSBs in BRCAdeficient cells is a likely explanation for the apparent
genomic instability observed in these cells. BRCA1- and
BRCA2-deficient cells have elevated sensitivity over
BRCA wild-type cells to DNA damaging agents that
cross-link DNA, such as MMC and to the platinumdrugs, cisplatin and carboplatin (Bhattacharyya et al.,
2000; Yu et al., 2000; Tutt et al., 2001; Fedier et al.,
2003). This increased sensitivity could therefore be
exploited for the treatment of BRCA-associated cancers,
as platinum drugs are already commonly used chemotherapeutic agents. Indeed, carboplatin is currently
being trialled as a therapeutic agent in BRCA1 or
BRCA2 mutational carriers (http://www.brcatrial.org;
Tutt et al., 2005).
The inherent DNA repair defect in BRCA-deficient
cells has also provided a rationale for another therapeutic approach. This approach involves inhibiting the
DNA repair protein PARP to generate specific lesions
that require BRCA1 or BRCA2 for their removal,
resulting in synthetic lethality. The concept of synthetic
lethality applies when the mutation of two genes causes
death, while mutation of either gene alone is viable
(Hartwell, 1997; Kaelin, 2005). Poly(ADP-ribose)polymerase-1 (PARP-1) is an enzyme involved in the repair
of SSBs by the BER pathway. PARP-1 deficiency causes
failure to repair SSB lesions effectively, which when
encountered by a DNA replication fork can lead to the
formation of DSBs (Dantzer et al., 2000; Hoeijmakers,
2001). Under normal circumstances these lesions would
be repaired by the error-free pathway of GC, but in a
BRCA defective background these DSBs are likely to be
repaired by more error-prone repair mechanisms,
causing chromosome aberrations and loss of viability.
PARP-1 inhibitors have indeed been shown to be
selectively lethal to cells deficient in BRCA1 or BRCA2
(Bryant et al., 2005; Farmer et al., 2005; McCabe et al.,
2005). In BRCA-deficient cells, PARP inhibitor treatment caused a cell cycle arrest in G2 phase of the cell
cycle with subsequent apoptosis resulting in reduced cell
survival. Cells that survived the treatment showed a
profound increase in complex chromosome rearrangements. Such aberrations are, to a lesser extent, also seen
in BRCA-deficient cells due to a defect in the repair of
DSBs by GC. However, and importantly, no toxicity
was evident in BRCA heterozygous cells, as these are the
normal cells in BRCA mutation carriers. Additionally,
BRCA-deficient cells are much more sensitive to PARP
inhibitors than to treatment with DNA cross-linking
agents. All this potentially creates a large therapeutic
window for safe and effective treatment of tumours in
carriers of a BRCA1 or BRCA2 mutation. PARP
inhibitors have recently been shown to be synthetically
lethal with defects in other HR-related proteins, including ATM, ATR and Fanconi Anemia genes, suggesting
a wider range of therapeutic targets for PARP inhibitors
than just for the treatment of BRCA-tumours (McCabe
et al., 2006). As defects in these genes are found in a
range of cancers, therapies designed to target the DNA
repair defects in BRCA-deficient cells may be more
widely applicable (Turner et al., 2004).
Acknowledgements
We are grateful to the members of the Ashworth laboratory
for useful comments during the writing of this review, with
special thanks to Nuala McCabe. Work in our laboratory is
funded by Breakthrough Breast Cancer and Cancer Research
UK.
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