Download Cellular functions of the BRCA tumour

S.J. Boulton1
DNA Damage Response Laboratory, Cancer Research UK, The London Research Institute, Clare Hall Laboratories, South Mimms EN6 3LD, U.K.
Colworth Medal Lecture
Delivered at the SECC, Glasgow,
on 26 July 2006
Simon Boulton
Abstract
Inherited germline mutations in either BRCA1 or BRCA2 confer a
significant lifetime risk of developing breast or ovarian cancer.
Defining how these two genes function at the cellular level is
essential for understanding their role in tumour suppression.
Although BRCA1 and BRCA2 were independently cloned over
10 years ago, it is only in the last few years that significant
progress has been made towards understanding their function
in cells. It is now widely accepted that both genes play critical
roles in the maintenance of genome stability. Evidence implicates BRCA2 as an integral component of the homologous recombination machinery, whereas BRCA1 is an E3 ubiquitin
ligase that has an impact on DNA repair, transcriptional regulation, cell-cycle progression and meiotic sex chromosome
inactivation. In this article, I will review the most recent advances and provide a perspective of potential future directions
in this field.
Introduction
Breast cancer is the second leading cause of cancer-related
deaths in women and represents the leading cause of cancer
Key words: BRCA1, BRCA2, DNA repair, DNA-damage sensing, tumour suppressor.
Abbreviations used: ATM, ataxia telangiectasia mutated; ATR, ATM- and Rad3-related; BAP,
BRCA1-associated protein; BARD1, BRCA1-associated RING domain 1; BRCT, BRCA1 C-terminus;
BRIP, BRCA1-interacting protein; Cdk, cyclin-dependent kinase; CtIP, C-terminal-binding-proteininteracting protein; DSB, double-strand break; dsDNA, double-stranded DNA; FA, Fanconi’s
anaemia; FANC, FA complementation group; GST, glutathione S-transferase; HR, homologous
recombination; MRN, Mre11–Rad50–Nbs1; MSCI, meiotic sex chromosome inactivation; NHEJ,
non-homologous end joining; OB, oligonucleotide/oligosaccharide-binding; SCF, Skp1/cullin/Fbox; siRNA, small interfering RNA; SSA, single-strand annealing; ssDNA, single-stranded DNA;
Ub, ubiquitin; Xi, inactive X chromosome; XIST, Xi-specific transcript.
1
email [email protected]
incidence. Of the total number of woman diagnosed with
breast cancer, approx. 10% have a reported history of the
disease. The fact that the disease has a high prevalence in
certain families led to the notion that this subset of breast
cancer cases may be attributed to inheritance of predisposing
mutations in cancer-susceptibility genes [1–3]. Indeed, in
1991, Mary-Claire King and co-workers used linkage analysis
to assign BRCA1, the first breast cancer-susceptibility gene,
to chromosome 17 [4]. Then, 3 years later, Mark Skolnick
and colleagues cloned the BRCA1 gene [5]. BRCA1 was subsequently shown to harbour truncating mutations in a large
number of familial breast cancer cases, and over 300 missense
variants of BRCA1 have been identified in patients to date
[5]. Search for a second breast cancer-susceptibility gene was
prompted by the absence of BRCA1 mutations in families
with a high incidence of male breast cancer [6]. Linkage analysis mapped BRCA2 to chromosome 13, and the identity
of the gene was reported in 1995 [7,8]. It is now widely
accepted that individuals that inherit a germline mutation
in BRCA1 or BRCA2 have a significantly increased lifetime
risk of developing breast or ovarian cancer. Approx. 80%
of individuals with a BRCA1 or BRCA2 mutation develop
breast cancer by the age of 70 years, whereas 40% of carriers with BRCA1 mutations and 20% with BRCA2 mutations develop ovarian cancer by a similar age. Consistent
with the paradigm of tumour-suppressor genes, tumours
arising in individuals with BRCA1 or BRCA2 mutations
consistently exhibit LOH (loss-of-heterozygosity) of the
wild-type second allele [1–3].
Colworth Medal Lecture
Cellular functions of the BRCA
tumour-suppressor proteins
BRCA1 and BRCA2 function in the
maintenance of genome integrity
Since their identification over a decade ago, the challenge of
defining the cellular functions of BRCA1 and BRCA2 has received considerable attention. Primarily, through the analysis
of chicken DT40 knockout cell lines, knockout mice and
tumour-derived human cell lines, it has become widely established that both BRCA1 and BRCA2 perform roles in the
maintenance of genome stability, where they function as
‘caretakers’ of the genome [9,10]. That is, cells that are defective for either BRCA1 or BRCA2 exhibit extensive genome instability. This phenotype is strikingly demonstrated
through the use of spectral karyotyping, a chromosome painting technique that reveals profound chromosomal translocation, duplications and aberrant fusion events between
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non-homologous chromosomes in BRCA1- and BRCA2deficient cells [11–15]. The genome instability phenotype
associated with the loss of BRCA1 or BRCA2 is a hallmark
of a number of human diseases, including FA (Fanconi’s anaemia) and Bloom’s syndromes [16,17]. These diseases share a
common underlying defect in DNA-repair or DNA-damagesensing processes. Indeed, the observation that BRCA1 and
BRCA2 proteins form nuclear foci upon treatment with
DNA-damaging agents led to the hypothesis that both genes
function in the maintenance of genome stability, potentially
through a direct role in DNA repair. This possibility received
further support by the findings that BRCA1 and BRCA2
foci co-localize with the DNA-repair protein Rad51 that is
essential for the accurate repair of DNA DSBs (double-strand
breaks) [18–20].
Eukaryotic cells possess at least three distinct pathways for
DSB repair including NHEJ (non-homologous end joining),
SSA (single-strand annealing) and HR (homologous recombination) [21,22]. NHEJ involves the direct re-ligation of the
broken DNA ends and can be error prone in nature owing
to the loss of terminal bases that may be removed in order
for ligation to occur efficiently. Also, NHEJ does not rely on
extensive sequence recognition for repair to proceed so has
the capacity to ligate DNA ends from non-homologous chromosomes. This type of aberrant repair may result in acentric
or dicentric chromosomes that will pose further problems
to the cell during chromosome segregation at mitosis. In the
case of acentric chromosomes, the remaining chromosome
fragment may be lost, whereas dicentric chromosomes may
undergo breakage at mitosis, followed by repair and further
aberrant rearrangements. Indeed, inappropriate utilization of
NHEJ is thought to be one of the major causes of DNA rearrangements and translocation in cells. SSA, on the other
hand, promotes DSB repair by annealing short stretches of sequence homology that flank the break site that are uncovered
following DSB resection. Repair between short stretches of
sequence homology results in the deletion of intervening sequences and loss of genetic information. In contrast with
NHEJ and SSA that are error-prone in nature, HR is the predominant mechanism employed to accurately repair DSBs in
S-phase and G2 -phase cells. HR is initiated by nucleolytic
processing of the DSB to generate recombination-proficient
ssDNA (single-stranded DNA) overhangs that are substrates
for the recombinase enzyme Rad51, the eukaryotic counterpart of RecA. In the presence of ATP, Rad51 forms a helical
nucleoprotein filament on ssDNA. Within the context of
the nucleoprotein filament, Rad51 is able to search for an
intact homologous template and then catalyse invasion of
the ssDNA into a donor sister chromatid or homologous
chromosome to form a joint molecule. The resulting joint
molecule acts as a primer for DNA synthesis to extend the
heteroduplex DNA that, following further processing and
resolution of the joint DNA molecules, leads to repair of the
DSB and restoration of DNA integrity [21,22].
The co-localization of Rad51, BRCA1 and BRCA2 at
nuclear foci implied that these proteins function together
in DNA-repair processes [19,20,23]. Although BRCA1 and
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BRCA2 have both been shown to associate with Rad51 in
pull-down experiments [19,20,23], the interaction between
BRCA1 and Rad51 appears to be indirect and the biological
relevance of this interaction has not been forthcoming.
However, BRCA2 does interact directly with Rad51 and is
an integral component of the HR machinery in human cells
(see below). Although a direct involvement for BRCA1 in
HR processes has remained elusive, BRCA1-defective cells
are compromised for homology-directed repair of DSBs [24],
although this defect is not as pronounced as that observed in
BRCA2-defective cells, such as CAPAN-1 [11].
One of the complicating issues with defining the cellular
function of BRCA1 is that its loss in cells confers a pleiotropic
phenotype that cannot be exclusively attributed to a defect in
DNA-repair processes. In fact, BRCA1 has been implicated
in a number of diverse cellular processes [25]. Xu et al.
[26] demonstrated that BRCA1 is required for S-phase and
G2 /M checkpoint arrest in response to DNA damage. ATM
(ataxia telangiectasia mutated)-dependent phosphorylation
of BRCA1 at Ser1423 is important for its role in the G2 /M
checkpoint, but not for its role in the S-phase checkpoint,
as mutation of this site compromised its G2 function [26].
Current evidence suggests that BRCA1 functions as a checkpoint mediator, similar to Mdc1, 53BP1 and claspin,
that collectively facilitates interactions between ATM/ATR
(ATM- and Rad3-related) and their substrates by mediating
the spatiotemporal assembly of damage-specific multiprotein
complexes at and around repair sites [27]. In BRCA1 exon 11
isoform-deficient cells, centrosome amplification is also observed that leads to the formation of multiple spindle poles
in a single cell [14]. BRCA1 also has an impact on transcriptional activation. Hartman and Ford [28] showed that
BRCA1 could enhance the expression of XPC (xeroderma
pigmentosum C), DDB2 (damage-specific DNA-binding
protein 2) and GADD45 (growth-arrest and DNA-damageinducible protein 45), independent of p53, but it is currently
unclear whether this effect is direct or an indirect consequence
of its roles in DNA repair and checkpoint signalling [28].
Links with transcription have also been proposed based on
the observation that BRCA1 can associate with the RNA
polymerase II holoenzyme complex via RNA helicase A and
by the finding that the BRCT (BRCA1 C-terminus) domains
at the C-terminus can function as a transcriptional activator
when fused to the DNA-binding domain of Gal4 [29–33].
BRCA1 is clearly dispensable for basal transcription function
and is not an essential component of RNA polymerase II,
so it remains to be seen whether the association of BRCA1
with transcription is biologically relevant. Ganesan et al.
[34] also identified a role for BRCA1 in supporting XIST
(inactive X chromosome-specific transcript) RNA concentration required for X chromosome inactivation. They
demonstrated that BRCA1 co-localizes with markers on the
inactive X chromosome (Xi) and binds to XIST RNA by
chromatin immunoprecipitation. In BRCA1-deficient cells,
the Xi chromatin structure is altered and leads to expression
of an otherwise Xi-silenced transgene tagged with GFP (green
fluorescent protein) [34]. A specialized role for BRCA1 on X
Cellular functions of the BRCA tumour-suppressor proteins
Figure 1 BRCA1: domains, interacting proteins, activities and functions
Schematic diagram of the BRCA1 protein depicting the position of the RING, nuclear localization signal (NLS) and BRCT
domains. The indicated BRCA1-interacting proteins are shown under the region of BRCA1 required for their association.
Interacting proteins important for BRCA1 function in vivo are shown in blue. Interacting proteins whose relevance to BRCA1
function remains unclear are shown in black. aa, amino acids. HDAC, histone deacetylase; pRB, retinoblastoma family protein.
and Y chromosomes has also been suggested by Turner et al.
[35]. During the pachytene stage of meiosis I, the X and Y
chromosomes form a condensed chromatin domain termed
the sex of XY body that is transcriptionally silent through
a process referred to as MSCI (meiotic sex chromosome
inactivation). ATR localization to the XY body is important
for the establishment of MSCI through H2AX histone phosphorylation. Both ATR localization and H2AX phosphorylation were shown to be abolished in BRCA1-deficient cells,
revealing a role for BRCA1 in MSCI [35]. The current
challenge to the field is to understand how BRCA1 has an
impact functionally on these different cellular processes.
The domain structure of BRCA1
The human BRCA1 gene comprises 24 exons and encodes an
1863-amino-acid protein (Figure 1). Sequence conservation
between mammalian species is weak, with the exception of
two highly conserved domains located in the N- and Cterminal regions of the protein, which include a RING domain located in the N-terminus and two tandem BRCT motifs at the extreme C-terminal end (Figure 1). The N-terminal
RING domain was identified as soon as the BRCA1 gene
was cloned on the basis of homology with similar domains
found in proteins that interact directly or indirectly with
DNA. The RING domain of BRCA1 encompasses the first
109 amino acids. Within this region of the protein a characteristic core of approx. 50 amino acids contains a conserved
pattern of seven cysteine residues and one histidine residue
arranged in an interleaved fashion to form a structure responsible for co-ordinating the binding of two Zn2+ ions [36].
Unlike some RING domains, this motif in BRCA1 does
not bind directly to DNA, rather it forms an interaction
surface responsible for heterodimerization with a structurally
related protein BARD1 (BRCA1-associated RING domain
1) [37]. Like BRCA1, BARD1 also possesses an N-terminal
RING domain and two BRCT motifs at the C-terminus,
in addition to three ankyrin repeats of unknown function.
Of the many protein interaction partners identified with
BRCA1, the significance of its association with BARD1 is
beyond question and is supported by numerous in vitro and
in vivo observations. Both proteins co-localize at repair
and S-phase foci and require each other for focus formation
[19]. BRCA1 and BARD1 mutant cell lines and knockout
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mice exhibit indistinguishable phenotypes indicative of a
shared functional role in vivo [13,38,39]. Three-dimensional
structural analysis of the BRCA1–BARD1 RING heterodimer complex indicates that these proteins interact through
an extensive four-helix bundle formed by helices that flank
the core RING motif (amino acids 24–64) [36,40]. Although
mutations in the BARD1 gene associated with breast
cancer predisposition are very infrequent compared with the
frequency of BRCA1 mutations, a number of tumour-derived
mutations have been found [41]. Despite the surprisingly
low frequency of BARD1 mutations found in tumours,
the interaction surface responsible for BRCA1–BARD1
heterodimerization is targeted for mutation in BRCA1.
Indeed, missense mutation in five of the critical Zn2+ binding residues in the BRCA1 RING have been found in
tumours and functional analysis has shown that many of these
missense mutations reduce or abolish heterodimerization
in vitro [9]. A number of studies also indicate that BRCA1–
BARD1 heterodimerization is important for stability of the
two proteins in vivo [42,43]. Collectively, these observations
indicate that BRCA1 functions as a heterodimer with BARD1
in vivo and this association and the structural integrity of
the RING domain is of critical importance for the tumoursuppression function of BRCA1.
Cancer-predisposing missense and truncating mutations in
BRCA1 are also found within the two BRCT motifs at the
C-terminus of the protein indicating that, like the RING
domain, the function of the BRCT motifs is also of critical
importance for tumour suppression [44]. The BRCT motif is
an approx. 100-amino-acid domain that is present in a number
of other DNA–repair and DNA-damage-response proteins,
including 53BP1, MDC1, XRCC1 and budding yeast Rad9
[45]. Structural analysis of the two BRCT motifs in BRCA1
reveals that the individual motifs form a similar structure to
each other that are packed together in a head-to-tail configuration. Many of the tumour-derived missense mutations
in this region of BRCA1 map to the interface between the
two BRCT motifs and result in destabilization of the BRCT
structure [45]. A number of BRCA1–interacting factors, such
as CtIP (C-terminal-binding-protein-interacting protein),
BRIP (BRCA1-interacting protein) and p300, interact via the
BRCT, supporting the notion that this part of the protein
functions as a multipurpose protein–protein interaction
module [46,47]. Indeed, recent studies have demonstrated
that BRCT motifs can function as phosphopeptide-binding
sites that can mediate protein–protein interactions with phosphoproteins [48,49]. Furthermore, BRCA1 is known to bind
to phosphorylated CtIP and BRIP/FANCJ (FA complementation group J) via its BRCT motifs [47]. The presence
of BRCT motifs in BRCA1 and BARD1 therefore raises
the possibility that binding to phosphoproteins may have
important functional implications.
BRCA1 function in a simple eukaryote
The importance of BRCA1 to DNA-damage-repair processes
is also highlighted by the conservation of certain cellular
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functions in the simple metazoan, Caenorhabditis elegans
[50]. The nematode orthologues of BRCA1 and BARD1
(CeBRC-1 and CeBRD-1 respectively) share extensive sequence and domain conservation with their counterparts
in higher eukaryotes, including RING, ankyrin and BRCT
domains. Although CeBRC-1 is shorter than human BRCA1,
it is highly related to the major human BRCA1 splice variant,
exon delta-11. CeBRC-1 and CeBRD-1 interact to form a
heterodimer via their respective RING domains [50]. As is
the case in Xenopus laevis and in humans [42,43], heterodimerization of CeBRC-1 and CeBRD-1 is required for
stable expression of each protein [51]. Cebrc-1 or Cebrd-1
mutants exhibit indistinguishable phenotypes consistent with
a shared role in vivo that are highly reminiscent of human
cell lines compromised for BRCA1. Specifically, Cebrc-1 or
Cebrd-1 mutants display enhanced p53-dependent germ cell
death, radiation sensitivity and chromosome fragmentation,
consistent with roles in DNA-damage-repair processes [50].
CeBRC-1 and CeBRD-1 also form foci at sites of DNA
damage where they co-localize with Rad51 [51]. The ability
of CeBRC-1 and CeBRD-1 to form repair foci is dependent
on the C. elegans MRN (Mre11–Rad50–Nbs1) complex, but
independent of Rad51 and CeBRC-2 (C. elegans BRCA2)
[51]. It is of notable interest that ionizing radiationinduced CeBRC-1/CeBRD-1 foci are restricted to the mitotic
compartment of the C. elegans germline, the only region of
the adult animal that is actively proliferating. This implies that
CeBRC-1 and CeBRD-1 do not simply respond to DSBs,
rather they are most likely to be recruited to sites of ionizing
radiation-induced replication stress. The distinction between
responding to replication stress and to DSBs is supported
further by the fact that CeBRC-1 and CeBRD-1 do not form
discrete foci at sites of SPO-11 induced meiotic DSBs and are
dispensable for meiotic recombination and the generation of
crossover products that are evident at diakinesis (results not
shown). This behaviour contrasts with CeBRC-2 and Rad51
that form foci at meiotic DSBs, are recruited to ionizing
radiation-induced repair foci throughout the germline (rather
than being restricted to nuclei in the mitotic region), and
are both essential for generating crossover products by
recombination [52,53].
Perhaps surprisingly, Cebrc-1 or Cebrd-1 mutants are
dispensable for checkpoint-dependent cell-cycle arrest following DNA damage. This contrasts with the failure to
induce cell-cycle arrest in atl-1 (C. elegans ATR) mutants, a
known component of the S-phase checkpoint, following hydroxyurea or ionizing radiation treatments [54]. The fact that
Cebrc-1 or Cebrd-1 is dispensable for checkpoint functions
indicates that the sensitivity of Cebrc-1 or Cebrd-1 mutants
to DNA-damaging agents is due to DNA-repair defects and
not due to a defect in checkpoint signalling. This observation
also indicates that the checkpoint function of human BRCA1
has been acquired late on in evolution. Finally, a role for
BRCA1 on the X chromosome may also be conserved,
as Cebrc-1 or Cebrd-1 mutants exhibit a Him phenotype
that arises due to non-disjunction of the X chromosome,
indicative of a problem in meiotic chromosome segregation
Cellular functions of the BRCA tumour-suppressor proteins
[50]. The precise function of CeBRC-1 and CeBRD-1 in
meiosis is currently unknown.
BRCA1 interaction proteins: true and false
It is possible that the diverse functions of BRCA1 manifest
through its ability to interact with many different proteins
(Figure 1). Indeed, BRCA1 has been reported to interact with
tumour-suppressor genes, oncogenes, transcriptional activators and repressors, DNA-damage checkpoint components,
cell-cycle regulators and ubiquitylation factors [9,10,25,47].
However, the biological significance of many of the reported
interactions are as yet unclear.
BARD1 and BRCA1 and their homologues in other species
interact to form a heterodimer, co-localize at repair foci,
require each other for protein stability and function together
as an E3 Ub (ubiquitin) ligase (see below). BARD1- and
BRCA1-deficient cells or animal knockouts exhibit strikingly
similar phenotypes. BARD1 is essential for BRCA1 function
in vivo. UbcH5c associates with a region that encompasses
the RING domain of BRCA1 and co-operates with BRCA1–
BARD1 in ubiquitylation reactions in vitro [42,55–59]. Depletion of UbcH5c in C. elegans or human cells abolished
BRCA1-mediated ubiquitylation in vivo [51]. UbcH5c is
therefore of critical importance to the ubiquitylation function
of BRCA1. BAP (BRCA1-associated protein) is a C-terminal
Ub hydrolase reported to interact with the N-terminal region
of BRCA1 and is predicted to function as a de-ubiquitylation
enzyme [60]. Although BRCA1 and BARD1 undergo autopolyubiquitylation in vitro, BAP1 does not catalyse their deubiquitylation [58]. Evidence that BAP performs a cellular
role that impacts on BRCA1 function has not been forthcoming. The MRN complex is one of the primary sensors of
DNA damage in eukaryotic cells and is implicated in DNA
repair and checkpoint signalling through its ability to tether
broken DNA ends in manner analogous to ‘molecular Velcro’
[61]. The MRN complex is essential for BRCA1 function
as it forms a stable association with BRCA1 in response to
DNA damage [62], its C. elegans counterpart is required for
efficient recruitment of CeBRC-1 and CeBRD-1 to repair
foci, and both C. elegans and human MRN complexes are
required for BRCA1-mediated ubiquitylation events at sites
of DNA damage in vivo [51]. The BRCC complex contains
BRCA1, BARD1, BRCA2, Rad51, BRCC36 and BRCC45
[56]. The significance of the interaction between BRCA1,
BRCA2 and Rad51 is not known, although these proteins
do co-localize at repair foci and similar interactions have
been reported for their C. elegans counterparts [20,51,63].
BRCC36 and BRCC45 have been reported to simulate
BRCA1–BARD1-mediated ubiquitylation reactions in vitro,
but the relevance of these proteins in vivo has not been
explored in any great detail [56]. BRIP/FANCJ and CtIP
are phosphoproteins that interact with the BRCT domains of
BRCA1 [47]. CtIP and BRCA1 are both required for efficient
activation of the S-phase checkpoint in response to DNA
damage, whereas BRIP/FANCJ are not [62]. The relevance
of the association between BRIP/FANCJ and BRCA1 has
been called into question by experiments in chicken DT40
cells [64]. It is possible that this interaction is only relevant
to BRCA1 function in human cells [46]. Phosphorylation of
γ H2AX is one of the earliest events that occurs in response
to DNA damage and is important for the recruitment
and/or retention of BRCA1 at repair foci [27,65,66]. It is
believed that this association is mediated through the binding
of phospho-γ H2AX by the BRCT domain in BRCA1.
Consistent with this role, cell lines deficient for γ H2AX
are compromised for BRCA1-mediated ubiquitylation events
at DNA-damage sites [51]. RNA polymerase II is essential
for transcription and is reported to interact with BRCA1
and is ubiquitylated by BRCA1–BARD1 in vitro [25,47].
Although, RNA polymerase II is ubiquitylated in vivo,
this modification still occurs in BRCA1-deficient cells, and
evidence to support an important biological connection
between these proteins remains ambiguous. E2F1, pRB, p53,
p300 and HDAC1/2 (histone deacetylase 1/2) are reported
to interact with BRCA1, but the functional relevance of their
association is not known at present [25,47] (Figure 1).
BRCA1–BARD1: an E3 Ub ligase
The RING domain present in a subset of proteins in the
RING domain family confers E3 Ub-ligase activity. Ubiquitylation is the stepwise process by which a target protein
is modified by covalent attachment of mono-Ub or polyUb chains [67]. This process occurs in an ATP-dependent
manner and is initiated by the formation of a thiol-ester bond
between a cysteine residue in the Ub-activating enzyme (E1)
and the C-terminal glycine residue of Ub. The second step
involves the transfer of Ub from the E1 enzyme to an E3
Ub-conjugating enzyme, with the formation of a new thiolester bond. Finally, an E3 Ub ligase catalyses the transfer
of Ub from the E2 to a lysine residue in the target protein.
Ubiquitylation is most commonly known for its role in proteolysis. In such cases, proteins modified with Lys48 poly-Ub
chains are recognized by the 26 S proteasome and targeted
for degradation. However, a role in protein turnover is not
the only function of ubiquitylation, as it is now known that
Ub chains can be formed by linkage through any one of the
seven lysine residues in Ub [68]. For example, Lys63 poly-Ub
chains impinge on DNA repair, translation and cellular transport independent of proteolysis. The consequence of ubiquitylation to form Lys6 , Lys11 , Lys27 , Lys29 , Lys33 or Lys63
chains on a given target protein remains to be defined, but
such modifications may alter cellular localization, change
enzymatic activity, regulate protein–protein interactions or
participate in signal transduction. The presence of a RING
domain in BRCA1 therefore prompted Lorick et al. [69] to
test for an associated E3 Ub-ligase activity. This study revealed that an N-terminal fragment of BRCA1 comprising the
first 788 amino acids possesses this activity when a recombinant fragment of BRCA1 fused to glutathione S-transferase
was incubated in the presence of E1 Ub-activating enzyme,
Escherichia coli lysate containing the human E2 Ub-conjugating enzyme, UbcH5b, and ATP [69]. A subsequent study
showed that a minimal region of 78 amino acids including the
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RING domain was sufficient for E3 Ub-ligase activity [59].
But of physiological importance, it was shown that this
activity is abolished by a tumour-derived mutation (C61G) in
one of the conserved cysteine residues within the RING
domain [59]. The fact that BRCA1 exists as a heterodimer
with BARD1 prompted subsequent studies of the ubiquitylation function of BRCA1 in combination with BARD1.
Hashizume et al. [57] found that the associated E3 Ub-ligase
activity of BRCA1 is dramatically enhanced when fragments
of BRCA1 (amino acids 1–304) and BARD1 (amino acids
25–189) proteins were combined in vitro [57]. The enhanced
E3 Ub-ligase activity of the BRCA1-BARD1 RING heterodimer is also abolished by the tumour-derived C61G mutation [57]. Collectively these studies demonstrated that the
BRCA1–BARD1 heterodimer catalyses ubiquitylation reactions in vitro when combined with E1 and E2 enzymes that
is dependent on the structural integrity of the BRCA1 RING
domain.
In contrast with HECT (homologous with the E6-associated protein C-terminus) domain E3 ligases that form a ubiquitylated intermediate as part of the transfer of Ub to a substrate, RING domain E3 ligases, such as BRCA1–BARD1,
are believed to stimulate the transfer of Ub from the E2
directly to the substrate [70,71]. In the initial studies of
BRCA1-mediated ubiquitylation, the ubiquitylated species
observed in the reaction was most likely to be the autoubiquitylation of BRCA1 and/or BARD1. Indeed, Mallery
et al. [58] demonstrated using purified full-length BRCA1–
BARD1 proteins that their associated E3 Ub-ligase activity
catalyses the formation of multiple poly-Ub chains on itself.
This auto-ubiquitylation reaction was found to stimulate the
E3 Ub-ligase activity of the heterodimer approx. 20-fold [58].
This suggested one mechanism through which this activity
may be regulated, but how auto-ubiquitylation is able to
simulate catalysis and whether this activation occurs in vivo
remains unclear. It has also been shown that BRCA1–BARD1
auto-ubiquitylation occurs through Lys6 in Ub, suggesting
that this enzyme may conjugate Ub through less conventional
linkages in vivo [72,73]. However, the Ub linkage(s) conjugated by BRCA1–BARD1 remains somewhat controversial, as
a number of independent studies have reached very different
conclusions. The majority of studies to determine the lysine
residue on Ub that is conjugated by a given E3 enzyme rely
on a panel of seven Ub mutants mutated for all but one of the
lysine residues able to form Ub chains. It is currently unclear
whether all of these seven different Ub mutants fold correctly,
so interpretation of data obtained with these mutant proteins
needs to be treated with caution.
The relevance of BRCA1-mediated ubiquitylation reactions in vivo was supported further by Dong et al. [56] who
demonstrated that a holoenzyme complex, termed BRCC,
that was purified from H1299 or HEK-293 (human embryonic kidney)-derived cell lines by virtue of a FLAG-tagged
BARD1, contained BRCA1, BRCA2, Rad51 and two uncharacterized proteins (BRCC36 and BRCC45) and possessed E3 Ub-ligase activity. Comparison of the recruitment
of recombinant BRCA1–BARD1 with that of the purified
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BRCC complex for E2 Ub-conjugating enzymes revealed a
preference for the E2 UbcH5c in vitro [56]. Through the utilization of an antibody that specifically recognizes conjugated
Ub, but not the free form of Ub, Morris and Solomon [73]
were able to visualize ubiquitylation events in vivo. In this
important study, it was shown that conjugated ubiquitylation
events are readily detected at stalled replication forks in
S-phase cells following treatment with hydroxyurea, an
inhibitor of ribonucleotide reductase, and at sites of DSB
repair following exposure to ionizing radiation. Strikingly,
ubiquitylation events at DNA-damage sites co-localized with
BRCA1 repair foci and through the use of siRNA (small
interfering RNA) technology, it was shown that depletion of
BRCA1 or BARD1 proteins abolished ubiquitylation events
at stalled replication forks and DSB repair sites in vivo [73]
(Figure 2). Furthermore, ectopic expression of the K6R
mutant of Ub also abolished ubiquitylation events at stalled
replication forks and DSB repair sites in vivo, consistent
with the fact that activation of BRCA1–BARD1 by autopolyubiquitylation requires Lys6 Ub chain conjugation [73].
This study established an assay to monitor BRCA1-mediated
ubiquitylation events at repair foci, but how this activity is
activated following DNA damage and the identity of the
ubiquitylated proteins at repair sites remains unknown.
Polanowska et al. [51,74] recently examined the role
of C. elegans BRCA1–BARD1 (CeBRC-1 and CeBRD-1;
CeBCD) in DNA-repair processes through the biochemical
characterization of the CeBCD complex purified from whole
C. elegans extracts before and after exposure to ionizing
radiation. Analogous to the ubiquitylation activity of the
human BRCC complex, the CeBCD complex possessed E3
Ub-ligase activity in vitro and utilizes the E2 Ub-conjugating
enzyme Ubc5(LET-70), the C. elegans orthologue of human
UbcH5c. The biological relevance to the DNA-damage repair
of CeBCD dependent ubiquitylation activity and Ubc5(LET70) was demonstrated by analysing ubiquitylation events
induced by damage in different genetic backgrounds in a
manner analogous to the study by Morris and Solomon [73].
Consistent with a conserved response in C. elegans and substantiating previous work in human cells, ionizing radiation
treatment was found to induce the formation of nuclear conjugated Ub foci specifically in the mitotic compartment of
the germline, the only region in the adult animal that is
actively proliferating. Importantly, these foci were abolished
in Cebrc-1, Cebrd-1 and ubc5(let-70) mutants, indicating that
ubiquitylation events at DNA-damage sites are dependent
on CeBCD [51]. The latter also provided the first in vivo
evidence that UbcH5c homologues, including UBC5(LET70), are the bona fide E2 Ub-conjugating enzymes required
for CeBCD-dependent ubiquitylation events. Consistent
with these data in C. elegans, siRNA knockdown for three
different E2 Ub-conjugating enzymes revealed that UbcH5c,
and not UbcH2 or Ube2K, is required for BRCA1-dependent
ubiquitylation at DNA-damage sites in human cells. Collectively, these data established UbcH5c homologues as the E2
Ub-conjugating enzymes that function with BRCA1 in vivo
[51] (Figure 2).
Cellular functions of the BRCA tumour-suppressor proteins
Figure 2 Regulation of BRCA1-mediated ubiquitylation at DNA-damage sites
Representative images of fixed MCF7 cells before and 1 h after 2 Gy of ionizing radiation, and then immunostained with
antibodies specific to conjugated Ub (FK2) and counterstained with DAPI (4 ,6-diamidino-2-phenylindole), are shown on the
left. Conjugated (Conj.) Ub foci are induced at DNA-damage sites. The pathway responsible for activating BRCA1-mediated
ubiquitylation events is shown on the right. Cells deficient for the MRN complex, ATM, ATR, γ H2AX, BRCA1 and UbcH5c (E2
Ub-conjugating enzyme) are defective for the formation of conjugated Ub foci are induced at DNA-damage sites [51]. Once
recruited and activated, BRCA1/BARD1–UbcH5c catalyses ubiquitylation of unknown factors at sites of DNA damage that are
predicted to function in DNA-damage signalling and/or repair.
Interplay between BRCA1 and the
DNA-damage checkpoint
Of particular interest was the finding that a stable complex
between CeBCD and Ubc5(LET-70) in C. elegans and between BRCA1 and UbcH5c in human cells was detected
specifically after DNA damage [51,74]. A role for the DNAdamage checkpoint in promoting this association was inferred
from the observation that the association between the respective C. elegans and human proteins, and consequently E3
Ub-ligase activity, was abrogated by treatment with caffeine,
an inhibitor of the DNA-damage checkpoint [51]. Moreover,
cell lines compromised for the DNA-damage checkpoint,
including Nbs1−/− , A-T (ataxia telangiectasia) and ATRSeckel cells (defective for Nbs1, ATM and ATR respectively)
were found to be defective for the damage-induced association between BRCA1 and UbcH5c. Consistent with these
observations, CeBCD-mediated ubiquitylation at DNAdamage sites in C. elegans was shown to depend on the checkpoint genes mre-11 and atl-1 (C. elegans ATR) and BRCA1mediated ubiquitylation at DNA-damage sites in human cells
was shown to require Mre11, Nbs1, ATM, ATR and
γ -H2AX. Collectively, these data revealed that CeBCD/
BRCA1-dependent ubiquitylation is a conserved response
to DNA damage that is activated by the checkpoint [51,74]
(Figure 2).
At least one way the DNA-damage checkpoint has an
impact on this response to DNA damage is by regulating the
association between BRCA1–BARD1 and its specific E2
Ub-conjugating enzyme, UbcH5c. The formation of a stable,
damage-specific, interaction between CeBCD/BRCA1 and
its E2 enzyme may therefore depend on a protein conformational change induced after damage or through association
with an adaptor protein. Indeed, human BRCA1 is phosphorylated directly by ATM and ATR in response to DNA
damage [75] and, although the functional consequence of this
interaction remains unclear, it is plausible that phosphorylation may stabilize the association between BRCA1 and
UbcH5c. Stabilization of the E2–E3 association could also occur following auto-polyubiquitylation of BRCA1–BARD1
[58]. UbcH5c may also be a direct target for checkpointdependent phosphorylation, but this is currently unknown.
Checkpoint-dependent phosphorylation could mark substrates for binding and subsequent ubiquitylation, analogous
to SCF (Skp1/cullin/F-box) Ub ligases that interact and
ubiquitylate their substrates in a phosphorylation-dependent
manner [76]. Substrate–E3 interaction could be mediated by
the phosphopeptide-binding activity of the BRCT domains
that would also bring the substrate into close juxtaposition
with the ubiquitylation activity of the RING domain. With a
total of four BRCT domains, it is tempting to speculate that
the BRCA1–BARD1 heterodimer may bind to more than one
phosphorylated protein at a time or have two interfaces for
defining substrate binding and specificity. It is also possible
that one of the BRCT domains may bind to γ -H2AX at
DNA-damage sites [65], while the other domain binds to its
substrate(s).
Elusive substrates
The data of Morris and Solomon [73], and Polanowska et al.
[51] strongly suggest that bona fide substrates for CeBCD/
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Figure 3 BRCA2: domains, interacting proteins, activities and functions
Schematic diagram of the BRCA2 protein depicting the position of the eight BRC motifs (blue), the DNA-binding region
[helical domain (yellow)], OB-folds (red) and the single Rad51-binding site (green) at the extreme C-terminus, regulated by
Cdk phosphorylation. The indicated BRCA2-interacting proteins are shown under the corresponding region of BRCA1 required
for their association. aa, amino acids; NLS, nuclear localization signal.
BRCA1-mediated ubiquitylation reside or are recruited to
sites of DNA damage and may therefore function in DNA
repair, checkpoint signalling and/or regulation of chromatin
dynamics [51,73] (Figure 2). Although the BRCA1–BARD1
E3 Ub ligase is able to ubiquitylate FANCD2 (FA complementation group D2), RNA polymerase II, nucleoplasmin,
p53, H2AX and the nucleosome core histones H2A, H2B,
H3 and H4 in vitro, bona fide targets in vivo remain elusive
[56,58]. Indeed, the BRCA1–BARD1 E3 Ub ligase is very
promiscuous in vitro in that it is able to ubiquitylate many
proteins that are highly unlikely targets in vivo. Therefore
the ability of BRCA1–BARD1 to mediate ubiquitylation of
a protein in vitro should be treated with caution, particularly
as none of the proteins so far shown to be ubiquitylated
in vitro by BRCA1–BARD1 have been shown to be substrates in vivo. Analogous to SCF Ub ligases, BRCA1–
BARD1 substrates are likely to be phosphorylated and in
this modified form may associate with the BRCT domains.
Thus substrates may be identified in screens designed to
pull out phosphoproteins that bind to the BRCT domains.
Alternatively, comparative proteomic approaches to identify
all of the proteins that are ubiquitylated on chromatin before
and after DNA damage has the potential to identify BRCA1
targets that should be distinguishable from alternative E3
enzyme targets by analysing the ubiquitylation status in
BRCA1-defective cells. Until a bona fide substrate is found,
it is impossible to predict the effect of BRCA1-mediated
ubiquitylation without knowing the type of lysine chain
linked to the substrate and mutation of the target lysine
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residue(s) in the substrate is the only conceivable way that the
effect of BRCA1-mediated ubiquitylation will be inferred.
In contrast with BRCA1, a wealth of evidence has implicated BRCA2 as an integral component of the HR pathway,
where it directly regulates the key recombinase Rad51 [9].
BRCA2 and Rad51 not only co-localize at DNA repair
foci, but also interact directly with one another in vitro and
by co-immunoprecipitation [18,23,63,77]. BRCA2-deficient
cells, such as CAPAN-1, exhibit predominantly cytoplasmic
Rad51 and are defective for recruitment of Rad51 to repair
foci. These data indicate that BRCA2 is required for nuclear
localization of Rad51 and for its efficient recruitment to sites
of DSB repair.
The domain structure of BRCA2
The human BRCA2 gene encodes a 3418-amino-acid protein
that is one of the largest polypeptides in the human proteome
(Figure 3). When the BRCA2 gene was cloned in 1995, no
obvious domain similarities were identified [7]. However, a
repeated motif termed the BRC domain was identified by
sequence comparison of the BRCA2 protein to itself [78]. The
BRC motif is approx. 70 amino acids in length with a core
sequence spanning 26 amino acids and is repeated eight times
over a region of 1000 amino acids within the second third
of the BRCA2 protein (Figure 3). The sequence of the BRC
repeats in BRCA2 are somewhat diverged from one another
with BRC1, BRC3, BRC4, BRC7 and BRC8 being most
related to one another. Of critical importance to the
Cellular functions of the BRCA tumour-suppressor proteins
understanding of the role of BRCA2 in DSB repair processes
was the finding that the BRC domains mediate direct binding
to the Rad51 recombinase [18,79]. Although all of the eight
BRC motifs in BRCA2 are able to bind to Rad51, in either
yeast two-hybrid or in vitro pull-down assays, they have very
different affinities for Rad51, with BRC3 and BRC4 having
high affinity, whereas BRC5 and BRC6 have very low affinity
[18,79]. It is currently believed that six of the eight mostly
highly conserved BRC motifs amongst mammalian species
bind to Rad51 in vivo, including BRC1–BRC4, BRC7 and
BRC8.
Structural insight into the way the BRC motif binds to
Rad51 came from Pellegrini et al. [80] who solved the cocrystal structure of the BRC4 motif bound to the RecA
homology domain of Rad51. Since Rad51 has a tendency to
form large heterogeneous multi-oligomeric aggregates, this
study required the generation of a covalent linkage between
BRC5 and Rad51 to facilitate purification of the two proteins as a single fusion. A number of hydrophobic residues
within a stretch of 28 amino acids within the BRC4 motif
ensure close contact with Rad51. Gly1523 and the sequence
F1524 HTASGK1530 , which forms a block of highly conserved
amino acids, form critical hydrophobic and polar interactions
with Rad51. Indeed, cancer-predisposing mutations in BRC4
corresponding to T1526A blocks the ability of BRC4 to bind
to Rad51. Similar tumour-derived mutations in equivalent
positions in BRC1 (T1011R), BRC2 (S1221P) and BRC7
(T1980I) have been reported. It was proposed that the BRC
motif functions as a Velcro strip in the way it binds to
Rad51, in that it possesses a large number of contacts that
are relatively independent of one another. Comparison of the
BRC4–Rad51 structure to the crystallographic RecA filament
revealed that BRC4 mimics the interface that is formed
between adjacent RecA molecules. That is, BRCA2 interacts
with Rad51 by mimicking a structural motif that enables
Rad51 to oligomerize [80]. It has been proposed that BRCA2
binding maintains Rad51 in a monomeric state that is primed
for deposition on to DNA to form a nucleoprotein filament
functional for DNA strand exchange. In addition to tumourderived mutations, the importance of the BRC domain to
the role of BRCA2 in HR was illustrated by the finding
that in vivo overexpression of the BRC4 motif blocked the
formation of Rad51 foci at DNA-damage sites [63].
Yang et al. [81] identified an 800-amino-acid region in
the final third of the BRCA2 protein that was predicted to
form extensive secondary structure. This region was shown
previously to associate with the highly acidic 70-aminoacid polypeptide DSS1 [82] that was originally identified as
one of three genes that map to a 1.5 Mb locus deleted in
the inherited developmental malformation syndrome, split
hand/split foot [83]. Co-expression of BRCA2 C-terminal
fragment with DSS1 was found to significantly improve solubility and subsequent protein purification that enabled the
generation of co-crystals with and without bound oligo(dT)
ssDNA [81]. Structural analysis revealed five distinct domains in the BRCA2 C-terminal fragment (Figure 3). The
first domain comprises 190 amino acids consisting of mainly
α-helices, termed the helical domain. This is followed by the
three structurally related domains of approx. 110 amino acids
(OB1, OB2 and OB3) that exhibit homology and structural
similarity to the OB (oligonucleotide/oligosaccharide-binding)-fold that is present in most prokaryotic and eukaryotic
ssDNA-binding proteins, including SSB (ssDNA-binding
protein) and RPA (replication protein A). A 130-amino-acid
insertion is also found in OB2, that is absent from other OBfold-containing proteins, which adopts a tower-like structure (tower domain) sticking out from the OB-fold. DSS1
was found to bind to the helical domain, OB1 and OB2
through extensive electrostatic interactions. The acidic nature
of DSS1 coupled with its association with the tower, OB1
and OB2, also led to the suggestion that DSS1 might
play a role as a DNA mimetic that could regulate the
DNA-binding activity of BRCA2. This possibility currently
remains unclear. Consistent with the presence of three OBfold domains, the BRCA2 C-terminal fragment was shown to
bind with high affinity to ssDNA, whereas the tower domain
was found to bind to dsDNA (double-stranded DNA) [81].
This led to the idea that BRCA2 could target Rad51 (via
interaction with the BRC motifs) to the dsDNA/ssDNA
junction at processed DSBs (through the combined DNAbinding affinities of the tower and OB-folds).
In addition to binding to Rad51 via the BRC motifs, a
second Rad51-binding site has also been identified at the
extreme C-terminus of the protein [84,85] (Figure 3). Using
a series of nine overlapping GST (glutathione S-transferase)fusion proteins spanning the entire coding region of BRCA2,
Esashi et al. [86] found that the extreme C-terminal GST–
BRCA2 fusion protein that is capable of binding Rad51 is
also phosphorylated in asynchronous HeLa extracts. Phosphorylation site mapping revealed that Ser3291 is the site
modified in BRCA2, the location of Rad51. This site is located
in a Cdk (cyclin-dependent kinase) consensus sequence and
was subsequently shown to be phosphorylated by Cdk.
Strikingly, it was shown that a non-phosphorylated peptide
containing the Rad51-binding sites efficiently bound Rad51,
whereas phosphorylation at Ser3291 abolished binding. Analysis of the phosphorylation status during the cell cycle revealed that phosphorylation at Ser3291 is low in S-phase when
recombination is active and is high as cells enter mitosis.
Moreover, under DNA-damaging conditions, phosphorylation at Ser3291 is rapidly removed, thus allowing Rad51 association. The biological relevance of this site is highlighted by
the existence of four independent tumour-derived mutations
in the Ser3291 -Phe3292 Cdk target sequence. Furthermore,
expression of a non-phosphorylated fragment in cells reduced
recombination rates. Collectively these results suggested that
phosphorylation at Ser3291 could act as a molecular switch to
regulate HR by modulating the interaction of BRCA2 with
Rad51 [86].
So far no obvious domains or functions have been assigned
to the first third of the BRCA2 protein. It is of notable interest
that Esashi et al. [86] also found that the first 906 amino acids
of BRCA2 contain at least two phosphorylation sites. However, the exact position of these phosphorylation sites and
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their relevance to the function of BRCA2 remains to be
determined. This region is clearly of great interest and will
be of considerable focus in the future.
BRCA2 homologues in simple eukaryotes
The presence of BRC and OB-fold motifs is a hallmark
of BRCA2-related proteins and has proved to be a useful
method for the identification of BRCA2 orthologues in
other species [87]. Surprisingly, the number of BRC motifs
in BRCA2-related proteins is highly variable with 15 BRC
motifs found in Trypanosoma brucei BRCA2 ranging to a
single BRC motif in Ustilago maydis BRCA2 (Brh2) and
C. elegans BRCA2 (CeBRC-2) [87]. Kojic et al. [88] were the
first to describe a non-mammalian BRCA2. Their analysis
of U. maydis Brh2 revealed an essential role in DNA repair,
allelic recombination and meiosis analogous to Rad51. Consistent with the presence of a single BRC domain, Brh2 was
also found to bind to Rad51 directly [88]. Subsequently, Siaud
et al. [89] described Arabidopsis thaliana BRCA2 with four
BRC motifs that was essential for meiotic recombination. In
the absence of Atbrca2, plants were sterile owing to a failure
to repair meiotic DSBs. Indeed, the aberrant recombination
events observed in Atbrca2 deficient plants were eliminated
by a mutation in spo11, the enzyme responsible for the generation of meiotic DSBs [89]. Martin et al. [53] also described
arguably the simplest BRCA2-related protein, which was
identified in C. elegans. CeBRC-2 is a 394-amino-acid protein, a little over one-tenth of the size of human BRCA2,
which contains a single BRC, a single OB-fold domain and a
nuclear localization signal. CeBRC-2 interacts directly with
Rad51 through its BRC motif and binds preferentially to
ssDNA through its OB-fold domain. It was shown that
Cebrc-2 mutants are defective for meiotic and ionizing
radiation-induced DSB repair owing to an inability to target
Rad51 to the nucleus and a failure to recruit Rad51 to sites of
DSBs [53]. Collectively, these studies substantiated the view
that BRCA2 and its related proteins function in DSB repair
via HR by directly regulating Rad51 functions.
BRCA2-interacting proteins
In addition to Rad51, a number of other BRCA2-associated
proteins have been described (Figure 3). DSS1 was originally
identified by Marston et al. [82] in a yeast two-hybrid screen
that bound to the region spanning amino acids 2472 and
2957 in BRCA2 (see above for details). The first convincing
demonstration that DSS1 is functionally related to BRCA2
came from studies of the DSS1 homologue in U. maydis.
UmDss1 was found to bind to Brh2 and mutants in this
gene conferred radiation sensitivity, defects in recombination
and genome instability [90]. Gudmundsdottir et al. [91] subsequently demonstrated that depletion of DSS1 in human cells
compromises DNA-damage-induced Rad51 focus formation
and genome maintenance, analogous to the defects observed
in the absence of BRCA2. Collectively, these data indicate
that DSS1 is a conserved component of the HR pathway that
somehow functions with BRCA2 to efficiently target Rad51
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to sites of DSBs. FANCD2, a component of the FA DNArepair pathway, also binds directly to BRCA2 in both yeast
two-hybrid assays and co-immunoprecipitation studies [92].
Moreover, biallelic inactivation of BRCA2 was found to result
in FA and was assigned to FANCD1 (FA complementation
group D1) [93]. Current evidence supports a role for
FANCD2, FANCD1/BRCA2 and the FA pathway in
orchestrating lesion repair via HR and/or translesion bypass
pathways. Indeed, a number of studies have reported that
FA-deficient cell lines are compromised for HR-mediated
DSB repair [94,95]. However, the precise mechanism through
which the FA pathway promotes DNA repair remains
unclear. EMSY was identified by Hughes-Davis et al. [96]
in a yeast two-hybrid screen as a factor that associates with
BRCA2 within the region encoded by exon 3. Of particular
significance was the finding that EMSY is amplified in breast
and ovarian cancers. Consistent with a role in DNA repair,
EMSY is recruited to DNA-damage sites; however, its precise
role in repair and its relevance to the role of BRCA2 in HR
repair processes remains unclear.
BRCA2: functional insights from
biochemistry
Assessing the influence of human BRCA2 on Rad51 activities
in vitro has been hampered by its low expression levels and
proteolytic degradation that have precluded the purification
of full-length BRCA2 protein. Consequently, biochemical
studies were initially restricted to the use of fragments. In
vitro, the BRC domain alone is capable of inhibiting the
formation of the Rad51 nucleoprotein filaments, whereas a
C-terminal fragment of BRCA2 containing the three OBfold domains that confer ssDNA binding was shown to
stimulate the strand exchange activity of Rad51 [81,97]. It
was not clear whether these effects were direct and reflected
the true biochemical function of human BRCA2 in vivo.
However, a recent study using the purified full-length U.
maydis BRCA2 homologue (Brh2) has shown that, when
present at substoichiometric amounts relative to Rad51, Brh2
facilitates Rad51 nucleation and filament formation from
a dsDNA/ssDNA junction and stimulates Rad51-mediated
recombination [98]. Subsequently, San Filippo et al. [99] have
reported that a hybrid recombinant BRCA2 fusion protein
consisting of BRC3/BRC4 and its DNA-binding domain is
able to stimulate Rad51 activities in vitro. A similar effect has
also been observed using purified C. elegans proteins (M.I.R.
Petalcorin and S.J. Boulton, unpublished work). Collectively,
these studies support a conserved role for BRCA2 proteins
as a recombination mediator.
The current model for BRCA2 function in DSB repair
by HR indicates that it is required for efficient nuclear
localization of Rad51, it mediates the recruitment of Rad51
to sites of DSBs, it binds to the ssDNA/dsDNA junction at a
processed DSB and promotes the nucleation of the Rad51
nucleprotein filament, and, once the Rad51 filament has
formed, BRCA2 stimulates Rad51-mediated strand exchange
and D-loop formation (Figure 4).
Cellular functions of the BRCA tumour-suppressor proteins
Figure 4 Model for the action of BRCA2 in DSB repair by HR
(1) BRCA2 is required for the recruitment of Rad51 to DSBs. (2) Following nucleolytic resection of the DSB to generate 3
ssDNA overhangs, BRCA2–DSS1 binds to the ssDNA/dsDNA junction and offloads Rad51 on to the ssDNA overhang, thereby
facilitating the nucleation of the Rad51 filament. (3) Once the Rad51 nucleoprotein filament has formed, BRCA2 stimulates
Rad51-mediated strand exchange and D-loop formation.
Conclusions
In the last decade, considerable progress has been made in
elucidating the cellular functions of BRCA1 and BRCA2.
On the basis of a number of recent studies, it is reasonable
to suggest that the role of BRCA1–BARD1 in such diverse
cellular processes as DNA repair, checkpoint signalling,
transcription and meiotic sex chromosome inactivation may
be achieved through ubiquitylation of specific target proteins.
This is clearly the case for DNA damage. Identification of
BRCA1 substrates will be the next major development in this
field, and their finding and characterization remains crucial to
our full understanding of how BRCA1 contributes to cancer
predisposition. On the other hand, the role for BRCA2 in
HR processes is well established, although the one area that
remains to be explored in detail is how BRCA2 function is
modulated in response to DNA damage.
Research in my laboratory is supported by Cancer Research UK and
grants from Breast Cancer Campaign.
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