CF-PPiD technology based on cell-free protein array and proximity biotinylation enzyme for in vitro direct interactome analysis.

Protein-protein interaction (PPI) analysis is a key process to understand protein functions. Recently, we constructed a human protein array (20 K human protein beads array) consisting of 19,712 recombinant human proteins produced by a wheat cell-free protein production system. Here, we developed a cell-free protein array technology for proximity biotinylation-based PPI identification (CF-PPiD). The proximity biotinylation enzyme AirID-fused TP53 and -IκBα proteins each biotinylated specific interacting proteins on a 1536-well magnetic plate. In addition, AirID-fused cereblon was shown to have drug-inducible PPIs using CF-PPiD. Using the human protein beads array with AirID-IκBα, 132 proteins were biotinylated, and then selected clones showed these biological interactions in cells. Although ZBTB9 was not immunoprecipitated, it was highly biotinylated by AirID-IκBα, suggesting that this system detected weak interactions. These results indicated that CF-PPiD is useful for the biochemical identification of directly interacting proteins.

CF-PA2Vtech: a cell-free human protein array technology for antibody validation against human proteins.

Antibodies are widely used for the detection of specific molecules such as peptides, proteins, and chemical compounds. The specificity of an antibody is therefore its most important feature. However, it is very difficult to confirm antibody specificity. Recently, we made a human protein array consisting of 19,712 kinds of recombinant human proteins produced by a wheat cell-free protein production system. Here, we demonstrate a novel protein array technology for antibody validation (CF-PA2Vtech). Full-length human cDNAs were fused to N-terminal FLAG-GST and then synthesized by the wheat cell-free system. To construct a 20 K human protein array, about 10 to 14 kinds of human proteins were mixed and captured in each well by glutathione-conjugated magnetic beads in 12 plates or one plate with 384- or 1536-well format, respectively, using a strong magnetic device. Using this protein array plate, commercially available anti-HA or anti-PD-1 antibody reacted to 13 or three human proteins, respectively. The cross-reactivity of these proteins was also confirmed by immunoblotting. These proteins have a similar epitope, and alanine mutations of these epitope candidates dissolved the reactivity. These results indicated that CF-PA2Vtech is very useful for validation of antibodies against human protein.

Structural Basis of Homology-Directed DNA Repair Mediated by RAD52.

RAD52 mediates homologous recombination by annealing cDNA strands. However, the detailed mechanism of DNA annealing promoted by RAD52 has remained elusive. Here we report two crystal structures of human RAD52 single-stranded DNA (ssDNA) complexes that probably represent key reaction intermediates of RAD52-mediated DNA annealing. The first structure revealed a "wrapped" conformation of ssDNA around the homo-oligomeric RAD52 ring, in which the edges of the bases involved in base pairing are exposed to the solvent. The ssDNA conformation is close to B-form and appears capable of engaging in Watson-Crick base pairing with the cDNA strand. The second structure revealed a "trapped" conformation of ssDNA between two RAD52 rings. This conformation is stabilized by a different RAD52 DNA binding site, which promotes the accumulation of multiple RAD52 rings on ssDNA and the aggregation of ssDNA. These structures provide a structural framework for understanding the mechanism of RAD52-mediated DNA annealing.

Functional analyses of the C-terminal half of the Saccharomyces cerevisiae Rad52 protein.

The Saccharomyces cerevisiae Rad52 protein is essential for efficient homologous recombination (HR). An important role of Rad52 in HR is the loading of Rad51 onto replication protein A-coated single-stranded DNA (ssDNA), which is referred to as the recombination mediator activity. In vitro, Rad52 displays additional activities, including self-association, DNA binding and ssDNA annealing. Although Rad52 has been a subject of extensive genetic, biochemical and structural studies, the mechanisms by which these activities are coordinated in the various roles of Rad52 in HR remain largely unknown. In the present study, we found that an isolated C-terminal half of Rad52 disrupted the Rad51 oligomer and formed a heterodimeric complex with Rad51. The Rad52 fragment inhibited the binding of Rad51 to double-stranded DNA, but not to ssDNA. The phenylalanine-349 and tyrosine-409 residues present in the C-terminal half of Rad52 were critical for the interaction with Rad51, the disruption of Rad51 oligomers, the mediator activity of the full-length protein and for DNA repair in vivo in the presence of methyl methanesulfonate. Our studies suggested that phenylalanine-349 and tyrosine-409 are key residues in the C-terminal half of Rad52 and probably play an important role in the mediator activity.