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Oncogene (2003) 22, 5784–5791
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Roles of BRCA1 and BRCA2 in homologous recombination, DNA
replication fidelity and the cellular response to ionizing radiation
Simon N Powell*,1 and Lisa A Kachnic2
1
Department of Radiation Oncology, Massachusetts General Hospital, Boston, MA, USA; 2Department of Radiation Oncology,
Boston Medical Center, Boston, MA, USA
Inheritance of one defective copy of either of the two
breast cancer susceptibility genes, BRCA1 and BRCA2,
predisposes individuals to breast and ovarian cancers.
Current progress in determining the function of these
genes suggests that they participate in a common pathway
to facilitate orderly homologous recombination and
thereby maintain genomic integrity. As a consequence of
this defect in homologous recombination, tumors that
arise in BRCA carriers are likely to be more sensitive to
ionizing radiation. This review summarizes recent investigations about the nature of the defect in DNA repair,
and highlights the unanswered questions about the tumor
suppressor paradox of BRCA genes. The unsolved
mystery is the other genetic changes that must occur to
turn a BRCA-deficient cell from a nonviable cell into a
tumor cell capable of endless growth.
Oncogene (2003) 22, 5784–5791. doi:10.1038/sj.onc.1206678
Keywords: breast cancer; BRCA genes; tumor suppressor genes; DNA repair; ionizing radiation; homologous
recombination; DNA replication
Introduction
The contribution of classical human genetics was
paramount in the identification of two breast cancer
susceptibility genes, BRCA1 and BRCA2. Current
progress in determining the function of these genes
suggests that they participate in a common pathway that
is involved in the control of DNA replication fidelity
and DNA damage repair. Familial breast and ovarian
cancer can be triggered by a mutation in one allele of
either BRCA1 or BRCA2. The sequence of events that
occur from the initial germline mutation to the
development of cancer is unclear, but loss of function
of the second allele is required. However, if both alleles
are knocked out, there is failure to develop or
proliferate. Solving this ‘tumor suppressor paradox’
and clarifying the function of the BRCA genes may
provide further insight on how inactivation leads to
tumorigenesis.
*Correspondence: SN Powell, Department of Radiation Oncology,
Massachusetts General Hospital, Cox 302, 100 Blossom Street,
Boston, MA 02114, USA; E-mail: [email protected]
Identification of the BRCA genes
The genetic basis for familial breast cancer predisposition has become established over the past decade with
the cloning of two major breast cancer susceptibility
genes, BRCA1 and BRCA2 (Miki et al., 1994; Wooster
et al., 1995). The BRCA1 gene was first cloned in 1994
after being mapped to chromosome 17q21 by genetic
linkage analysis. The BRCA1 protein consists of 1863
amino acids and is expressed in most proliferating cells
(Figure 1). Within the cell, BRCA1 is part of a large
protein complex up to 3 MDa in size. The C-terminus
of BRCA1 contains an amino-acid sequence motif,
now known as a BRCT domain, recognized in many
DNA repair proteins. This appears to be a site of
protein–protein interactions, perhaps with other DNA
repair proteins to build a large protein complex. In the
N-terminus, there is a ring-finger domain, also allowing
for protein–protein interactions, but thought to be
involved in protein ubiquitination. The BRCA2 gene,
a very large protein mapped to chromosome 13q12-13,
contains 3418 amino acids and encodes a protein of
approximately 385 kDa molecular weight (Figure 1).
BRCA2 is characterized by a very large exon 11, which
encodes peptide motifs required for interaction with the
RAD51 protein (Chen et al., 1999b).
Together, mutations in BRCA1 and BRCA2 account
for the majority of families with hereditary susceptibility
to breast and ovarian cancer (Hall et al., 1990; Hofmann
and Schlag, 2000; Paakkonen et al., 2001). Carriers of
the BRCA gene mutations are heterozygous (one
normal copy and one mutated copy) within the germ
line. Recent epidemiologic studies indicate that BRCA1
mutation carriers have a high lifetime risk of breast
cancer of up to 80% (Satagopan et al., 2001). The breast
cancer risk for the BRCA2 mutation carriers approaches
that of BRCA1, however, with a later age of disease
onset (Schubert et al., 1997). In addition to the breast
cancer risk, women with BRCA1 mutations have an
increased risk of ovarian cancer (Ford et al., 1994).
BRCA2 carriers are also at an increased risk for
developing ovarian cancer as well as other solid tumors:
prostatic, pancreatico-biliary, gastric and colon cancers,
as well as melanoma (Peto et al., 1999). The BRCA2
mutation appears to confer higher risks for male breast
cancer (Ford et al., 1998). There are subtle histologic
differences between BRCA1- and BRCA2-linked breast
cancers (Lakhani et al., 1998). The breast cancers that
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classified in BRCA-associated murine breast cancers,
which failed to suppress transformation but retained
other wild-type functions of P53 (Smith et al., 1999a).
Additionally, P53 inactivation partially rescued the
developmental arrest in BRCA1 and BRCA2 null mice,
further supporting the role of the BRCA genes in DNA
damage repair and the hypothesis that P53 checkpoint
loss is associated with BRCA gene loss in cancer
development (Jacks and Weinberg, 1998).
The BRCA tumor suppressor paradox
Figure 1 A schematic structure of BRCA1 and BRCA2, showing
the key features and domains of both proteins. They are unrelated
proteins. BRCA1 is characterized by the BRCT motif at the Cterminus, the SQ cluster region (the site of phosphorylation by
ATM and ATR) and the ring domain in the N-terminus (with E3
ubiquitin ligase structure). BRCA2 is characterized by the very
large exon 11, containing the eight BRC peptide motifs, important
for the interaction with RAD51. A ssDNA binding domain is
found within the C-terminus of BRCA2 (Figure 1)
arise in BRCA1 carriers tend to have distinctive pathological features: invasive ductal, high grade, abundant
lymphocyte infiltration, and usually estrogen receptor
negative (Lakhani et al., 2000). In contrast, tumors
arising in BRCA2 carriers do not differ significantly
from noncarriers (Lakhani et al., 2002). Many aspects of
the function of BRCA1 and BRCA2 suggest a common
pathway of DNA recombinational repair; so the reasons
for this subtle difference in pathology are not clear.
Knockout mice
BRCA1 and BRCA2 null embryos (loss of both functional
alleles) die early in development (approximately day 8
or 9), with a severe growth deficit. The loss of BRCA gene
function in embryogenesis has been shown to cause
an elevated expression of the cell cycle inhibitor, P21,
and subsequent growth arrest (Hakem et al., 1996, 1998;
Suzuki et al., 1997). The P53-dependent growth arrest
associated with a deficiency in BRCA proteins may
represent a checkpoint response to DNA damage. Conversely, loss of such DNA-damage responsive checkpoint
function may collaborate with loss of BRCA-mediated
function in the development of tumors. Additional
observations suggest that the embryos arising in BRCA1
or BRCA2 knockouts are radiation sensitive, as observed
by apoptosis of the inner cell mass following exposure to
8 Gy of ionizing radiation (Sharan et al., 1997).
In the clinical setting, breast cancers that arise in
carriers of BRCA1 or BRCA2 mutations are associated
with a high incidence of inactivation of P53, perhaps
greater than 90% (Crook et al., 1997, 1998; Smith et al.,
1999b). In the mouse model, when BRCA1 gene
inactivation was achieved with the use of a CRE-lox
vector system (Xu et al., 1999a), the mice developed late
onset breast cancer with frequent P53 mutations. Reexamination of these mice on a P53 þ / background
showed an increased frequency and earlier onset of
breast cancer. A novel class of P53 mutants was
It is generally accepted that a cancer results from the
accumulation of genetic changes in a target cell. More
than 30 years ago, Knudson hypothesized that carcinogenesis results from the occurrence of a second mutation
in a somatic target cell, so that the difference between
hereditary and nonhereditary cancers is the timing of the
first mutation (prezygotic vs postzygotic). The second
mutation can affect the remaining normal allele of the
same gene (recessive trait) or one copy of a different
gene (dominant trait). The first evidence substantiating
this ‘two-hit’ hypothesis or recessive model was the identification of the retinoblastoma tumor-suppressor gene.
Breast cancer represents an exception to this two-hit
model, as it does not show the predicted relation
between hereditary and nonhereditary tumorigenesis.
Tumorigenesis in individuals with germline BRCA
mutations requires inactivation of the remaining wildtype allele, suggesting that the BRCA genes are tumorsuppressor genes. However, unlike other tumor suppressor genes, no disease-causing dominant BRCA1 or
BRCA2 mutations have been detected in sporadic breast
or ovarian cancers. Epigenetic regulation of the expression of BRCA1 has been reported in human breast
cancers, especially in high-grade tumors (Wilson et al.,
1999). There remains the possibility that a BRCAdependent ‘pathway’ can be inactivated not just by loss
of BRCA1 or BRCA2, but by other components of the
BRCA-regulated response network, and these may play
a role in sporadic breast cancer.
The ‘caretaker’ gene model of Kinzler and Vogelstein
(1997) could explain this BRCA tumor-suppressor
paradox. The inactivation of a caretaker gene may
indirectly promote cancer by causing genomic instability. This hypothesis would suggest that mutations in
BRCA1 or BRCA2 might predispose to cancer development, and the loss of other genes that cooperate with the
loss of BRCA function is necessary for the full
development of cancer. Whether heterozygosity at the
loci for the BRCA genes is associated with a haploinsufficiency state is not yet clear. The next step from
germline heterozygosity to cancer development is critical
to understanding the function of these genes in vivo.
Response to ionizing radiation
In the last decade, major strides have been made toward
understanding the genetic determinants of the response
to both ionizing radiation treatment and the use of
cytotoxic chemotherapy. It has been known for some
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Execution of NHR
Execution of HR
Ku70/80
end processing
homology search
Ku translocation
DNA-PKcs
Artemis
Rad52
Hold and ligate
XRCC4
Ligase IV
single-strand
invasion
Rad51 + paralogs,
Rad54, RPA, BRCA2
Figure 2 Outline of the two principal pathways of DNA doublestrand break repair. NHEJ involves six known proteins, in a
coordinated sequence to ensure ligation of the broken ends. Loss of
DNA sequence occurs at the site of repair. HR involves multiple
proteins in a complex to allow strand exchange and resolution. The
process of HR can be an error-free process
time that the primary determinant of radiation sensitivity relates to the efficiency of DNA double-strand break
(DSB) repair. However, it has only more recently been
appreciated that the molecular mechanisms of DSB
repair can be broadly divided into two classes of repair:
nonhomologous end-joining (NHEJ) and homologous
recombination (HR). Figure 2 shows the genetic and
mechanistic difference of these two DSB repair pathways, and shows a summary of genetic factors that
determine radiation sensitivity. The vast majority of
single gene defects that cause radiation sensitivity can be
categorized as a defect in either of these two pathways of
DSB repair. In addition to DNA DSB repair, other
determinants of radiation sensitivity are radiationinduced cell cycle checkpoints, in particular the S and
G2 cell cycle checkpoints, which appear in some cases to
have a clear link to radiation sensitivity. Loss of
function of the BRCA1 gene is associated with a defect
in both the S phase checkpoint and the G2/M transition.
These checkpoints are both rapid in response and
transient, reaching a peak effect within 1–2 h of
irradiation and resolving within 4–6 h. There are also
cellular determinants of programmed cell death (apoptosis), which can modulate the radiation response in
certain situations, frequently in a tissue-specific manner.
A particular feature of BRCA1-deficient cells, as well
as many cells showing a defect in homologous recombination, is sensitivity to the DNA crosslinking agent
mitomycin C (Moynahan et al., 2001a). The reasons for
this feature are thought to be the requirement for
homologous recombination in the repair of DNA
interstrand crosslinks, but mitomycin C is also a potent
inducer of oxidative stress. Further work is needed to
clarify the precise reasons for the striking sensitivity to
this drug seen in BRCA1-deficient cells.
Role of BRCA1 and BRCA2 in homologous recombination
After 9 years of research since the BRCA1 gene was
cloned, the precise function of this protein is not yet
determined. BRCA1 binds to numerous cellular proteins
in vivo and appears to have multiple functions depending on the cellular context (Deng and Brodie, 2000;
Oncogene
Parvin, 2001). Based on a relatively small number of cell
culture experiments, cells deficient in the BRCA1 protein
do appear to be radiation sensitive. This radiation
sensitivity is accompanied by a defect in the measurable
rate of homologous recombination (Snouwaert et al.,
1999; Moynahan et al., 2001b). The measurement of
homologous recombination in cells is not straightforward and usually requires the establishment of cell
clones in which a plasmid-based artificial DNA recombination substrate is integrated into the genome of the
test cells (Taghian and Nickoloff, 1997). With this type
of procedure, it can be determined that the loss of the
BRCA1 protein results in a fivefold reduction in the
frequency of homologous recombination events (Moynahan et al., 2001b). It is difficult to relate this change in
homologous recombination to the change in radiation
sensitivity; however, correction of BRCA1 function in a
deficient cell line resulted in correction of both the defect
in homologous recombination and radiation sensitivity.
Recent unpublished data from our own laboratory
have shown that the BRCA-mediated stimulation of
homologous recombination can be abrogated by key
single amino-acid mutations in the ring domain (C64G),
the CHK2 site S988A in the so-called DNA-binding
domain and in the BRCT domain (P1749R). Interestingly, mutations of the ATM sites (S1423A, S1524A)
had no effect on homologous recombination, even
though the G2/M cell cycle checkpoint was defective.
The BRCA2 protein is a very large protein containing
3418 amino acids, which encodes a protein of approximately 385 kDa molecular weight (see Figure 1). At
present, there is only one known function of the BRCA2
protein, which is its role in facilitating homologous
recombination (Moynahan et al., 2001b; Xia et al.,
2001), as was also found for the BRCA1 protein. In
contrast to BRCA1, there appears to be no defect in
either NHEJ or cell cycle checkpoints (see below) (Yu
et al., 2000; Xia et al., 2001). The BRCA2 protein is
known to interact with major proteins involved in DNA
repair by homologous recombination and, in particular,
the RAD51 protein (Chen et al., 1998a, b). This protein
is critical in catalyzing the primary reaction required for
homologous recombination, and its association with
BRCA2 is required to facilitate this effect (Davies et al.,
2001). Expressing a short sequence of amino acids (a
peptide) from the region of BRCA2 that interacts with
RAD51 can result in a disruption of the connection
between these two proteins, leading to both a reduction
in homologous recombination and an increase in
radiation sensitivity (Chen et al., 1999a; Xia et al., 2001).
Two recent publications have highlighted structural
aspects of BRCA2, and both studies offer some insight
into the structural basis of BRCA2’s role in recombinational repair. RAD51 interacts with the BRC repeat
regions of BRCA2, encoded by the very large exon 11.
There are eight BRC repeats, and BRC3 and BRC4
have the strongest interaction with RAD51. Pellegrini
et al. (2002) have solved the crystal structure of the
BRC4 peptide sequence (amino acids 1517–1551) of
BRCA2 bound to RAD51 (D1–96). This structure
demonstrates a series of hydrophobic and hydrophilic
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BR
C
BR
r ep
CA
ea t
s
Rad51
2
OB folds
RP-A
C-terminus
Figure 3 Interaction between RAD51 and BRCA2. RAD51
interacts with the BRC repeat region of BRCA2, which is shown
as the projections between RAD51 monomers. The proposed
model is that the BRC repeat provides an assembly line of RAD51
monomers, and that the C-terminus of BRCA2 binds ssDNA,
providing a means to displace RPA and allow the orderly assembly
of the RAD51 filament. In the absence of BRCA2, critical events in
the initiation of homologous recombination are impaired, and
repair (and replication) errors accrue rapidly with each cell cycle
interactions involving the hairpin structure of BRC4
created by the short antiparallel b-sheets and an a-helix
(Figure 3). Contact between BRC4 and RAD51 is maintained from Leu1521 to Glu1548 of the BRC4 sequence.
The structure of the BRC4 peptide is similar to the
monomer–monomer interaction domain of RecA (the
bacterial homolog of RAD51) that is involved in filament
formation. The expression of BRC4 (or BRC3) peptides in
cells expressing GFP-tagged RAD51 blocks the ability of
RAD51 to form nuclear foci (presupposing that filament
formation and focus formation go hand-in-hand).
Previous in vitro work had suggested that BRC3 or
BRC4 peptides could inhibit the formation of RAD51
complexes (Davies et al., 2001). However, in living cells,
the effect of full-length BRCA2 is to promote homologous recombination (Moynahan et al., 2001b), expression of the BRC4 peptide inhibits BRCA2-stimulated homologous recombination (Xia et al., 2001), and,
the formation of RAD51 foci appears to require BRCA2
(Yuan et al., 1999) (and BRCA1), especially after
exposure to ionizing radiation. These observations
present a quandary: why does the BRC4 peptide inhibit
filament formation, but the whole BRCA2 protein
appears to promote filament formation? The use of the
BRC4 peptide presents an excess of the RAD51 interacting moiety, whereas the location of BRC4 (and other
BRC repeats) within the whole BRCA2 protein may
dictate a more highly controlled interaction with RAD51.
The quandary may be resolved by proposing a model of
BRCA2 as a loading factor for RAD51 on ssDNA by
means of its BRC peptide interaction. As the RAD51
bound to BRCA2 finds its position adjacent to a RAD51
monomer on ssDNA, the monomer–monomer interaction becomes favored over the BRCA2 interaction (see
Figure 3). We know that RAD51-dependent homologous
recombination and RAD51 filament formation in S phase
do occur in the absence of BRCA2. However, the
presence of BRCA2 is required to prevent major errors,
such as chromosome aberrations, which occur in
BRCA2-deficient cells (Patel et al., 1998). Whether these
errors reflect utilization of an alternative pathway of
repair, such as NHEJ (Moynahan et al., 2001b), or occur
as a result of error-prone homology-directed repair (Tutt
et al., 2001), remains to be solved.
The second paper providing structural insights into
the function of the BRCA2 protein (Yang et al., 2002)
solved the crystal structure of the C-terminal 800 amino
acids of BRCA2, completely separate from the BRC
repeat region of the protein, bound to ssDNA (an oligo
dT sequence). The BRCA2 structure delineated in this
study reveals a series of oligonucleotide-binding folds
(OB1/OB2/OB3), which bind ssDNA and a ‘tower’
region between OB2 and OB3 containing a three-helix
bundle with a structure very similar to the Hin recombinase. The structure of the OB2/OB3 domains has significant alignment with replication protein A (RPA), a
ssDNA-binding protein. Two tempting models are
raised by these observations. In one, BRCA2 could
play a role in displacing RPA from ssDNA and allowing
the formation of a RAD51 filament; alternatively,
BRCA2 could primarily bind ssDNA and directly facilitate the formation of RAD51 filaments. What is clear
is that BRCA2’s major role is to help organize RAD51
function and facilitate homologous recombination.
Nonhomologous end joining
When cells are irradiated, the immediate effect of
radiation is to cause fragmentation of double-stranded
DNA into smaller-sized pieces. Using pulse-field gel
electrophoresis, the size of these DNA fragments can be
observed to increase with time after radiation, which
reflects the process of DNA repair. It is thought that this
assay predominantly reflects the NHEJ type of DNA
DSB repair. Cells deficient in BRCA1 protein were
reported to show defects in the rejoining of fragmented
DNA after ionizing radiation, as assessed by pulse-field
gel electrophoresis (Scully et al., 1999). More recent
experiments (Wang et al., 2001) have suggested that the
magnitude of the difference in NHEJ between BRCA1deficient cells and BRCA1-proficient cells is relatively
small in comparison with the additional impact that can
be obtained with the use of the drug wortmannin.
Wortmannin is known to inhibit the activity of DNAdependent protein kinase (DNA-PK) (DiBiase et al.,
2000) and thereby suppress NHEJ. The observation that
wortmannin produces a significant effect in BRCA1deficient cells strongly suggests that there is relatively
little loss of function of the NHEJ pathway.
To date, there is no evidence that BRCA2-deficient
cells have any defect in NHEJ. Both pulsed-field gel
electrophoresis and a plasmid-based assay of V(D)J
recombination showed no differences between Capan-1,
a human pancreatic carcinoma with one deleted copy of
BRCA2 and a truncation mutation in the second, and a
BRCA2-corrected clone of Capan-1 (Xia et al., 2001).
Nuclear protein foci
When cells are exposed to ionizing radiation, aggregates
of repair proteins (now commonly known as IRIFs)
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appear within their nuclei. Depending on the protein,
the time elapsed before the IRIFs appear can vary from
less than 1 h to more than 6 h. Furthermore, the phase
of the cell cycle can influence their appearance, and
some nuclear protein foci (e.g., BRCA1, RAD51) occur
spontaneously within S phase. Investigators studying the
cellular response to ionizing radiation (IR) frequently
use IRIFs to study signaling mechanisms. However, the
precise nature of the IRIFs is still unclear, and therefore
their value is more as an indicator that some component
of the damage response pathway has changed, for
example with a genetic manipulation of the test cell.
BRCA1 and BRCA2 both aggregate into IRIFs,
usually evident within 6–8 h after exposure. Furthermore, both proteins appear to associate with RAD51,
presumably in the same multi-protein complex. BRCA2
is required for the formation of RAD51 IRIF (Yuan
et al., 1999), but in BRCA1-deficient cells, RAD51 foci
still form, implying that BRCA2 is the more critical
protein in relation to RAD51. Our own observations
have suggested that RAD51 IRIFs are seen in the
absence of BRCA1, but that more foci (both number of
foci per cell, and the percentage of cells with foci) are
seen when wtBRCA1 is re-expressed in the deficient
cells. These observations are in keeping with a prevailing
theme for the BRCA proteins – that BRCA2 directly
controls the function of RAD51, and that BRCA1 can
regulate the behavior of both proteins. IRIFs containing
the MRE11/RAD50/NBS1 (MRN) protein complex are
observed to be mutually exclusive with RAD51 containing foci, although the mechanistic reasons for this
observation are unclear. Initially, BRCA1 was reported
to interact with the MRN complex and was required for
the formation of MRN IRIFs (Zhong et al., 1999).
However, it subsequently became clear that MRN foci
form at least normally in BRCA1-deficient cells (Wu
et al., 2000), and our own observations support this
view. Indeed, our unpublished observations suggest that
MRN foci are more abundant in BRCA1-deficient cells.
DNA binding
There are reports for both BRCA1 and BRCA2 that
DNA-binding activity can be observed. For BRCA1,
limited evidence suggests that binding to complex DNA
structures, such as a four-strand structure, can be
observed in gel-shift assays (Paull et al., 2001). As a
consequence of this binding, the nucleolytic activity of
the MRN complex was inhibited. These observations are
intriguing, and may contribute significantly to BRCA1’s
function in vivo. However, the in vivo demonstration of
DNA binding has not been shown, and although
BRCA1 is known to be part of large protein complexes
(Wang et al., 2000), we do not know whether DNA
binding is critical to achieve these complexes.
For BRCA2, the recent crystal structure of an 800
amino-acid region of the C-terminus has shown ssDNA
binding, with the potential for regions of second-strand
binding, very suggestive of a protein with recombination
as its prime function (Yang et al., 2002). However, to
make a soluble complex capable of crystallizing, the
Oncogene
fragment of BRCA2 was mixed with a putative BRCA2
interacting protein, DSS-1. The resultant structure of
this region of BRCA2 was a motif-like RP-A, which is
highly capable of ssDNA binding. Even though the
preparation of the BRCA2 crystal structure was
somewhat manipulated the results usually prove to be
accurate. The idea that BRCA2’s main role is an orderly
formation of RAD51 filaments fits all the prevailing
evidence (Powell et al., 2002).
Ubiquitination
The significance of the E3-ubiquitin ligase structure of the
N-terminus of BRCA1 is unclear (Baer and Ludwig,
2002). This same region interacts with the BARD1
(BRCA1 associated Ring Domain) protein, whose Nterminus has a similar E3-ubiquitin ligase motif. To date,
there are no known targets of this ubiquitin ligase: present
speculation wonders whether their prime target is each
other, as one mechanism to regulate levels of the proteins.
However, there is no evidence for ubiquitination of these
proteins (BRCA1 or BARD1) and no evidence for
proteolytic degradation. Monoubiquitination could be a
form of signaling and may be transient and more difficult
to detect. Even though the extent of knowledge about
BRCA1 and ubiquitination is limited, it seems highly
likely that this is a critical part of the function of BRCA1.
BRCA2 has no role in ubiquitination.
Cell cycle checkpoints
Recently, Kastan and colleagues reported that BRCA1deficient cells show a defect in the arrest of DNA
synthesis after ionizing radiation damage (Xu et al.,
2001). This phenotype, the so-called radioresistant DNA
synthesis (RDS), is also a feature of ataxia telangiectasia
(AT) cells, but the precise relation between RDS and
radiation sensitivity is not clear. There are AT cell clones
in which the cells no longer show RDS, but they remain
radiation sensitive (Lehmann et al., 1986), thereby
dissociating these two features of AT cells. BRCA1deficient cells also show a defect in the G2/M phase cell
cycle checkpoint (Xu et al., 1999b, 2001), and this
phenotype has long been associated with radiation
sensitivity. The implication is that if cells cannot halt
progression into mitosis following irradiation, then
there is insufficient time to repair DNA damage prior
to entering mitosis leading to the production of lethal
DNA damage. Recent investigations to study the
relation between cell cycle checkpoint abnormalities
seen in BRCA1-deficient cells and their relation to
radiation sensitivity have suggested that the two endpoints can be dissociated. Single amino-acid mutations
in BRCA1, such as S1423A, can abrogate the G2/M
checkpoint but do not impact radiation sensitivity.
Similarly, S1387A can abrogate the S phase checkpoint
but does not affect radiation sensitivity (Xu et al., 2002).
The reports that BRCA2-deficient cells have a defect
in cell cycle checkpoints are more difficult to interpret,
since these cells show a more severe replicative failure,
with persistent DNA damage (Patel et al., 1998).
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Disruption of the interaction between BRCA2 and
RAD51 leads to radiation and chemosensitivity and a
reported defect in the G2/M transition (Chen et al.,
1999a). BRCA2 has also been found to associate with
known mitotic checkpoint proteins, such as hBUBR1
(Futamura et al., 1531), and with new proteins, such as
BRAF35, in close association with phosphorylated
histone H3 (Marmorstein et al., 2001). The S phase
checkpoint was also found to be defective in a Chinese
hamster ovary cell line mutant, recently found to be
defective in BRCA2 (Kraakman-van der Zwet et al.,
2002). Post-translational modifications of BRCA2, such
as those found for BRCA1 after ionizing radiation, are
less well documented with no clear role demonstrated as
yet in vivo (Milner et al., 2000).
Transcription-coupled repair
In addition to the function of BRCA1 in DSB repair and
cell cycle checkpoints, BRCA1 is also reported to show a
role in transcription-coupled repair of oxidative DNA
damage, presumably via base-excision repair (Gowen
et al., 1998; Le Page et al., 2000). Transcription-coupled
repair is the repair of single-stranded DNA damage,
utilizing base-excision repair and nucleotide-excision
repair, when the DNA damage occurs within transcribed genes. This feature of BRCA1-deficient cells is of
uncertain significance at the present time and does not
result in sensitivity to ultra-violet light radiation.
Cisplatin, a chemotherapeutic agent that causes intrastrand and interstrand crosslinks, sensitizes cells deficient in BRCA1 (Bhattacharyya et al., 2000). Whether
this is due to the defect in homologous recombination or
transcription-coupled repair of nucleotide excision
repair is not clear at this time. Similarly, BRCA2deficient cells have been reported to have some of the
same defects (Le Page et al., 2000), but the full
significance of these observations has not been determined. A curious feature of BRCA1 is that the majority
of the protein in a cell is found in association with RNA
polymerase II (Scully et al., 1997; Anderson et al., 1998;
Schlegel et al., 2000), and that DNA damage disrupts
that association (Li et al., 1999). BRCA1 can stimulate
transcriptional activation in vitro (Haile and Parvin,
1999) and overexpression studies have suggested a role
for BRCA1 as a transcriptional coactivator (Harkin
et al., 1999; Somasundaram et al., 1999; MacLachlan
et al., 2000). However, the significance of the overexpression studies to the in vivo function of BRCA1
remains unclear: is post-translational modification of
BRCA1 required for its transcriptional role or to inhibit
it? More definitive studies about the role of BRCA1 in
regulating transcription are awaited.
Apoptosis
Wild-type BRCA1 can promote IR-induced apoptosis in
breast and ovarian cancer cell lines (Deng and Brodie,
2000). This function of BRCA1 is dependent on the
H-RAS/MEKK4/JNK pathway and is independent of
P53 status (Thangaraju et al., 2000). The significance
of BRCA1-mediated apoptosis in regard to response of
BRCA1-deficient breast cancers to radiation and cytotoxic chemotherapy still needs to be elucidated. The role
of BRCA1 in the apoptotic process is presumably well
upstream, in that there is no direct connection between
BRCA1 and the direct regulators of apoptosis. There is
a complex sequence of events that follow DNA damage
in cells, in which DNA repair, cell cycle checkpoints and
programmed cell death (apoptosis) all contribute to the
ultimate outcome of whether a cell lives or dies. What is
clear is that cancerous cells have lost many of these
protective mechanisms, including apoptosis, and the
result is genetically unstable, but viable cells.
DNA replication
The endogenous source of double-stranded DNA
damage is DNA replication. It has been known for
some time, from bacterial studies, that homologous
recombination is required for every cycle of DNA
replication and that recombination is a normal protective mechanism for processing stalled replication forks
(Goodman, 2000). Similarly, inducible knockout of
RAD51 in vertebrate cells (Sonoda et al., 1998) results
in chromosomal fragility. The replication-dependent
signaling protein, ATR, is known to modify, posttranslationally, the BRCA1 protein (Tibbetts et al.,
2000; Gatei et al., 2001). ATR only becomes an active
kinase within S phase, and thus the most important
function of BRCA1 and BRCA2 may be in coordinating
these protective mechanisms in S phase. The BRCA1
protein becomes phosphorylated in the SQ cluster
domain within S phase, although the precise difference
between these sites of phosphorylation and those from
exogenous DNA damage is not known. When replication stalls, due to a variety of reasons such as
endogenous oxidative DNA damage, the cell has a
number of choices of how to restart replication. One
method is to partially rewind the replication fork
(utilizing a specialized helicase such as the BLM protein)
to create a four-strand structure, and template switching
can allow repair synthesis to bypass the region of the
blocking lesion. This may work for some but not all
lesions. When cleavage of the parental strand by
excision repair at the time the replication fork arrives
produces an effective DSB, or when a stalled replication
fork is deliberately cleaved to help bypass the block, the
restart of replication would require homologous recombination. In yeast, the mus81 gene is a specialized
endonuclease that can cleave stalled replication forks
(Haber and Heyer, 2001), and if mus81 and the BLM
homologe, sgs1, are both deleted, the yeast is a synthetic
lethal (i.e., it dies in DNA replication). In other words,
cells need mechanisms to successfully negotiate stalled
replication forks and have at least two genetically
defined options. The suggested primary role of the
BRCA1-/BRCA2-defined pathway is to facilitate homologous recombination in the bypass of stalled replication
forks. As to the question of how cancer arises in cells
with deficiencies in BRCA1 or BRCA2, it seems likely
that the impaired function of homologous recombination is a key step, since this is the only known defect in
Oncogene
BRCA1 and BRCA2 in DNA replication fidelity
SN Powell et al
5790
BRCA2-deficient cells. A ‘mutator’ phenotype can be
triggered by a defect in the regulation of homologous
recombination, either by making the process errorprone or by shunting repair events into a NHEJ
pathway that is inherently mutagenic. Additional mutational steps, to bypass the proliferative block, are also
necessary to allow a net growth in the number of cancer
cells. However, what remains, is the unsolved mystery of
why this described defect in homologous recombination
leads to the tissue-specific cancers of the breast and
ovary. Proliferation in breast or ovarian epithelium may
be associated with higher levels of endogenous DNA
damage, relative to other tissues, which leads to
replication stalling and requires homologous recombination for replication restart.
Maintenance of genomic integrity
Defining the physiological function of BRCA1 that
maintains genomic integrity has proven to be extremely
complicated. Studies have ascribed roles for BRCA1 in
an overwhelming number of processes, including DNA
replication, cell cycle checkpoint control, DNA repair,
regulation of transcription, protein ubiquitination,
apoptosis, and chromatin remodeling. On the other
hand, there has been only one function delineated so far
for BRCA2 – as a cofactor in homologous recombination that requires its interaction with RAD51 (Davies
et al., 2001; Xia et al., 2001). As such, the role of
BRCA2 in regulating RAD51-dependent homologous
recombination may be the key to understanding the
cancer-prone phenotype of both BRCA1 and BRCA2
mutations.
Based on all the evidence presented in this review, it
seems likely that the role of BRCA1 and BRCA2 in
facilitating orderly homologous recombination is the
key to maintaining genomic integrity. In the absence of
these key proteins, replication errors produce a mutator
phenotype that is a key feature of carcinogenesis. The
cooperation between loss function of BRCA1/BRCA2
and other key genetic changes that are required for
maintaining cell viability is the key remaining question.
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