From the Departments of Genetics and Medical Genetics
and the
Department of Biochemistry and Biomolecular Structure
Center, University of Washington, Seattle, Washington 98195-7742
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ABSTRACT |
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Breast cancer 1 (BRCA1) and BRCA1-associated RING
domain 1 (BARD1) are multidomain proteins that interact in
vivo via their N-terminal RING finger motif regions. To
characterize functional aspects of the BRCA1/BARD1 interaction, we have
defined the structural domains required for the interaction, as well as
their oligomerization state, relative stability, and possible nucleic
acid binding activity. We have found that the RING finger motifs do not
themselves constitute stable structural domains but are instead part of
larger domains comprising residues 1-109 of BRCA1 and residues 26-119
of BARD1. These domains exist as homodimers and preferentially form a
stable heterodimer. Shorter BRCA1 RING finger constructs do not
interact with BARD1 or with longer BRCA1 constructs, indicating that
the heterodimeric and homodimer interactions are mediated by regions outside the canonical RING finger motif. Nucleic acid binding is a
generally proposed function of RING finger domains. We show that
neither the homodimers nor the heterodimer displays affinity for
nucleic acids, indicating that the proposed roles of BRCA1 and BARD1 in
DNA repair and/or transcriptional activation must be mediated either by
other regions of the proteins or by additional cofactors.
Unraveling the cellular basis for BRCA1-mediated tumor suppression
is a challenging goal that is complicated, in part, by the
multifunctional nature of the protein. In large multifunctional proteins, such as BRCA1, different functions are often dispersed among
discrete structural regions or domains. In such cases, functional studies can be facilitated by characterization of the properties of
individual domains or subunits. Unfortunately, in the case of BRCA1, a
large protein of 1863 amino acids, clues regarding the location or
extent of independent functional domains are not readily available on
the basis of sequence homologies. A noteworthy exception is the
RING1 finger consensus motif
located at the N-terminal end of the protein (1). The RING finger is a
zinc-binding motif defined by a conserved pattern of cysteine and
histidine residues that is found in a wide variety of proteins of
diverse origin and function (2). Although more than 80 RING
finger-containing proteins have been identified to date, a specific
cellular function has not yet been associated with the motif (3).
Nevertheless, the importance of the RING finger to BRCA1 function is
supported by the following observations: 1) the first 100 residues,
including the RING finger motif (residues 24-64), are the most highly
conserved regions among known BRCA1 genes (4, 5); 2) several
cancer-predisposing mutations have been identified within this region
of BRCA1 (6, 7); and 3) yeast two-hybrid studies have identified two
novel proteins that interact with this region of BRCA1 (8, 9).
One of the proteins found to associate with the BRCA1 N-terminal
region, BARD1 (8), also contains an N-terminal RING finger motif
(residues 50-86). Yeast two-hybrid studies showed that the N-terminal
RING finger regions of both BRCA1 and BARD1 are required for specific
heterocomplex formation between the proteins. Subsequent cellular
localization studies demonstrated that BRCA1 and BARD1 localize to
discrete nuclear foci (dots) during S phase and disperse during other
phases of the cell cycle (10). In addition, complexes containing BRCA1
and BARD1 have been detected on damaged, replicating DNA structures
(10). Taken together, these observations suggest that a multiprotein
complex involving BRCA1 is responsive both to the cell cycle and to DNA
damage and identify an important cellular interaction among the proteins.
Despite identification of heterologous protein interactions, the
cellular basis of BRCA1-mediated tumor suppression remains elusive.
When initially identified, it was suggested that the BRCA1 RING finger
may play a role in specific DNA binding (1). Indeed, there has been
considerable speculation regarding a general function for RING finger
proteins in nucleic-acid interactions. To date, published data on the
DNA binding properties of RING fingers are conflicting and equivocal:
there are reports that particular RING finger proteins or domains bind
synthetic oligonucleotides or DNA cellulose, whereas other reports
provide evidence to the contrary (11-14). In no case, however, has
specific nucleic acid binding been demonstrated. Nonetheless, BRCA1 has
been reported associated with certain chromatin structures in
vivo and linked to the RNA polymerase II holoenzyme complex (15,
16). These findings raise the possibility that BRCA1 might associate
directly with nucleic acids either alone or upon formation of a complex with BARD1.
In order to characterize the functional significance of the BRCA1/BARD1
interaction further, we have sought to define the extent of the
structural region encompassing the RING finger consensus motifs for
both proteins. We find that the RING finger motifs do not themselves
constitute stable structural units but are instead part of larger
structural domains. Interactions and properties that may contribute to
the function of these proteins, including their oligomerization states,
relative stabilities, and nucleic acid binding activities are described herein.
Materials--
3,5-Dimethoxy-4-hydroxycinnamic acid (sinapinic
acid) and 2,2':6',2"terpyridine were obtained from Aldrich and used
without further purification. Limited proteolysis studies used
sequencing grade endoproteinase Lys-C (Pierce) and V8 endo proteinase
(Worthington). Bovine serum albumin, ovalbumin, Preparation and Cloning of BRCA1 and BARD1
Constructs--
BC-76, BC-112, and BC-172 constructs were prepared as
described previously (17). BARD1 constructs were prepared in a similar fashion using primers to obtain the desired BARD1 polymerase chain reaction products from human lymphoblast cDNA. The primers were synthesized with NheI and BamHI endonuclease
restriction sites in the 5'- and 3'-ends, respectively. The polymerase
chain reaction products and pET11a vector (Novagen) were then digested
with NheI and BamHI, ligated, and transformed
into Escherichia coli BL21 cells, and clones were sequenced
as described previously for BRCA1 constructs (17). Because of the
restriction sites used, each construct contains two extra amino acids
(Ala and Ser) at the N terminus. BC-112 also contains two additional
residues at its C terminus (Gly and Lys) as a result of the cloning
procedure. Construct and fragment designations refer to the number and
position of BRCA1 or BARD1 residues.
Expression and Purification of BRCA1 and BARD1 N-terminal
Constructs--
BRCA1 constructs were expressed and purified as
described previously (17). Purification of BARD1 constructs was
identical to that of BRCA1 constructs with one minor modification. The
BARD1-containing insoluble fraction was re-solubilized into 6 M guanidine-HCl as before (17), diluted to 2 M
guanidine-HCl by dropwise addition of Tris buffer (50 mM
Tris, 10 mM dithiothreitol, pH 7.6) and purified to
homogeneity by reverse phase chromatography. Protein purity and
concentration was determined as described previously for the BRCA1
constructs (17). Approximate extinction coefficients at 280 nm for
BARD1 constructs, calculated on the basis of amino acid composition,
are 19.55, 13.86, and 13.86 mM Fluorescence Spectroscopy--
Fluorescence spectra were
measured on a Spex Fluorolog 2 fluorometer using a 1 × 0.5-cm
cuvette at 25 °C. BARD1 homodimer and BRCA1/BARD1 heterodimer sample
(1.0 ml) concentrations were adjusted to 1 µM BARD1
monomer in 25 mM Tris, 200 mM NaCl buffer at pH
7.6. Spectra were collected using an excitation wavelength of 290 nm
and scanning the emission spectrum between 300 and 450 nm.
Stoichiometry Determination and Limited
Proteolysis--
Gel-filtration chromatography, glutaraldehyde
cross-linking, limited proteolysis, and MALDI-TOF spectroscopy
experiments were performed as described previously (17).
Nucleic Acid Binding--
BRCA1, BARD1, and the ADR1 zinc finger
domain constructs were purified by gel-filtration chromatography in 25 mM Tris, 50 mM NaCl, pH 7.6 (Buffer 1). Peak
fractions (approximately 10 µM by absorbance) were loaded
onto double- and single-stranded DNA cellulose (calf thymus DNA, Sigma)
and synthetic RNA (poly(A), poly(C), poly(G), and poly(U)) agarose
(Sigma) columns at a flow rate of 0.2 ml/min in Buffer 1. Columns were
washed with 5 column volumes of Buffer 1 to remove nonspecifically
bound material. The bound material was eluted with a 50-350
mM NaCl gradient (5 column volumes), followed by a 350-600
mM NaCl gradient (2 column volumes), and a final wash with
2 M NaCl to remove tightly bound protein. Each column
(Perseptive Biosystems) was packed with approximately 1 ml of nucleic
acid resin. The identity of eluted material was verified by analysis of
peak fractions on 14% SDS-PAGE.
The BRCA1 RING Finger Structural Domain Extends beyond the RING
Finger Homology Region--
Three different sized BRCA1 RING finger
constructs (termed BC-76, BC-112, and BC-172; Table
I) were tested for their ability to
interact with a BARD1 RING finger construct. The BARD1 construct (BD
26-152) encompasses residues 26-152 and was chosen on the basis of
reported two-hybrid studies
(8).2 We used gel-filtration
chromatography to assess the oligomeric states of the constructs. Fig.
1A illustrates gel-filtration
elution profiles for BC-172, BD 26-152, and a 1:1 mixture of the two
constructs. Individually, each construct elutes primarily as a single
peak at an elution volume that corresponds to a molecular weight
roughly twice the expected value, suggesting that both BC-172 and BD
26-152 homodimerize in solution. An equimolar mixture of the two
constructs elutes as a single sharp peak with a width comparable to
those of standard proteins used for column calibration. The
intermediate elution volume relative to the two individual species is
indicative of a heterodimeric complex between BC-172 and BD 26-152.
Similar results were obtained with BC-112 and BD 26-152 (data not
shown).
Glutaraldehyde cross-linking of peak fractions collected from
gel-filtration columns, followed by SDS-PAGE, confirmed the oligomerization state of each species. Fig. 1B shows results
of cross-linking experiments performed on the BC-172, BC-172/BD
26-152, and BD 26-152 peak fractions. Each cross-linking reaction
produced a new band not present in the starting material (Fig.
1B, compare lanes 1, 3, and 5 with
lanes 2, 4, and 6, respectively). The
cross-linked bands in lanes 1 and 5 run at
molecular masses that correspond to the homodimeric form of BC-172 and
BD 26-152 (~40 kDa and ~29 kDa, respectively), whereas the
cross-linked band in lane 3 exhibits an intermediate
molecular mass, which corresponds to that of the BC-172/BD 26-152
heterodimer (~34.5 kDa). Similar to the results from the
gel-filtration analysis, the cross-linking experiments demonstrate that
the heterodimer is formed preferentially relative to the homodimeric
species in solution. Again, similar cross-linking results were obtained
with BC-112 and BD 26-152 (data not shown).
BD 26-152 contains three tryptophans, whereas BC-112 contains no
tryptophans (see Fig. 6 for sequences), allowing the use of
fluorescence to distinguish the BD 26-152 homodimer from the BC-112/BD
26-152 heterodimer. The fluorescence spectrum of the BD 26-152
homodimer exhibits an emission maximum at 347 nm (
We have previously demonstrated that the short BRCA1 construct, BC-76,
forms a homodimer (17). However, using the approaches described above,
we do not detect heterodimer formation with BC-76 and BD 26-152 (data
not shown). Furthermore, mixtures of BC-76 and BC-172 elute as two
separate peaks from the gel-filtration column at volumes corresponding
to the expected molecular weights of BC-76 and BC-172 homodimers (Fig.
1C). Similar results were obtained with mixtures of BC-76
and BC-112 (data not shown). Thus, the BC-76 RING finger construct does
not dimerize with either BC-112, BC-172, or BD 26-152, indicating that
specific and high affinity BRCA1 homodimerization and
heterodimerization with BARD1 requires BRCA1 residues C-terminal to
position 76.
Determination of the Structural BRCA1 and BARD1 Domains
Required for Heterodimerization--
To identify the structural
domains of BRCA1 and BARD1 required for heterodimerization, we
used limited proteolysis in conjunction with matrix-assisted laser
desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. The
premise behind this experimental approach is that regions of
proteins that are structured are less susceptible to proteolysis than
regions of proteins that are flexible or disordered. This
approach has also been used to map regions of proteins involved in
mediating interactions with other molecules (18).
The BC-112/BD 26-152 heterodimer was probed with endoprotease Endo Lys
C, which cleaves on the C-terminal side of lysine residues. Fig.
3 shows MALDI-TOF mass spectra that were
collected at various times after the addition of protease. Although
there are potential cleavage sites throughout the primary sequence of
both BC-112 and BD 26-152 (see Fig. 6), cleavage only occurs in a
limited number of sites. Twenty-five minutes after the addition of
protease, BC-112 is cleaved mainly at Lys109, generating
fragment BC 1-109 (Fig. 3B). BC 1-109 is quite resistant to further proteolysis, persisting even 60 min into the reaction (Fig.
3C). Cleavage also occurs at Lys55 of BC-112,
generating fragments 1-55 and 56-109, which are also resistant to
further proteolysis. Our previous studies showed that this same site is
also accessible in the BC-112 homodimer (17). BD 26-152 quickly loses
22 residues from its C terminus, generating a BD 26-130 fragment (Fig.
3B), which is very stable: no additional cleavage occurs
within the fragment even after 60 min of proteolysis (Fig.
3C).
Limited proteolysis was also performed with V8, which cleaves on the
C-terminal side of glutamate residues (see Fig. 6 for sequences). Sixty
minutes after the addition of protease, BC-112 remained intact, whereas
BARD1 was cleaved at residue 119, generating a stable BD 26-119
fragment (see Fig. 5B). Taken together, these results show
that BRCA1 and BARD1 RING finger motifs are part of larger structural
domains, comprising the first 109 residues of BRCA1 and residues
26-119 of BARD1.
Differences in Proteolytic Susceptibility between Homodimeric and
Heterodimeric Complexes--
Proteolytic susceptibilities of the
homodimers and the heterodimer were compared to probe for
distinguishing features among the species. Two shorter BARD1
constructs, BD 26-131 and BD 26-140 (Table I), engineered based on
the proteolysis results reported above, were used with BC-112 in these
experiments. As illustrated in Fig. 4,
the mass spectra of Endo Lys C reactions of the two homodimers and the
heterodimer show striking differences. Twenty-five minutes after
digestion, the only species of BD 26-140 observed in the heterodimer
reaction was the large BD 26-130 fragment (Fig. 4B). In
contrast, cleavage occurred at virtually every lysine in the BD 26-140
homodimer reaction (Fig. 4C).
The differences in susceptibility of BC-112 in the homodimer
versus heterodimer are less striking than those of BD
26-140. Most of the same cleavages occur (Fig. 4, A and
B) in the two forms, with one clear exception: cleavage
occurs at an N-terminal lysine, Lys20, in the homodimer,
but not in the heterodimer. Thus, the N-terminal region of BC-112
becomes protected upon dimerizing with BD 26-140.
To further probe differences in the N-terminal region of BRCA1, limited
proteolysis was performed with V8. The mass spectra shown in Fig.
5 illustrate a pronounced difference in
the proteolytic susceptibility between the BRCA1 homodimer and
heterodimer to V8. The enzyme readily cleaved the N-terminal region of
the homodimer, generating fragments BC 11-112, BC 11-100, and BC
11-75 (Fig. 5A). In contrast, BC-112 remained completely
intact in the heterodimer (Fig. 5B). Taken together, the
proteolysis results, summarized in Fig.
6, clearly illustrate that in each case
the homodimers are more susceptible to proteolysis than the
heterodimer. Similar results were observed with the slightly shorter BD
26-131 construct (data not shown).
Neither Homodimeric nor the Heterodimeric Complex Binds Nucleic
Acids--
We tested the ability of our RING finger constructs to bind
nucleic acids using double- and single-stranded DNA cellulose and
single-stranded RNA agarose (poly(A), poly(C), poly(G), and poly(U))
columns. Homodimeric (BC-112, BD 26-131, and BD 26-140) and
heterodimeric (BC-112/BD 26-131 and BC-112/BD 26-140) complexes were
applied to each nucleic acid column and eluted with a NaCl gradient.
The zinc finger domain from ADR1 (a known DNA-binding domain from a
yeast transcription factor) was used as a positive control (19). Fig.
7 shows the elution profiles for BC-112, BD 26-140, BC-112/BD 26-140, and the ADR1 zinc finger domain from a
double-stranded DNA column. Whereas the ADR1 zinc finger was retained
on the column, eluting at ~400 mM NaCl, the RING finger complexes showed no affinity for double-stranded DNA, eluting in the
void volume (50 mM NaCl). A general property of nucleic acid-binding proteins is that they exhibit detectable affinity to
nonspecific nucleic acids. The sequence-specific DNA-binding ADR1 zinc
finger domain displays this property. ADR1 bound to every column
assayed, eluting from single-stranded DNA, poly(A), poly(C), poly(G),
and poly(U) RNA columns at NaCl concentrations of ~350, ~325,
~200, ~400, and ~150 mM, respectively. In direct contrast, neither homodimer nor the heterodimer displayed any affinity
for the columns tested, with the protein eluting in the 50 mM NaCl wash in every case (data not shown). Similar
results are observed with the BD 26-131 construct (data not
shown).
Although BRCA1 has been implicated in the maintenance of genome
integrity (10, 15) and in transcriptional activation (16, 20), little
is known about its specific function in either of these processes. In
an approach aimed at characterizing functional properties of BRCA1, we
have focused on its interactions with BARD1, a protein shown to
interact with BRCA1 in vivo (8). Our results demonstrate
that, individually, BRCA1 and BARD1 RING finger constructs exist as
homodimers and that they preferentially form heterodimers. Limited
proteolysis analysis defines the RING finger structural domains
required for heterodimerization as comprising residues 1-109 of BRCA1
and residues 26-119 of BARD1. The heterodimer is remarkably stable,
existing at concentrations well below 10 The RING finger motifs correspond to residues 24-64 and residues
50-86 in BRCA1 and BARD1, respectively. Thus, our results demonstrate
that these motifs are part of a larger structural domain, rather than
representing stable autonomous domains themselves. A similar situation
exists for the recombination-activating gene 1 (RAG1) protein, the RING
finger motif of which is also contained within a larger domain that
exists as a homodimer in solution (21). The three domains share little
homology aside from the consensus RING finger motif residues, and RAG1
does not share additional sequence homology with either BRCA1 or BARD1.
There are several reports of studies in which constructs that consist
of a minimal RING finger motif sequence have been characterized, including a metal binding study of the RING finger (residues 22-77) of
BRCA1 (22) and solution structure studies of the RING fingers from
promyelocytic leukemia proto-oncoprotein and immediate-early equine
herpesvirus-1 protein (11, 23). In contrast to BRCA1, BARD1, and RAG1,
the short RING finger constructs used in the structure determinations
were reported to be monomeric. In each case, however, the authors
observed low solubility and a tendency of the constructs to aggregate.
This is consistent with our experience with shorter RING finger
constructs of BRCA1.4
Although results derived from studies involving minimal RING finger
constructs may provide interesting information, the use of suboptimal
domains may not reflect properties of the native proteins. In contrast,
longer constructs of BRCA1 and BARD1 have good solubility properties
and are well behaved in solution, which are general properties of
stable, folded structural domains.
Our studies and others (8, 9) demonstrate that the N-terminal domain of
BRCA1 is capable of high affinity protein-protein interactions with
heterologous partners. There has been much speculation in the
literature, however, concerning the role of RING finger domains as
potential DNA-binding modules (3). The conflicting reports on the DNA
binding ability of RING finger domains, along with studies that
implicate BRCA1 and BARD1 in DNA repair and transcriptional activation,
led us to test the nucleic acid binding properties of our well behaved
and well characterized RING finger constructs. Despite the fact that
all the constructs are positively charged at neutral pH, with
calculated pI values ranging from 7.7 to 8.4, neither homodimer nor the
heterodimer exhibited any nucleic acid binding activity. Consequently,
if BRCA1 and BARD1 play a role in DNA repair and/or transcriptional
activation, interactions with DNA must be mediated either by other
regions of the proteins or by additional cofactors.
Proteolysis mapping and fluorescence experiments provide general
structural insights as a preliminary step toward building a picture of
the BRCA1/BARD1 heterodimer. The strong protection from proteolysis of
regions outside the RING finger motif afforded in the heterodimer
suggests that these regions may play a direct role in the heterodimer
interface. In contrast, Lys55, a residue within the BRCA1
RING finger motif, is susceptible to proteolysis both in the homodimer
and the heterodimer, suggesting that it is surface-accessible in both
forms. Therefore, the loop that contains Lys55, which is
highly variable both in sequence and structure among known RING
fingers, does not appear to play a direct role in heterodimer formation
and may be accessible for interactions with other molecules. The
significant blue shift and intensity enhancement of intrinsic tryptophan (Trp) fluorescence observed upon heterodimer formation indicates that at least one of the three BD 26-152 Trp residues becomes buried. Of the three Trp residues in BD 26-152 (see Fig. 6),
Trp146 is very near the C-terminal end of the construct and
is readily removed by proteases in the heterodimer, making it unlikely
that Trp146 is responsible for the observed fluorescence
change. The two other Trp residues (Trp34 and
Trp91) flank the RING finger motif and are close to sites
that become protected to proteolysis in the heterodimer, suggesting
that one or both of these Trps undergo a change in environment.
Results from gel-filtration (Fig. 1A), cross-linking (Fig.
1B), and fluorescence (Fig. 2) studies convincingly show
that the heterodimer is the preferred species in solution.
Additionally, limited proteolysis results indicate that homodimers are
consistently more susceptible to proteolysis than the heterodimer. BD
26-140 is cleaved by Endo Lys C at virtually every lysine residue in the homodimer, whereas cleavage occurs only at Lys130 in
the heterodimer (Fig. 6). Likewise, BC-112 is susceptible to
proteolysis at its N-terminal region in its homodimeric state, whereas
the same region is protected in its heterodimeric state (Fig. 6). The
added proteolytic protection afforded to the heterodimer is in good
agreement with the observation that it is the preferred species in
solution and suggests that it is a functionally relevant species in the cell.
A major challenge in the study of breast cancer and other diseases is
the determination of the functions of the proteins involved. An
important early step in such efforts is the identification of
structural domains and binding partners. Such a characterization can
greatly aid in biochemical and functional studies of large multidomain,
multifunctional proteins. The studies reported here are a first step in
our goal to identify and understand the structure and function of the
N-terminal regions of BRCA1 and BARD1.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
-chymotrypsinogen,
and ribonuclease A, and glutaraldehyde were purchased from Sigma.
1
cm
1 for BD 26-152, BD 26-140, and BD 26-131, respectively.
RESULTS
Description of BRCA1 and BARD1 N-terminal constructs
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Fig. 1.
Characterization of the oligomeric states of
BRCA1 and BARD1 N-terminal constructs. A, analytical
gel-filtration elution profiles for BC-172 (dotted trace),
BD 26-152 (solid trace), and an equimolar mixture of
BC-172/BD 26-152 (dashed trace). Positions for the protein
standards bovine serum albumin (66 kDa), ovalbumin (43 kDa),
-chymotrypsinogen (23 kDa), and RNase (13.7 kDa) are indicated by
arrows. B, SDS-PAGE of glutaraldehyde
cross-linking reactions of BC-172 homodimer (lanes 1 and
2), BC-172/BD 26-152 heterodimer (lanes 3 and
4), and BD 26-152 homodimer (lanes 5 and
6) complexes. Molecular mass standards are shown in
lane 7. Each complex was incubated for 5 min in the presence
(lanes 1, 3, and 5) or absence (lanes 2, 4, and 6) of 0.01% (v/v) glutaraldehyde. The position
of molecular mass markers (right) and the monomeric
(M), homodimeric (D), and heterodimeric
(H) complexes are indicated (left). C,
analytical gel-filtration chromatography of BC-76 (solid
trace) and an equimolar mixture of BC-76 and BC-172 (dashed
trace) RING finger domain constructs. The mixture elutes as two
separate peaks from the gel-filtration column at volumes corresponding
to the expected molecular weights of BC-76 and BC-172 homodimers,
demonstrating that the two constructs do not interact.
ex = 290 nm). The emission maximum of an equimolar mixture of BC-112 and BD
26-152 is blue-shifted to 335 nm with a corresponding increase in
intensity (Fig. 2). Under the conditions
used in the fluorescence experiments, BD 26-152 is a homodimer.
Therefore, the shift and increase in emission intensity indicate that
at least one tryptophan in BD 26-152 experiences a more hydrophobic
environment in the heterodimer relative to the homodimer. The
fluorescence spectrum of the heterodimer isolated by gel-filtration is
virtually identical to the spectrum obtained by direct mixing of the
proteins (data not shown).
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Fig. 2.
Change in BARD1 tryptophan fluorescence upon
heterodimer formation. Tryptophan fluorescence emission spectra
( ex = 290 nm) of BD 26-152 homodimer (solid
trace) and an equimolar mixture of BC-112 and BD 26-152
(dashed trace) are shown. The BD 26-152 homodimer exhibits
an emission maximum at 347 nm, whereas the emission maximum of the
BC-112/BD 26-152 heterodimer blue-shifts to 335 nm with a concomitant
increase in fluorescence intensity.
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Fig. 3.
Determination of BRCA1 and BARD1 N-terminal
structural domains. MALDI-TOF mass spectra for the limited
proteolytic digest of the BC-112/BD 26-152 heterodimer with Endo Lys C
are shown. Samples were collected before the addition of protease
(A) and 25 (B) and 60 (C) min after
the addition of protease. Peaks labeled +2 indicate doubly protonated
fragments that arise from the MALDI process. Peaks corresponding to
singly charged fragments are labeled with residue numbers as determined
from the measured mass.
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Fig. 4.
Comparison of proteolytic susceptibility of
BRCA1 and BARD1 homodimers and heterodimer. MALDI-TOF mass spectra
for the limited proteolytic digest of BC-112 (A), BC-112/BD
26-140 (B), and BD 26-140 (C) complexes with
Endo Lys C. Spectra shown are of samples collected 25 min after the
addition of protease.
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Fig. 5.
Comparison of susceptibility of BRCA1 homo-
and heterodimer complexes to V8 proteolysis. MALDI-TOF mass
spectra of BC-112 homodimer (A) and BC-112/BD 26-140
heterodimer (B) complex samples collected 60 min after the
addition of V8 protease. The asterisk denotes the presence
of known sample impurities.
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Fig. 6.
Summary of limited proteolysis results.
BC-112 and BD 26-152 constructs are aligned according to their
conserved residues. RING finger motif conserved residues are
underlined and boldface. Arrowheads
above and below each sequence indicate susceptible cleavage sites in
homodimeric (small arrowheads) and heterodimeric
(large arrowheads) complexes. The superscript
number on the first and last conserved RING finger cysteine residues in
each motif corresponds to the amino acid number in the sequence of each
protein. Tryptophan residues are shown in green, and
potential Endo Lys C and V8 cleavage sites are shown in blue
and red, respectively.
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Fig. 7.
BRCA1 and BARD1 N-terminal complexes do not
bind nucleic acids. Elution profiles of the BC-112 homodimer
(dotted trace), BD 26-140 homodimer (solid
trace), BC-112/BD 26-140 heterodimer (dashed trace),
and the ADR1 zinc finger domain (solid trace) from the
double-stranded DNA-cellulose column are shown. Samples were applied in
equilibration buffer containing 50 mM NaCl. Every RING
finger species tested failed to bind, eluting in the void volume,
whereas the ADR1 zinc finger domain (a known DNA-binding domain) was
retained by the DNA column and eluted at [NaCl] ~400
mM. The arrowheads below the elution profiles
indicate the NaCl concentration gradient used.
DISCUSSION
7
M.3
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants RO1 GM46701 and RO1 CA79953 (to R. E. K.) and RO1 CA27632 (to M.-C. K.) and a generous contribution from the Boeing Foundation.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.
§ Supported by a Research Supplement for Underrepresented Minorities from the National Institutes of Health (RO1 CA27632-18S1).
¶ These authors contributed equally to this work.
** Supported in part by an American Cancer Society postdoctoral fellowship.
To whom correspondence should be addressed: Dept. of
Biochemistry and Biomolecular Structure Center, Univ. of Washington, Box 357742, Seattle, WA 98195-7742. Fax: 206-543-8394; E-mail: klevit{at}u.washington.edu.
2 BARD1 contains two potential initiator methionines (residues Met1 and Met26). The yeast two-hybrid studies showed that the first 25 residues of BARD1 are not required for the BRCA1/BARD1 interaction.
3 Serial dilution experiments failed to detect dissociation of the dimers by either fluorescence or gel-filtration to the lowest concentrations at which a signal could be measured (J. Meza, P. Brzovic, and R. Klevit, unpublished observation).
4 J. Meza, P. Brzovic, and R. Klevit, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: RING, really interesting new gene; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; Endo, endoproteinase.
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REFERENCES |
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