Recombination is important for the repair of DNA damage and for

Recombination is important for the repair of DNA damage and for chromosome segregation during meiosis; it has also been shown to participate in the regulation of cell proliferation. the yeast epistasis group are required for both homologous recombination and the repair of double strand-breaks (DSBs) (10). Mutations in these genes result in severe cellular sensitivity to ionizing radiation and alkylating brokers (e.g., methyl methanesulfonate [MMS]), reduced spontaneous and DNA damage-induced mitotic recombination, and the production of inviable spores in meiotic recombination (36). Biochemical data suggest that some products of the epistasis group (Rad51, Rad52, Rad54, Rad55, Rad57, and replication protein A [RPA]) assemble-disassemble on DNA. The Rad51 protein is a key component of this complex. It has significant sequence and functional similarity to RecA protein, the crystal structure of which has been determined (47). The two proteins share a region of 30% identity, comprising amino acid residues 154 to 374 of Rad51 and 33 to 240 of RecA, corresponding to a large middle domain essential for recombination. Indeed, Rad51 protein also possesses some of the RecA functional activities, e.g., binding of single-stranded DNA (ssDNA) and double-stranded DNA, ATP hydrolysis, formation of nucleoprotein filaments, and formation of heteroduplex DNA (51, 54). Rad51 interacts with itself, with Rad52 (9, 43), with Rad54 (7, 17), and with Rad55, which in turn associates with Rad57 (15, 18). In accordance with the biochemical and two-hybrid data obtained for these interactions, there are also many genetic data supporting their cellular relevance (7, 11, 41). The importance of the N-terminal a part of Rad51 has been exhibited in Rad51 self-association and in the conversation with Rad52 (31). The details of these two interactions have not been explored further. Recently, much attention has been paid to the biochemical function of Rad51 and its associated proteins, Rad51, Rad52, Rad54, and the Rad55-Rad57 heterodimer. Rad52 shows annealing activities (32, 50) and promotes the exchange of RPA for Rad51 protein on ssDNA (28, 52), and human Rad52 binds double-strand breaks (56). Rad54 belongs to a SWI2/SNF2 protein family, whose users modulate chromatin structure (57). Biochemical studies show that Rad54 forms a dimer or oligomer on DNA and promotes Rad51-dependent homologous DNA pairing through changes in DNA double-helix conformation (37). Both and are sequence homologs of genes is usually conserved in a wide variety of eukaryotic organisms, suggesting their importance to eukaryotic cellular function in general. An interesting feature of Rad51p is usually its crucial role in the mouse, where the mutant displays early embryonic lethality (24) but also impairs spontaneous and DSB-induced conservative recombination without affecting cell viability (22). The physical conversation of HsRad51 with several tumor suppressor genes, namely, p53, BRCA1, and BRCA2, implies its possible role(s) in tumorigenesis (26, 48). Here we describe a new approach to dissect protein interactions within the multiprotein complex and the application of this technique to the 63902-38-5 yeast recombination-repair complex. By this strategy, mutations launched into one component of a two-hybrid conversation pair can be readily and simultaneously screened for effects on interactions with each of several desired partner proteins, thus directly exposing different patterns of effects and defining the residues involved. We have used this approach to investigate the interactions of yeast Rad51 with Rad52, Rad54, Rad55, and Rad51 itself by isolating mutants which abolish specific interactions within the Rad51 SLC7A7 complex without affecting others. Such analysis was not possible using the conventional two-hybrid system. Localization of these mutations in a homology model of the Rad51 protein and the Rad51 filament 63902-38-5 reveals possible conversation interfaces. The mutants defective in specific interactions also show a decrease in MMS-induced DSB repair, exposing new data around the importance of protein-protein interactions in recombination and repair. Possible compensatory mutations that activate protein interactions were also recognized. This mutagenic two-hybrid strategy can be used to dissect other multiprotein complexes or mechanisms and can help us understand the development of compensatory mutations as well as define conversation regions de novo. MATERIALS AND METHODS Media and plasmids. Yeast 63902-38-5 and bacterial media, as well as all the standard yeast genetic methods, were used as explained previously (2). 5-Fluoroorotic acid medium was prepared by the method of Boeke et al. (5). The vectors pGBT9 and pGAD10 have been described elsewhere (6). Coding sequences of were amplified from genomic clones by PCR using the primers scRAD51-FOR plus scRAD51-REV, scRAD52-FOR plus scRAD52-REV, and scRAD54-FOR plus scRAD54-REV, respectively (Table ?(Table1).1). The PCR products were digested with mutations, were constructed by inserting the complementation studies is usually a derivative of W303 (gene was replaced.