Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.
The highly conserved Saccharomyces cerevisiae Rad51 protein plays a central role in both mitotic and meiotic homologous DNA recombination. Seven members of the Rad51 family have been identified in vertebrate cells, including Rad51, Dmc1, and five Rad51-related proteins referred to as Rad51 paralogs, which share 20 to 30% sequence identity with Rad51. In chicken B lymphocyte DT40 cells, we generated a mutant with RAD51B/ RAD51L1, a member of the Rad51 family, knocked out. RAD51B؊/؊ cells are viable, although spontaneous chromosomal aberrations kill about 20% of the cells in each cell cycle. Rad51B deficiency impairs homologous recombinational repair (HRR), as measured by targeted integration, sister chromatid exchange, and intragenic recombination at the immunoglobulin locus. RAD51B؊/؊ cells are quite sensitive to the cross-linking agents cisplatin and mitomycin C and mildly sensitive to ␥-rays. The formation of damage-induced Rad51 nuclear foci is much reduced in RAD51B ؊/؊ cells, suggesting that Rad51B promotes the assembly of Rad51 nucleoprotein filaments during HRR. These findings show that Rad51B is important for repairing various types of DNA lesions and maintaining chromosome integrity. Double-strand DNA breaks (DSBs) occur during DNA replication and are produced by ionizing radiation. Since DSBs are so deleterious to the cell, it is not surprising that there are two DSB repair pathways: nonhomologous end joining (NHEJ) and homologous recombination repair (HRR). Repair of DSBs by HRR requires the presence of homologous duplex DNA elsewhere in the genome, i.e., either a homologous chromosome or, more likely, a sister chromatid. NHEJ simply acts to process and ligate broken ends without a requirement for extensive homology. These pathways are conserved from the yeast Saccharomyces cerevisiae to humans (5,8,9,19,49,53,64). While HRR is the primary mechanism of DSB repair in yeast, vertebrate cells use both the NHEJ and HRR pathways extensively (28,34,35,44). The analysis of radiosensitive yeast mutants has revealed a number of genes involved in HRR, which comprise the RAD52 epistasis group (reviewed in references 4, 29, and 51).Among the members of the RAD52 epistasis group, the structure and function of Rad51 have been conserved to a remarkable degree among all eukaryotes. Rad51 is structurally and functionally related to the Escherichia coli recombination protein RecA (reviewed in reference 32). The functional forms of both RecA and Rad51 are multimeric helical nucleoprotein filaments that form on single-stranded DNA ends produced at DSBs (41). These filaments are involved in the search for homologous sequence, DNA pairing, and strand exchange. Recombination intermediates produced in this way are then processed further in reactions that involve DNA synthesis, branch migration, resolution of Holliday junctions, and ligation (reviewed in reference 4). The conservation of the RAD52 epistasis group genes from yeast to vertebrate cells suggests that the basic mechanism of HRR is maintained during evolution. Howe...
Five Rad51-like proteins, referred to as Rad51 paralogs, have been described in vertebrates. We show that two of them, Rad51B and Rad51C, are associated in a stable complex. Rad51B-Rad51C complex has ssDNA binding and ssDNA-stimulated ATPase activities. We also examined the functional interaction of Rad51B-Rad51C with Rad51 and RPA. Even though RPA enhances Rad51-catalyzed DNA joint formation via removal of secondary structure in the ssDNA substrate, it can also compete with Rad51 for binding to the substrate, leading to suppressed reaction efficiency. The competition by RPA for substrate binding can be partially alleviated by Rad51B-Rad51C. This recombination mediator function of Rad51B-Rad51C is likely required for the assembly of the Rad51-ssDNA nucleoprotein filament in vivo. Studies in Saccharomyces cerevisiae have identified a large number of genetic loci required for mitotic and meiotic recombination. These genes, comprising RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, RDH54/TID1, MRE11, and XRS2 are collectively known as the RAD52 epistasis group. The RAD52 group of genes are also intimately involved in the repair of DNA double-strand breaks induced by exogenous agents such as ionizing radiation (Paques and Haber 1999;Sung et al. 2000) and for telomere maintenance in the absence of telomerase.Cloning, genetic, and biochemical studies have indicated that the structure and function of the RAD52 group genes are highly conserved among eukaryotes, from yeast to humans Thompson and Schild 2001). Interestingly, in mammals, the efficiency of recombination and DNA double-strand break repair is contingent upon the integrity of the tumor suppressors BRCA1 and BRCA2 (Dasika et al. 1999;Moynahan et al. 1999Moynahan et al. , 2001Thompson and Schild 2001), underscoring the importance for deciphering the mechanistic basis of the recombination machinery.In recombination processes that involve the formation of a DNA double-strand break, the ends of the DNA break are processed to yield single-stranded DNA tails. These DNA tails are utilized by the RAD52 group recombination factors for the formation of DNA joints with a homologous DNA template, contained within the sister chromatid or the chromosomal homolog. The nascent DNA joints are then extended in length by branch migration, followed by resolution of DNA intermediates to complete the recombination process (Paques and Haber 1999;Sung et al. 2000).The RAD51 encoded product is the functional homolog of Escherichia coli RecA protein, and like RecA, possesses the ability to promote the homologous DNA pairing and strand exchange reaction that forms heteroduplex DNA joints. In mediating homologous DNA pairing and strand exchange, Rad51 must first assemble onto ssDNA as a nucleoprotein filament, in which the DNA is held in a highly extended conformation (Ogawa et al. 1993;Benson et al. 1994;Sung and Robberson 1995). Assembly of the Rad51-ssDNA nucleoprotein filament is rate-limiting and strongly inhibited by secondary structure in the ssDNA template. The removal of seconda...
The advent of systems biology necessitates the cloning of nearly entire sets of protein-encoding open reading frames (ORFs), or ORFeomes, to allow functional studies of the corresponding proteomes. Here, we describe the generation of a first version of the human ORFeome using a newly improved Gateway recombinational cloning approach. Using the Mammalian Gene Collection (MGC) resource as a starting point, we report the successful cloning of 8076 human ORFs, representing at least 7263 human genes, as mini-pools of PCR-amplified products. These were assembled into the human ORFeome version 1.1 (hORFeome v1.1) collection. After assessing the overall quality of this version, we describe the use of hORFeome v1.1 for heterologous protein expression in two different expression systems at proteome scale. The hORFeome v1.1 represents a central resource for the cloning of large sets of human ORFs in various settings for functional proteomics of many types, and will serve as the foundation for subsequent improved versions of the human ORFeome.
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