We describe a method that detects proteins capable of interacting with a known protein and that results in the immediate availability of the cloned genes for these interacting proteins. Plasmids are constructed to encode two hybrid proteins. One hybrid consists of the DNA-binding domain of the yeast transcriptional activator protein GAL4 fused to the known protein; the other hybrid consists of the GAL4 activation domain fused to protein sequences encoded by a library of yeast genomic DNA fragments. Interaction between the known protein and a protein encoded by one of the library plasmids leads to transcriptional activation of a reporter gene containing a binding site for GAL4. We used this method with the yeast SIR4 protein, which is involved in the trinscriptional repression of yeast mating type information. (a) We used the twohybrid system to demonstrate that SIR4 can form homodimers.(ii) A small domain consisting of the C terminus of SIR4 was shown to be sufficient to mediate this interaction. (iii) We screened a library to detect hybrid proteins that could interact with the SIR4 C-terminal domain and identified SIR4 from this library. This approach could be readily extended to mammalian proteins by the construction of appropriate cDNA libraries in the activation domain plasmid.Specific interactions between proteins form the basis of many essential biological processes. Additionally, transforming proteins of tumor viruses in many cases exert their effect through their interactions with cellular proteins; for example, the simian virus 40 (SV40) large tumor (T) antigen binds to the cellular proteins p53 and Rb (1, 2). Consequently, considerable effort has been made to identify those proteins that bind to proteins of interest. Typically, these interactions have been detected by using coimmunoprecipitation experiments in which antibody to a known protein is used to precipitate associated proteins as well. Such biochemical methods, however, result only in the identification ofthe apparent molecular mass of the associated proteins; obtaining cloned genes for these proteins is often a difficult process. In one approach, this problem has been circumvented by the use ofpurified proteins as probes against bacterial expression libraries, where a positive signal for an interacting protein is accompanied by the availability of the corresponding gene (3).We have described a method by which a protein-protein interaction is identified in vivo through reconstitution of the activity ofa transcriptional activator (4). The method is based on the properties of the yeast GAL4 protein, which consists of separable domains responsible for DNA-binding and transcriptional activation (5). Plasmids encoding two hybrid proteins, one consisting of the GAL4 DNA-binding domain fused to protein X and the other consisting of the GAL4 activation domain fused to protein Y, are constructed and introduced into yeast. Interaction between proteins X and Y leads to the transcriptional activation of a reporter gene containing a binding site fo...

Recent work has shown that the murine BRCA2 tumor suppressor protein interacts with the murine RAD51 protein. This interaction suggests that BRCA2 participates in DNA repair. Residues 3196 -3232 of the murine BRCA2 protein were shown to be involved in this interaction. Here, we report the detailed mapping of additional domains that are involved in interactions between the human homologs of these two proteins. Through yeast two-hybrid and biochemical assays, we demonstrate that the RAD51 protein interacts specifically with the eight evolutionarily conserved BRC motifs encoded in exon 11 of brca2 and with a similar motif found in a Caenorhabditis elegans hypothetical protein. Deletion analysis demonstrates that residues 98 -339 of human RAD51 interact with the 59-residue minimal region that is conserved in all BRC motifs. These data suggest that the BRC repeats function to bind RAD51.Germline mutations in the brca1 and brca2 tumor suppressor genes account for approximately 5-10% of all breast cancer cases (1-4). In addition, deleterious alleles of brca1 or brca2 are responsible for almost all familial ovarian cancer, and deleterious alleles of brca2 are involved in hereditary male breast cancer (1-4). Currently the mechanism of action of these two genes remains largely undefined. The brca1 and brca2 genes encode large proteins, 1863 and 3418 amino acids, respectively (1, 3). A search of the public sequence data bases has revealed little sequence homology to previously identified proteins. However, analysis of the protein sequence has revealed several statistically significant repeated motifs in these genes (5, 6). For example, eight internal repeats, known as BRC motifs, are found clustered in exon 11 of the human BRCA2 protein. These motifs are not found in BRCA1 but are conserved in all mammalian BRCA2 proteins that have been sequenced. A similar motif is also present in the hypothetical protein encoded by the Caenorhabditis elegans gene T07E3.5 (5, 7).A number of studies have been conducted in the past year to elucidate the biological roles of the brca1 and brca2 genes.Knock-outs of the mouse homologs of these genes indicate that they are essential during development. The ablation of either brca1 or brca2 results in an embryonic lethal phenotype characterized by failure of proliferation during approximately days 6 -8 of gestation (8, 9). Levels of RNA expression from brca1 and brca2 are coordinately regulated during proliferation and differentiation in mammary epithelial cells (11,12). Furthermore, their expression varies at different stages of the cell cycle, with RNA levels peaking at the G 1 /S boundary (13,14). These findings provide preliminary evidence that these breast cancer genes participate in a common functional pathway. Three independent studies have now established that the RAD51 DNA repair protein is linked to the BRCA1 and BRCA2 pathways (9,10,15). Immunoprecipitation experiments reveal that RAD51 forms a complex with BRCA1, and immunofluorescence analysis shows that both proteins are co-localize...

ABSTRACTp53 is a tumor-suppressor proton that can activate and repress trnscription. Using