Identification of proteins that interact with BRCA1 by Far-Western library screening.
Protein-protein interactions control numerous biological processes. In the case of a protein with no known function, identification of interacting proteins may lend insight into its cellular function. Protein-protein interactions are often detected by yeast two-hybrid screening which is based on a transcriptional read-out. One limitation of this technique is that transcription factors, when used as bait, frequently impair the effectiveness of this screen because they give rise to high levels of false positives. The carboxyl terminus of the breast cancer tumor suppressor gene, BRCA1, contains two BRCT motifs, a motif found in several DNA repair and cell cycle checkpoint proteins. This region of BRCA1 also exhibits an intrinsic transcriptional transactivation activity when bound to DNA as a fusion protein, thereby limiting its use in yeast two-hybrid screen. In order to isolate proteins that interact with this domain of BRCA1, we utilized a Far-Western screen, a method based on direct protein binding. We used recombinant histidine-tagged BRCT as the primary protein probe. We isolated eight cDNAs that bind to the BRCT domain of BRCA1. Further analysis demonstrated that two of the clones encode for proteins that interact directly with the BRCT domain of BRCA1.
Yarden
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Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Brody
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D019313:BRCA1 Protein / Q000378:metabolism*; D015153:Blotting, Western / Q000379:methods*; D003001:Cloning, Molecular; D015723:Gene Library; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D010446:Peptide Fragments / Q000235:genetics; D010920:Placenta / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics; D014407:Tumor Cells, Cultured
D019313:BRCA1 Protein; D010446:Peptide Fragments; D012333:RNA, Messenger