RAD51B and RAD51C are two of five known paralogs of the human RAD51 protein that are thought to function in both homologous recombination and DNA double-strand break repair. This work describes the in vitro and in vivo identification of the RAD51B/RAD51C heterocomplex. The RAD51B/RAD51C heterocomplex was isolated and purified by immunoaffinity chromatography from insect cells co-expressing the recombinant proteins. Moreover, co-immunoprecipitation of the RAD51B and RAD51C proteins from HeLa, MCF10A, and MCF7 cells strongly suggests the existence of an endogenous RAD51B/RAD51C heterocomplex. We extended these observations to examine the interaction between the RAD51B/RAD51C complex and the other RAD51 paralogs. Immunoprecipitation using protein-specific antibodies showed that RAD51C is central to a single large protein complex and/or several smaller complexes with RAD51B, RAD51D, XRCC2, and XRCC3. However, our experiments showed no evidence for the inclusion of RAD51 within these complexes. Further analysis is required to elucidate the function of the RAD51B/RAD51C heterocomplex and its association with the other RAD51 paralogs in the processes of homologous recombination and DNA double-strand break repair.The human RAD51 protein functions in homologous recombination and DNA double-strand break repair (1-4). Five paralogs of human RAD51 have been identified: RAD51B (hREC2, RAD51L1), RAD51C (RAD51L2), RAD51D (RAD51L3), XRCC2, and XRCC3; these proteins share ϳ25% amino acid sequence identity with one another and RAD51 (5-9). DT40 chicken cell knockouts have been generated for each paralog, all of which exhibit a lack of RAD51 foci formation as well as enhanced radiation and cisplatin sensitivity, consistent with a deficiency in recombinational repair (10, 11). These results are consistent with previous work, which showed that the XRCC3 knockout CHO cell line (irs1SF) is deficient in RAD51 foci formation, suggesting that XRCC3 is required for RAD51 function (12). Similarly, XRCC2-defective cell lines also fail to form damage-dependent RAD51 foci (13). Furthermore, both XRCC2 and XRCC3 have been shown to be required for repair of double-strand breaks by homologous recombination in vivo (14 -16). Initially by sequence and now by association and function, it has become increasingly evident that these RAD51 paralogs participate together in recombination and repair processes.Extensive yeast two-and three-hybrid analysis suggests that there are a variety of putative protein-protein interactions between the RAD51 paralogs. These include XRCC2/RAD51D (17, 18) and XRCC3/RAD51(19). Moreover, interactions have been suggested between RAD51C/RAD51B, RAD51C/RAD51D, RAD51C/XRCC3, and RAD51C/RAD51 (8, 18). More recent biochemical evidence has corroborated the interaction between XRCC2 and RAD51D by co-purification of the recombinant proteins and co-elution of the native proteins by gel filtration from mammalian cell extracts; RAD51D was further shown to be a DNA-stimulated ATPase (17). In addition, a stable heterocomplex was demons...

The development of high-throughput methods for gene discovery has paved the way for the design of new strategies for genome-scale protein analysis. Lawrence Livermore National Laboratory and Onyx Pharmaceuticals, Inc., have produced an automatable system for the expression and purification of large numbers of proteins encoded by cDNA clones from the IMAGE (Integrated Molecular Analysis of Genomes and Their Expression) collection. This high-throughput protein expression system has been developed for the analysis of the human proteome, the protein equivalent of the human genome, comprising the translated products of all expressed genes. Functional and structural analysis of novel genes identified by EST (Expressed Sequence Tag) sequencing and the Human Genome Project will be greatly advanced by the application of this high-throughput expression system for protein production. A prototype was designed to demonstrate the feasibility of our approach. Using a PCR-based strategy, 72 unique IMAGE cDNA clones have been used to create an array of recombinant baculoviruses in a 96-well microtiter plate format. Forty-two percent of these cDNAs successfully produced soluble, recombinant protein. All of the steps in this process, from PCR to protein production, were performed in 96-well microtiter plates, and are thus amenable to automation. Each recombinant protein was engineered to incorporate an epitope tag at the amino terminal end to allow for immunoaffinity purification. Proteins expressed from this system are currently being analyzed for functional and biochemical properties.

Normal human bronchial epithelial (NHBE) cells are the putative progenitor cells of all types of lung cancer. NHBE cells immortalized by SV40 T-antigen retain many characteristics of the primary cells and are a useful model for investigating the role of oncogenes, tumor suppressor genes, and certain chemical carcinogens in the molecular pathogenesis of lung cancer. In this study, SV40 T-antigen-positive cells (BEAS-2B) were characterized for their metabolic functions and were shown to continue to express epoxide hydrolase, glutathione S-transferase pi, glutathione peroxidase, and catalase. To increase their metabolic activity towards human procarcinogens, human cytochrome P450 1A2 (CYP1A2) was stably expressed by introducing CYP1A2 cDNA into BEAS-2B cells either by infection with a high-titer recombinant retrovirus (pXT-1A2) or by transfection with a CYP1A2 expression vector (pCMV1A2), which produced the cell lines B-1A2 and B-CMV1A2, respectively. Cell lines established with either expression system expressed enzymatically active CYP1A2 protein and were 50- to 400-fold more sensitive to the cytotoxic effect of the carcinogen aflatoxin B1 (AFB1) than the corresponding control cell lines. The cytotoxic effects of AFB1 were paralleled by increased metabolism of AFB1 and enhanced formation of the AFB1-N7 guanine adduct in B-CMV1A2 cells. Cytotoxicity and adduct formation correlated with a significantly higher protein expression of CYP1A2 by the cytomegalovirus promoter-driven plasmid. Since this human epithelial cell line is the precursor cell type of lung cancer, has normal phase II enzymes, and exhibits highly reproducible expression of phase I enzymes, this in vitro model should aid in the evaluation of putative human carcinogens and anticarcinogens.

290 EAE = experimental autoimmune encephalomyelitis; ELISA = enzyme-linked immunosorbent assay; hnRNP = heterogeneous nuclear ribonucleopro-teins; IDDM = insulin-dependent diabetes mellitus; RA = rheumatoid arthritis; SLE = systemic lupus erythematosus; Sm/RNP = Smith ribonucleo-proteins; Th = T helper cell.