LiteFold/DisProt huggingface dataset:
Protein: DP00238 (UniProt P38398)
Region: residues 498	to 663
Annotation: DNA binding (Molecular function)
Evidence: electrophoretic mobility shift assay evidence used in manual assertion
Supporting statements:
Electrophoretic mobility shift assay and intrinsic tryptophan fluorescence experiments also demonstrate that, although intrinsically disordered, polypeptides from the central region are able to mediate interactions with DNA and p53 with affinities in the low micromolar range. This supports a model in which the central region may act as a long flexible scaffold for intermolecular interactions, thereby helping to integrate multiple signals in the DNA damage response pathway.
The region previously reported to bind non-specifically to DNA (residues 452–1079) is relatively large (>600 residues), yet appears to be largely disordered. Since DNA binding domains are often small (∼60–150), we therefore suspected that the DNA binding region might actually comprise an even smaller region of BRCA1. Each of the 20 soluble fragments were assayed for binding to a 31 bp double-stranded DNA (Figure 3). Fragments #5–#7, #10, #11, #17, #18, #20 and #21 demonstrated DNA binding at protein concentrations in the low micromolar range. Accounting for the overlapping nature of the constructs, these binding experiments implicate two regions in DNA binding, residues 498–663 (DB1) and 936–1057 (DB2).
The region previously reported to bind non-specifically to DNA (residues 452–1079) is relatively large (>600 residues), yet appears to be largely disordered. Since DNA binding domains are often small (∼60–150), we therefore suspected that the DNA binding region might actually comprise an even smaller region of BRCA1. Each of the 20 soluble fragments were assayed for binding to a 31 bp double-stranded DNA (Figure 3). Fragments #5–#7, #10, #11, #17, #18, #20 and #21 demonstrated DNA binding at protein concentrations in the low micromolar range. Accounting for the overlapping nature of the constructs, these binding experiments implicate two regions in DNA binding, residues 498–663 (DB1) and 936–1057 (DB2).
Protein: DP00238 (UniProt P38398)
Region: residues 936	to 1057
Annotation: DNA binding (Molecular function)
Evidence: electrophoretic mobility shift assay evidence used in manual assertion
Supporting statements:
Electrophoretic mobility shift assay and intrinsic tryptophan fluorescence experiments also demonstrate that, although intrinsically disordered, polypeptides from the central region are able to mediate interactions with DNA and p53 with affinities in the low micromolar range. This supports a model in which the central region may act as a long flexible scaffold for intermolecular interactions, thereby helping to integrate multiple signals in the DNA damage response pathway.
The region previously reported to bind non-specifically to DNA (residues 452–1079) is relatively large (>600 residues), yet appears to be largely disordered. Since DNA binding domains are often small (∼60–150), we therefore suspected that the DNA binding region might actually comprise an even smaller region of BRCA1. Each of the 20 soluble fragments were assayed for binding to a 31 bp double-stranded DNA (Figure 3). Fragments #5–#7, #10, #11, #17, #18, #20 and #21 demonstrated DNA binding at protein concentrations in the low micromolar range. Accounting for the overlapping nature of the constructs, these binding experiments implicate two regions in DNA binding, residues 498–663 (DB1) and 936–1057 (DB2).
The region previously reported to bind non-specifically to DNA (residues 452–1079) is relatively large (>600 residues), yet appears to be largely disordered. Since DNA binding domains are often small (∼60–150), we therefore suspected that the DNA binding region might actually comprise an even smaller region of BRCA1. Each of the 20 soluble fragments were assayed for binding to a 31 bp double-stranded DNA (Figure 3). Fragments #5–#7, #10, #11, #17, #18, #20 and #21 demonstrated DNA binding at protein concentrations in the low micromolar range. Accounting for the overlapping nature of the constructs, these binding experiments implicate two regions in DNA binding, residues 498–663 (DB1) and 936–1057 (DB2).
Protein: DP00238 (UniProt P38398)
Region: residues 219	to 498
Annotation: p53 binding (Molecular function)
Evidence: fluorescence evidence used in manual assertion
Supporting statements:
Electrophoretic mobility shift assay and intrinsic tryptophan fluorescence experiments also demonstrate that, although intrinsically disordered, polypeptides from the central region are able to mediate interactions with DNA and p53 with affinities in the low micromolar range. This supports a model in which the central region may act as a long flexible scaffold for intermolecular interactions, thereby helping to integrate multiple signals in the DNA damage response pathway.
A small region of BRCA1 binds weakly to p53
Another part of central BRCA1 that has been reported to interact with other molecules is the region from residues 224 to 500. Zhang et al.31 mapped its interaction, in vitro, to the C-terminal domain of p53 and suggested that such physical association could regulate gene expression through BRCA1-dependent stimulation of p53-mediated transcription. To investigate the strength of the interaction, we measured quenching of the intrinsic tryptophan fluorescence of BRCA1 fragment #19 (residues 219–498) upon binding to various p53 C-terminal domains (residues 311–393, 310–360 and 355–393).
As shown in the titration curves in Figure 4 both the p53 C terminus (residues 311–393), which contains the tetramerization domain, and the p53 basic region (residues 355–393) bound to BRCA1 with relatively weak affinity (Kd values of ∼15 μM and ∼68 μM, respectively).
Analysis of p53 binding to BRCA1 by intrinsic tryptophan fluorescence. BRCA1 (residues 219–498) which includes four tryptophan residues was incubated with increasing amounts of p53 peptides 311–393 (●), 355–393 (▴) or 310–360 (□) which all lack a tryptophan residue. Binding was assessed by measuring the total tryptophan fluorescence at 354 nm, using an excitation wavelength of 295 nm. The change in fluorescence signal, as compared with BRCA1 (residues 219–498) alone, was plotted as a function of target peptide concentration.
Protein: DP00238 (UniProt P38398)
Region: residues 175	to 303
Annotation: protein binding (Molecular function)
Evidence: deletion mutation phenotypic evidence used in manual assertion
Supporting statements:
We next generated a series of Gal4BD-BRCA1 deletion mutants to map the regions required for interaction with c-Myc. As shown in Figure 1, a small segment of BRCA1 (amino acid residue 433-511, as in pGalBD-BRMS) is both required and sufficient for binding to c-Myc. We designated this region MB1 (for Myc-binding region 1). An N-terminal Myc-binding region (termed MB2) of BRCA1 requires amino acid residues 175-303.
This region corresponds to the first binding site of c-Myc, MB1.
Protein: DP00238 (UniProt P38398)
Region: residues 433	to 511
Annotation: protein binding (Molecular function)
Evidence: deletion mutation phenotypic evidence used in manual assertion
Supporting statements:
We next generated a series of Gal4BD-BRCA1 deletion mutants to map the regions required for interaction with c-Myc. As shown in Figure 1, a small segment of BRCA1 (amino acid residue 433-511, as in pGalBD-BRMS) is both required and sufficient for binding to c-Myc. We designated this region MB1 (for Myc-binding region 1). An N-terminal Myc-binding region (termed MB2) of BRCA1 requires amino acid residues 175-303.
This region corresponds to the second binding site of c-Myc, MB2.
Protein: DP00238 (UniProt P38398)
Region: residues 304	to 394
Annotation: protein binding (Molecular function)
Evidence: glutathione S-transferase pull-down assay evidence used in manual assertion
Supporting statements:
We further found that the BRCA1 protein complexes with the hypophosphorylated form of pRb. This binding is localized to amino acids 304–394 of BRCA1 protein and requires the ABC domain of pRb.
The NH2-terminal 394 aa (ScC7 fragment, amino acids 1–394) also showed stable binding to GST-Rb ABC protein (Fig.6B). However, further truncation of BRCA1 (N-R, amino acids 1–303) completely eliminated the interaction with GST-Rb ABC.
To further demonstrate the binding of pRb to BRCA1, we reversed the probe and target proteins by using GST fusion proteins with BRCA1 fragments GST-N-R (amino acids 1–303) and GST-R-K (amino acids 304–772 shown in Fig. ​Fig.66A) as probe proteins and pRb as target. As before, in vitro-translated Rb protein bound the GST-R-K fragment containing previously identified pRb binding domain in exon 11, but not with the GST-N-R protein or GST alone (Fig. ​(Fig.66C).
BRCA1 Protein Binds to the Hypophosphorylated Form of pRb.
Protein: DP00238 (UniProt P38398)
Region: residues 304	to 394
Annotation: negative regulation of cell growth (Biological process)
Evidence: deletion mutation phenotypic evidence used in manual assertion
Supporting statements:
BRCA1-associated growth arrest is RB-dependent
Deletion of Amino Acids 303–394 from BRCA1 Protein Inactivates Its Growth-Suppressive Property.
To test the biological role of Rb-BRCA1 interaction, we performed an in-frame deletion of the 92 aa (amino acids 303–394) from BRCA1 which comprised the putative binding region for Rb protein. When used in colony formation assay, BRCA1-delta90 had no effect on growth control (Fig.8). A construct generating the COOH-terminal truncation of BRCA1 was also ineffective in inhibiting colony formation.
A mutant BRCA1 protein bearing a deletion of the Rb binding domain does not suppress cell growth. Colony formation assay was performed in U2OS cells (as in Fig.1) with wild-type BRCA1 or BRCA1 bearing an in-frame deletion of 90 aa (amino acids 303–394) or a COOH-terminal truncation. Each transfection was done in triplicate in three independent experiments.
Protein: DP00238 (UniProt P38398)
Region: residues 758	to 1064
Annotation: protein binding (Molecular function)
Evidence: qualitative western immunoblotting evidence used in manual assertion
Supporting statements:
BRCA1 residues 758-1064 alone formed Rad51-containing complexes in vitro. Rad51 is also specifically associated with developing synaptonemal complexes in meiotic cells, and BRCA1 and Rad51 were both detected on asynapsed (axial) elements of human synaptonemal complexes. These findings suggest a functional interaction between BRCA1 and Rad51 in the meiotic and mitotic cell cycles, which, in turn, suggests a role for BRCA1 in the control of recombination and of genome integrity.
Protein: DP00238 (UniProt P38398)
Region: residues 758	to 1064
Annotation: localization (Biological process)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
BRCA1 and Rad51 Colocalize in Discrete Nuclear Foci
Here, we report that BRCA1 and hRad51 colocalize in S phase cells, interact physically, and, in keeping with previous reports of the behavior of hRad51 (2, 53), share common space on the surfaces of zygotene and pachytene meiotic chromosomes.
This suggested that the colocalization of BRCA1 and Rad51 is conditional or transient, even in S phase cells. Similar colocalization results were obtained in WI38 and CV-1 cells.
Protein: DP00238 (UniProt P38398)
Region: residues 740	to 1083
Annotation: protein binding (Molecular function)
Evidence: yeast 2-hybrid evidence used in manual assertion
Supporting statements:
Among the known FANC proteins, we find evidence for direct interaction only between the FANCA protein and BRCA1. The evidence rests on three different tests: yeast two-hybrid analysis, coimmunoprecipitation from in vitro synthesis, and coimmunoprecipitation from cell extracts. The amino terminal portion of FANCA and the central part (aa 740-1083) of BRCA1 contain the sites of interaction. The interaction does not depend on DNA damage, thus FANCA and BRCA1 are constitutively interacting. The demonstrated interaction directly connects BRCA1 to the FA pathway of DNA repair.
FANCA and BRCA1 interact in the yeast two-hybrid test
Protein: DP00238 (UniProt P38398)
Region: residues 740	to 1083
Annotation: protein binding (Molecular function)
Evidence: co-immunoprecipitation evidence used in manual assertion
Supporting statements:
Among the known FANC proteins, we find evidence for direct interaction only between the FANCA protein and BRCA1. The evidence rests on three different tests: yeast two-hybrid analysis, coimmunoprecipitation from in vitro synthesis, and coimmunoprecipitation from cell extracts. The amino terminal portion of FANCA and the central part (aa 740-1083) of BRCA1 contain the sites of interaction. The interaction does not depend on DNA damage, thus FANCA and BRCA1 are constitutively interacting. The demonstrated interaction directly connects BRCA1 to the FA pathway of DNA repair.
FANCA and BRCA1 coimmunoprecipitate in vitro
Coimmunoprecipitation of FANCA and BRCA1 in cell extracts
Protein: DP00238 (UniProt P38398)
Region: residues 1343	to 1440
Annotation: protein binding (Molecular function)
Evidence: deletion mutation phenotypic evidence used in manual assertion
Supporting statements:
JunB potentiates function of BRCA1 activation domain 1 (AD1) through a coiled-coil-mediated interaction
BRCA1 is involved in the regulation of multiple nuclear events including transcription. AD1, one of the two trans-activation domains in BRCA1, stimulates transcription in a cell context-dependent manner. Here, it is shown that BRCA1 interacts with Jun proteins via a coiled-coil motif in AD1 and the basic leucine zipper (bZIP) region of the Jun proteins. The Jun-interacting domain in BRCA1 is critical for AD1-mediated transcriptional activation. In particular, the strength of AD1 in transcriptional activation is limited by the JunB level and ectopic expression of JunB potentiates the transcriptional activity of AD1. Furthermore, JunB mRNA expression is down-regulated in many ovarian tumor tissues examined. Thus, the coiled-coil-mediated cooperation between BRCA1 and JunB may facilitate the function of these proteins in tissue-specific transcriptional regulation and tumor suppression.
AD1 is necessary and sufficient for BRCA1 binding to the Jun proteins
Further deletional studies within AD1 (Fig.3, constructs 13–16) revealed a minimal JunB-binding domain (Fig.3, construct 15; amino acids 1343–1440), which consists of the coiled-coil motif and an ∼60 amino acid upstream sequence. This finding indicates that the AD1 region is both necessary and sufficient for BRCA1 binding to JunB.
Protein: DP00238 (UniProt P38398)
Region: residues 1407	to 1414
Annotation: positive regulation of transcription, DNA-templated (Biological process)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
JunB potentiates function of BRCA1 activation domain 1 (AD1) through a coiled-coil-mediated interaction
BRCA1 is involved in the regulation of multiple nuclear events including transcription. AD1, one of the two trans-activation domains in BRCA1, stimulates transcription in a cell context-dependent manner. Here, it is shown that BRCA1 interacts with Jun proteins via a coiled-coil motif in AD1 and the basic leucine zipper (bZIP) region of the Jun proteins. The Jun-interacting domain in BRCA1 is critical for AD1-mediated transcriptional activation. In particular, the strength of AD1 in transcriptional activation is limited by the JunB level and ectopic expression of JunB potentiates the transcriptional activity of AD1. Furthermore, JunB mRNA expression is down-regulated in many ovarian tumor tissues examined. Thus, the coiled-coil-mediated cooperation between BRCA1 and JunB may facilitate the function of these proteins in tissue-specific transcriptional regulation and tumor suppression.
Substitution of two such leucines in the coiled-coil region of BRCA1 (L1407 and L1414) abrogated the BRCA1–JunB interaction (Fig.4A,B, lanes 3–5). Importantly, the same mutations also impaired the transcriptional activity of GAL4–AD in the luciferase reporter assay (Fig.4C, lanes 3–5).
Thus, the affinity of BRCA1 for the Jun proteins strongly correlates with the strength of the trans-activation domain in transcriptional stimulation.
Protein: DP00238 (UniProt P38398)
Region: residues 758	to 1064
Annotation: intracellular non-membrane-bounded organelle (Cellular component)
Evidence: immunofluorescence confocal microscopy evidence used in manual assertion
Supporting statements:
BRCA1 immunostaining reveals discrete, nuclear foci during S phase of the cell cycle.
Here, we report that BRCA1 and hRad51 colocalize in S phase cells, interact physically, and, in keeping with previous reports of the behavior of hRad51 (2, 53), share common space on the surfaces of zygotene and pachytene meiotic chromosomes.
These and other results not shown here indicate that the BRCA1 nuclear dot pattern is S phase–specific.
Protein: DP00240 (UniProt P35611)
Region: residues 430	to 737
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Circular dichroism indicated that COOH-terminal tail regions of a- and f3-adducin are unstructured, with no evidence for a-helix or f3-sheet based on signals at 208 and 222 nm, and a negative peak at 200 nm which is indicative of a random coil (Fig. 4B).
Protein: DP00240 (UniProt P35611)
Region: residues 430	to 737
Annotation: disorder (Structural state)
Evidence: cleavage assay evidence used in manual assertion
Supporting statements:
The COOH-terminal region of both the α- and β-adducin subunits is hydrophilic and highly protease sensitive, in contrast to the stable
globular NH2 terminus (Fig. 3).
Protein: DP00240 (UniProt P35611)
Region: residues 430	to 737
Annotation: disorder (Structural state)
Evidence: sodium dodecyl sulfate polyacrylamide gel electrophoresis evidence used in manual assertion
Supporting statements:
Isolated COOH-ter-minal constructs migrated at an apparent 60 kDa, substantially higher than predicted molecular mass of 34 and 33 kDa based on amino acid sequence.
Protein: DP00240 (UniProt P35611)
Region: residues 430	to 737
Annotation: protein dimerization activity (Molecular function)
Evidence: radioligand binding assay evidence used in manual assertion
Supporting statements:
α- and β-adducin tail domains associated with tails of both subunits, as well as the intact erythrocyte adducin protein (panels B and D). This binding is specific, as evidenced by the displacement of binding when 1.3 μM unlabeled α- or β-adducin tail is included in the incubation (panels C and E ). Individual subunit tails were able to form α-α, α-β, and β-β contacts. Neither the α- nor β-tails bound to the NH2-terminal core.
Protein: DP00241 (UniProt P35612)
Region: residues 409	to 726
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Circular dichroism indicated that COOH-terminal tail regions of α- and β-adducin are unstructured, with no evidence for α-helix or β-sheet based on signals at 208 and 222 nm, and a negative peak at 200 nm which is indicative of a random coil (Fig. 4B).
Protein: DP00241 (UniProt P35612)
Region: residues 409	to 726
Annotation: disorder (Structural state)
Evidence: sodium dodecyl sulfate polyacrylamide gel electrophoresis evidence used in manual assertion
Supporting statements:
"Isolated COOH-ter-minal constructs migrated at an apparent 60 kDa, substantially higher than predicted molecular mass of 34 and 33 kDa based on amino acid sequence.
Protein: DP00241 (UniProt P35612)
Region: residues 409	to 726
Annotation: disorder (Structural state)
Evidence: cleavage assay evidence used in manual assertion
Supporting statements:
The COOH-terminal region of both the α- and β-adducin subunits is hydrophilic and highly protease sensitive, in contrast to the stable globular NH2 terminus (Fig. 3).
Protein: DP00241 (UniProt P35612)
Region: residues 409	to 726
Annotation: protein dimerization activity (Molecular function)
Evidence: radioligand binding assay evidence used in manual assertion
Supporting statements:
α- and β-adducin tail domains associated with tails of both subunits, as well as the intact erythrocyte adducin protein (panels B and D). This binding is specific, as evidenced by the displacement of binding when 1.3 μM unlabeled α- or β-adducin tail is included in the incubation (panels C and E ). Individual subunit tails were able to form α-α, α-β, and β-β contacts. Neither the α- nor β-tails bound to the NH2-terminal core.
Protein: DP00243 (UniProt P23025)
Region: residues 166	to 179
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The sheet-helix-loop region is composed of an antiparallel β-sheet (strands β3–5), helix α1, and two long loops, L1 and L2. Loop L1 (residues 148–163) connects the α1 helix and the β4 strand, and loop L2 (residues 166–179) connects strands β4 and β5. While the structure of loop L1, except for residues 156–162, has been relatively well defined by hydrophobic contacts with residues in helices α1 and α2, loop L2 apparently does not form a single definite structure based on the observation of few long-range NOEs.
Since we observe few medium- and no long-range NOEs for the C-terminal sequence (residues 211–219) this region was excluded from the structure calculations.
Protein: DP00243 (UniProt P23025)
Region: residues 166	to 179
Annotation: flexible linker (Disorder function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The sheet-helix-loop region is composed of an antiparallel β-sheet (strands β3–5), helix α1, and two long loops, L1 and L2. Loop L1 (residues 148–163) connects the α1 helix and the β4 strand, and loop L2 (residues 166–179) connects strands β4 and β5. While the structure of loop L1, except for residues 156–162, has been relatively well defined by hydrophobic contacts with residues in helices α1 and α2, loop L2 apparently does not form a single definite structure based on the observation of few long-range NOEs.
Protein: DP00243 (UniProt P23025)
Region: residues 210	to 219
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Since we observe few medium- and no long-range NOEs for the C-terminal sequence (residues 211–219) this region was excluded from the structure calculations.
Steady-state heteronuclear 15N{1H}-NOE values19 smaller than 0.68 were observed for the residues 157–162 in loop L1, for all the residues in loop L2 except residues 171–174 whose signals could not be observed in the HSQC spectra, and for the nine C-terminal residues of the central domain (data not shown). This indicates that L2, part of L1 and the C-terminal flanking sequence are highly mobile in solution.
Since we observe few medium- and no long-range NOEs for the C-terminal sequence (residues 211–219) this region was excluded from the structure calculations.
Protein: DP00243 (UniProt P23025)
Region: residues 1	to 101
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The N-terminal 100 residues of XPA, which interact with RPA32, are disordered (Fig. 1a).
The Electron Microscopy evidences of the Core7-XPA-DNA complexes, shows this region is disordered. 
Cross-references: PDB:8EBU
PDB:8EBY
EMDB:27998
EMDB:28002
Protein: DP00246 (UniProt P21758)
Region: residues 1	to 50
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The results in phosphate buffer (pH 7.0) shown in Fig. 3 indicated that neither peptide formed a definite secondary structure.