From the Department of Biochemistry, 437 Medical Sciences Building, University of Alberta, Edmonton T6G 2H7, Canada
Received for publication, September 30, 2002, and in revised form, October 23, 2002
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ABSTRACT |
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The integrity of the carboxyl-terminal BRCT
repeat region is critical for BRCA1 tumor suppressor function; however,
the molecular details of how a number of clinically derived BRCT
missense mutations affect BRCA1 function remain largely unknown. Here
we assess the structural response of the BRCT tandem repeat domain to a
well characterized, cancer-associated single amino acid substitution, Met-1775 Germ line mutations within the breast and ovarian cancer
susceptibility gene BRCA1 predispose carriers to early onset
breast and ovarian cancer, and it is estimated that ~5-10% of all
breast cancers are caused by inheritance of dominant disease genes (1). Various lines of evidence suggest that the BRCA1 protein product is involved in the regulation of multiple nuclear functions including transcription, recombination, DNA repair, and checkpoint control (for
reviews, see Refs. 2-4). BRCA1 is an 1863-amino acid nuclear phosphoprotein that includes an amino-terminal RING finger domain and
two tandem carboxyl-terminal repeats, termed the BRCT domain. The
importance of the conserved RING and BRCT domains to the tumor suppressor function of BRCA1 is demonstrated by the fact that the
majority of known cancer-causing BRCA1 mutations localize to these
domains (5-9).
The extreme carboxyl-terminal region of BRCA1 contains two ~90-100
amino acid sequence repeats called
BRCT1 (BRCA1
carboxyl-terminal) repeats that are the
prototypical members of a protein fold superfamily that includes many
proteins associated with DNA repair (10-12). The recently determined
x-ray crystal structures of the human (13) and rat (14) BRCA1 BRCT
repeats provide a framework for the interpretation of BRCT mutations
identified in patients from breast cancer screening programs. The two
structurally similar BRCT repeats resemble the structures of the
isolated BRCT domains from XRCC1 (15) and DNA ligase III (16) and are
composed of a central four-stranded, parallel Several cancer-associated BRCT missense mutations (1, 6) have been
characterized in functional detail. Two extensively studied variants,
A1708E and M1775R, ablate double-strand break repair and transcription
function of BRCA1 (17-19) and inhibit BRCT interactions with histone
deacetylases (20), the DNA helicase BACH1 (21), and the transcriptional
co-repressor CtIP (22, 23). Both of these mutations occur at the
interface between the amino- and carboxyl-terminal BRCT repeats and are
predicted to affect the way in which the two repeats interact. The
enhanced sensitivity of the A1708E mutant to proteolytic digestion
indicates that this mutation has profound structural consequences (13). In contrast, the M1775R mutant displays a proteolytic sensitivity that
is intermediate between that of the wild type protein and the A1708E
mutant, suggesting that the M1775R mutation results in a milder
structural defect.
In the present study we employed limited proteolysis, CD spectroscopy,
and x-ray structural analysis to probe the structural consequences of
the M1775R mutation. Our results show that the methionine-arginine
substitution leads to a rearrangement of the BRCT repeat interface,
alterations in the surface of the protein, and global destabilization
of the BRCT domain.
Proteolysis--
For proteolytic assays, BRCT-WT and BRCT-M1775R
were expressed from the T7 expression vector pLM1 and labeled with
[35S]methionine using the TNT-Quick in vitro
transcription/translation system (Promega). Immediately before
digestion, proteins were translated at 30 °C for 2 h. The
reticulocyte lysates were then centrifuged for 2 min at 10000 × g to remove insoluble material; 3 µl of the lysate
supernatants containing the labeled translation products were added to
12 µl of digestion buffer (150 mM NaCl, 50 mM
potassium phosphate, pH 7.5) containing increasing concentrations of
1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK)-treated chymotrypsin (Sigma). Following digestion at 20 or 37 °C for 12 min, the
reactions were stopped with phenylmethylsulfonyl fluoride. Digestion
products were electrophoresed on 15% SDS-PAGE gels and visualized with a phosphorimaging plate and an Amersham Biosciences Typhoon scanner. Quantification of the reaction products within ImageQuant (Amersham Biosciences) used a local average background correction.
Protein Expression and Purification--
Expression and
purification of recombinant human BRCA1-(1646-1859) (BRCT-WT)
and BRCA1-(1646-1859)-M1775R (BRCT-M1775R) was performed essentially
as described (13), with some changes. When expressed at 30 and
37 °C, yields of BRCT-M1775R were greatly reduced relative to
BRCT-WT, likely because of degradation. To obtain large quantities of
BRCT-M1775R protein for crystallization studies, growth of cells and
induction of protein expression were carried out at 25 °C.
Purification using a combination of ammonium sulfate precipitation,
hydrophobic interaction, gel filtration, and anion exchange
chromatography yielded 4-5 mg of protein/liter Escherichia
coli culture for both proteins. Matrix-assisted laser desorption
ionization time-of-flight (MALDI-TOF) mass spectrometric analysis
confirmed mutation of residue Met-1775 to arginine.
CD Spectroscopy--
For circular dichroism measurements,
proteins were dialyzed into 400 mM NaCl, 50 mM
potassium phosphate (pH 7.5), 0.1% Crystallization of BRCT-M1775R--
Protein concentrations for
crystallization were determined using the BCA protein assay (Pierce).
Unlike the wild type protein, storage of the mutant at concentrations
greater than 5 mg/ml resulted in irreversible aggregation within days.
To attain high protein concentrations suitable for crystallization,
BRCT-M1775R at 1 mg/ml was dialyzed into Protein buffer (400 mM NaCl, 5 mM Tris-HCl, pH 7.5) and
concentrated to 20 mg/ml immediately prior to crystallization. Crystals
were grown at room temperature (20-22 °C) using the hanging drop
vapor diffusion technique. 2 µl of 20 mg/ml BRCT-M1775R in Protein
buffer was mixed with 2 µl of well solution (1.4 M
ammonium sulfate, 100 mM MES, pH 6.7, 10 mM
CoCl2) to produce hexagonal crystals within 1 to 2 days
(see Table I).
Data Collection, Structure Solution, and Refinement--
For
data collection, crystals were gradually transferred to a
cryoprotectant solution containing 1.2 M ammonium sulfate,
100 mM MES, pH 6.7, 10 mM CoCl2,
26% glycerol over the course of 45-60 min. Diffraction data to 2.8 Å resolution were collected from a single crystal at 100 K at the
Structural Biology Centre (Argonne National Laboratory)-APS beamline
19-ID. Intensity data were processed using the HKL2000 package (24)
(see Table I). Crystallographic phase information for BRCT-M1775R was
obtained using molecular replacement with the native human BRCT model
(RCSB, 1JNX) (13). All data between 20 and 2.8 Å were used in
crystallographic refinement, and 7% of this data was allocated for
cross-validation. Initial rigid body fitting of the model in the
Crystallography and NMR System (CNS) (25) lowered the
Rfactor/Rfree to
36.2/38.6%. Side chains of regions of the protein that change with
mutation of the BRCT were built using O (26) into Native Packing Environment of Met-1775--
The tandem BRCT
repeat structure is composed of two Proteolytic Sensitivity of M1775R--
Mutant M1775R conferred
moderate sensitivity to digestion with trypsin at 20 °C (13).
Because Arg-1775 immediately precedes a proline residue, this
substitution is not expected to introduce a new trypsin cleavage site;
thus the enhanced trypsin sensitivity is an indication of a subtle
structural change in the M1775R mutant. To further assess the stability
of the M1775R mutant, we compared its chymotrypsin sensitivity to that
of the wild-type protein (Fig. 2,
A and B). BRCT-WT and BRCT-M1775R both show
resistance to digestion at 20 °C for all protease concentrations.
Elevating the temperature to 37 °C enhanced cleavage of the mutant
by chymotrypsin at all protease concentrations, whereas the wild type
showed only a slight increase in cleavage efficiency at the highest
concentration. The fact that the BRCT-M1775R exhibits modest but
significant increases in sensitivity to digestion by two proteases with
different cleavage specificities, especially at elevated temperatures,
indicates that M1775R results in a folding defect in the BRCT
domain.
CD Analysis--
To further probe the extent of protein
destabilization induced by the M1775R substitution, we compared the
secondary structure and thermal stability of BRCT-M1775R to the wild
type protein using CD spectroscopy (Fig.
3). The mutant exhibits a far-UV CD absorbance spectrum that is characteristic of a mixed X-ray Structure of Missense Variant M1775R--
To gain structural
insights into the destabilizing effect of M1775R, we crystallized and
determined the structure of BRCT-M1775R at 2.8 Å (see Fig.
4A, Table
I, and "Experimental Procedures"). 2Fo
Introduction of an arginine at position 1775 creates a clustering of
three positively charged residues, Arg-1699, -1775, and -1835. In the
native structure, Arg-1699 from the amino-terminal BRCT repeat
participates in the sole conserved inter-BRCT repeat salt bridge with a
pair of carboxyl-terminal BRCT acidic residues, Asp-1840 and Glu-1836
(Fig. 5A). Arg-1835 in human BRCA1 normally participates in
a hydrogen bonding network with Gln-1811, thereby helping to orient the
The adjustments in the positions of Arg-1775 and -1835 described above
result in the displacement of the aliphatic portions of these side
chains from the protein core (Fig. 5C). In response to these
movements, Leu-1780 shifts to maintain van der Waals contact with the
arginine residues. These movements are accomplished without the
creation of large, energetically costly cavities within the hydrophobic
core (33-35). The repacking of the core, however, in combination with
instability created by hydrogen bonding changes and charge-charge
repulsion of basic residues at the BRCT-BRCT interface may collectively
help account for the 5 kcal/mol destabilization reported for the
BRCT-M1775R protein (36).
Few crystallographic examples documenting protein structural responses
to the introduction of charged residues into the hydrophobic core are
available. Structural investigation of a comparable, highly
destabilizing mutation in T4 lysozyme, Met-102 Destabilization of the BRCT Abrogates BRCA1-M1775R
Function--
At the surface of the protein, a hydrophobic groove near
Met-1775 is apparent in the wild type structure (Fig. 5D).
With the movement of Arg-1775 in the mutant, this cleft becomes
occluded with charged atoms (Fig. 5E). This raises the
possibility that, in addition to the destabilizing effect of the
mutation, the substitution may disrupt association of BRCA1 with its
interaction partners by directly modifying an exposed protein binding
site. However, this site does not overlap with a BACH1 binding site
that maps to the
Measurement of the steady state levels of BRCA1 missense variants
M1775R, P1749R, and A1708E reveals they are similar to the wild type,
suggesting that these proteins are not destabilized to the degree that
they are degraded significantly in vivo (17, 23). Although
it is difficult to predict individual energetic contributions of the
M1775R rearrangements to BRCT destabilization, it is clear that the
cumulative changes affecting the structure across the BRCT-BRCT
interface result in a temperature-sensitive tandem BRCT domain (Figs. 2
and 3) that is defective in BRCA1 protein interaction, transcription,
and DNA repair functions. In analogous studies, the quantitative
measurement of the thermodynamic stability of the p53 tumor suppressor
has shown that many of the common cancer-causing mutations are
destabilizing. Consequently, p53 is inactivated by mutation, and cell
cycle control is lost (38). Because many of the BRCT missense variants
destabilize the BRCT to the same extent or more than M1775R (13,
36),2 we might expect these
variants to possess molecular and disease phenotypes similar to
M1775R. Examination of the effects of other missense mutations
on BRCA1 cellular function will be necessary to correlate BRCT loss of
structure with BRCA1 loss of function and will further establish
protein misfolding as a basic molecular mechanism of disease.
Arg-1775. The structure of BRCT-M1775R reveals that the
mutated side chain is extruded from the protein hydrophobic core,
thereby altering the protein surface. Charge-charge repulsion, rearrangement of the hydrophobic core, and disruption of the native hydrogen bonding network at the interface between the two BRCT repeats
contribute to the conformational instability of BRCT-M1775R. Destabilization and global unfolding of the mutated BRCT domain at physiological temperatures explain the pleiotropic molecular and
genetic defects associated with the BRCA1-M1775R protein.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-sheet flanked by a
single
-helix on one side (
2), with a pair of
-helices (
1
and
3) and a short 310-helix on the opposite side. The
two repeats are connected by a relatively flexible linker and pack
together in a specific, head-to-tail manner that is conserved not only
between human and rat BRCA1 but also in the BRCT domain of the
p53-interacting protein, 53BP1 (13, 14).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-mercaptoethanol, in a Millipore
Ultrafree-10 concentration unit. Protein concentrations for molar
ellipticity calculations were derived from amino acid analysis. Far-UV
(195-255 nm) CD spectra were determined using 0.2 mg/ml solutions of
protein and were acquired using a Jasco J-720 spectrapolarimeter that
was interfaced with Jasco software and equipped with a 0.1-cm quartz
cuvette cell. For denaturation experiments, temperature within the cell
was regulated using a Peltier thermal control unit. Assuming a
two-state unfolding model, denaturation midpoints were determined by
following the change in molar ellipticity at 222 nm as a function of
temperature. Measurements were taken at 0.5 °C intervals, and the
temperature was increased at a rate of 30 °C/h. Ellipticity readings
were normalized to the mole fraction of protein folded
(ff) or denatured (fu)
using the standard equations
(Eq. 1)
where [
]n and
[
]u are the molar ellipticity for the fully
folded and fully denatured protein species. [
] is the observed
ellipticity at each temperature. The midpoint of the
temperature-dependent folding-unfolding transition for both
proteins was reproducible, but this transition was irreversible and
characterized by precipitation within the cell under the conditions used.
-A weighted
model-phased Fo
Fc and
2Fo
Fc electron density
maps calculated with CNS. Maximum likelihood targets, bulk solvent
correction, and overall anisotropic B-factor scaling were applied
throughout the refinement process. Further refinement involved
iterative cycles of manual building and restrained refinement with
translation, libration, and screw rotation group anisotropic
thermal parameter modeling as implemented in REFMAC (v5.0.32) (27, 28).
The quality of the model was assessed using PROCHECK (Ref. 29, Table
I). The final model (see Table I) has good stereochemistry and a
working R-factor of 27.3% (Rfree,
29.8%) for all data in the resolution range 20-2.8 Å. Structural
diagrams were created using BOBSCRIPT (30) and rendered with POVRAY
(www.povray.org) (Figs. 1, 4, and 5, A and B)
or RASTER3D (31) (Fig. 5, C-E). Molecular surfaces were
drawn with GRASP (32).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
/
-fold BRCT domains that
interact end-to-end, with helices
1' and
3' from the
carboxyl-terminal BRCT intimately contacting
2 from the
amino-terminal repeat in a three-helix bundle-like packing arrangement
(Fig. 1A) (13, 14). Residue
Met-1775 is largely buried within the interface between the two repeats
and lies in a pocket formed by Leu-1701 and Phe-1704 from the
amino-terminal repeat and Leu-1780, Met-1783, Arg-1835, and Leu-1839
from the carboxyl-terminal repeat. Sequence and structural conservation of Met-1775 and its contacting residues among mammalian, xenopus, and
avian BRCA1 homologues highlights the importance of Met-1775 in
orienting the two BRCT repeats (Fig. 1B) (13, 14). With the
exception of residue Arg-1835, which is replaced by a tryptophan in the
mouse and rat BRCT domains, the residues forming this pocket are highly
conserved. This substitution within the pocket is significant, however,
as proximity of the positively charged residue Arg-1835 to Met-1775 in
the human protein influences the response of the structure to the
cancer-linked substitution, M1775R.
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Fig. 1.
Evolutionary conservation of the Met-1775
packing environment. A, ribbons diagram of the BRCT repeat
region of BRCA1. Met-1775 (red) in BRCT-WT is
positioned between the two BRCT fold repeats. Met-1775 (red
surface) lies in a hydrophobic pocket created by Leu-1701,
Phe-1704, Leu-1780, Met-1783 (behind Met-1775), Arg-1835, and Leu-1839
(gray surfaces). B, multiple sequence alignment
of BRCA1 homologues for the BRCT regions surrounding Met-1775. Met-1775
is red, and contacting amino acids are blue.
Numbering is for human BRCA1.
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Fig. 2.
Protease sensitivity of BRCT-M1775R.
A, reticulocyte lysates containing 35S-labeled,
in vitro-translated BRCT-WT and BRCT-M1775R were digested at
the indicated temperatures for 12 min using chymotrypsin at
concentrations of 0, 6, 60, 600 µg/ml (lanes 1-4).
Reaction products were analyzed by SDS-PAGE and autoradiography.
B, quantification of chymotryptic digestions. The fraction
remaining is the percent of starting protein present following
digestion with the indicated concentrations of chymotrypsin. Data
points are the mean value of digestions performed in triplicate with
error bars reflecting the S.D.
/
protein and is similar to the wild type, indicating that the overall fold of
the M1775R domain is maintained in solution at 20 °C. BRCT-M1775R, however, is less stable, having a midpoint of thermal denaturation of
~41 °C, 11 °C less than the wild type protein (see Fig. 3, inset and "Experimental Procedures"). Consistent with
these findings, solvent denaturation measurements of the thermodynamic
stability of the BRCT tandem domain indicates that M1775R destabilizes
the BRCT fold by 5.0 kcal/mol at 20 °C (36). Taken together, these results suggest that functional defects associated with
BRCA1-M1775R may be attributed to destabilization of the BRCT domain
at physiological temperatures.
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Fig. 3.
CD spectra. The far-UV CD spectra for
the wild type ( ) and mutant protein (
) are similar at 20 °C.
Thermal denaturation of BRCT-WT and BRCT-M1775R. Molar ellipticity at
222 nm was measured, and the values were used to calculate a mole
fraction of denatured molecules for each temperature
(inset). Midpoints of the transitions (WT, 52 °C; M1775R,
41 °C) are marked by dotted lines.
Fc and
Fo
Fc difference
electron density indicate that structural rearrangements in response to
the mutation are confined to residues within an ~5 Å radius of
Arg-1775. Paired 4
-positive and -negative electron density features
in the Fo
Fc map near
residue 1775 reveal that the substituted arginine is displaced from the
hydrophobic core of the protein (Fig. 4A). The mutation is
further accommodated by an ~1 Å shift of a neighboring residue,
Pro-1776, in the
1'-
1' connecting loop and a series of side chain
rearrangements that perturb the native inter-BRCT hydrogen bonding and
van der Waals contact networks (Fig. 5,
A-C).
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Fig. 4.
M1775R structure determination.
A, stereoview of an overlay of BRCT-WT (gray) and
BRCT-M1775R (gold) for the region surrounding M1775R. The
2.8 Å positive (blue) and negative (red) -A
weighted Fo
Fc electron
density (phased with the wild type model, RCSB, 1JNX) is
contoured at ± 2.9
. Two large positive peaks, modeled as
sulfate molecules S1 and S2, are found coplanar to Arg-1775.
B, a stereoview of model phased
-A weighted 2 Fo
Fc map contoured at
1.0
is displayed for the final model of BRCT-M1775R. Electron
density maps were calculated with CNS (A) or REFMAC
(B).
Crystallographic data and refinement statistics
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Fig. 5.
Structural rearrangements accommodate
M1775R. A, native hydrogen bonding interactions
proximal to Met-1775. Hydrogen bonds are indicated by dashed
lines. B, hydrogen bonding, salt bridging for mutant
M1775R. Arg-1775 participates in the coordination of two solvent
anions, S1 and S2, and has been flipped out from the hydrophobic pocket
where Met-1775 normally packs. C, cutaway view of the
hydrophobic core of the BRCT. Structural overlay of WT (gray with
red surface) and M1775R (gold with gray surface)
hydrophobic core residues that move upon mutation. D, charge
potential GRASP surface for BRCT-WT. Blue surface reflects
positive charge potential, and red is negative. The
arrow indicates a hydrophobic groove near Met-1775.
E, charge potential GRASP surface for BRCT-M1775R.
Green spheres mark the positions of bound anions.
1'-
1' loop (Fig. 5A). The positively charged guanidinium group of variant Arg-1775 is found positioned between two
large positive peaks in our electron density maps that we have
interpreted to be sulfate molecules, the highest concentration anion in
the crystallization mother liquor (Figs. 4 and 5B). In the
mutant, Arg-1699 retains the salt bridge with Asp-1840 but no longer
contacts Glu-1836 and instead coordinates an anion. Arg-1835 rotates
away from Gln-1811 and forms a new salt bridge with Glu-1836. Thus, it
appears that electrostatic stabilization of this positive charge
cluster is achieved through ordering of solvent anions and a cascade of
hydrogen bonding alterations.
Lys-102, reveals the
position of the lysine side chain changes little when compared with
native Met-102 packing (37). Instead, the mutation is accompanied by
increased mobility of flanking residues, including a helix that
normally packs against the substituted methionine. This motion allows
access of the buried, basic side chain to solvent via a
folding-unfolding transition of neighboring structured regions. The
structural rearrangement that we observe in BRCT-M1775R is less
dramatic but nevertheless results in the expulsion of the charged side
chain from the hydrophobic protein core.
3-
2 connecting loop (Fig. 1) of the
amino-terminal BRCT repeat (14), which is greater than 20 Å away from
Met-1775. BACH1 binding is impaired by the M1775R substitution,
suggesting that global structural defects resulting from the mutation,
rather than localized structural and electrostatic perturbation, are responsible for the binding defect. Moreover, variant M1775R is defective in many biochemical assays that assess BRCA1 function and is
unable to interact with several other proteins that associate with the
wild type BRCT domain (Table II). It is
unlikely that all of these proteins target a common surface near
Met-1775 that is changed by the mutation. We therefore suggest that the
wide spectrum of molecular defects reported for variant M1775R is
explained by the destabilization of the domain.
Defects associated with BRCA1-M1775R
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ACKNOWLEDGEMENTS |
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We thank Bob Luty for help with spectroscopy measurements, Jason S. Lamoureux and Brian L. Mark for assistance with x-ray data collection, and Youngchang Kim and the Structural Biology Centre 19-ID beamline staff for excellent technical support and discussion during synchrotron data collection.
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FOOTNOTES |
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* This work was supported by funding from the Canadian Breast Cancer Research Initiative, the Canadian Institutes of Health Research, the National Cancer Institute of Canada, and the Alberta Heritage Foundation for Medical Research. Use of the Argonne National Laboratory Structural Biology Center beamlines at the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Biological and Environmental Research under Contract No. W-31-109-ENG-38.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1N5O) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
To whom correspondence should be addressed. Tel.: 1-780-492-2136;
Fax: 1-780-492-0886; E-mail: mark.glover@ualberta.ca.
Published, JBC Papers in Press, November 8, 2002, DOI 10.1074/jbc.M210019200
2 R. S. Williams and J. N. M. Glover, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: BRCT, BRCA1 carboxyl-terminal domain; CTIP, CtBP interacting protein; MES, 4-morpholineethanesulfonic acid.
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