(from the application): Events that are triggered by DNA fragmentation factor (DFF) during the terminal stages of apoptosis include internucleosomal DNA fragmentation and chromatin condensation. In its inactive form, DFF is a heterodimer composed of 45-kDa and 40-kDa protein subunits (also called ICAD and CAD/CPAN, respectively). Upon caspase-3 or -7 cleavage of recombinant DFF in vitro, DFF45 is cut, releasing DFF40, which forms homo-oligomers that possess potent endonuclease activity. Released DFF40 also initiates intranuclear chromatin condensation. Here we propose to elucidate the substructure and mechanisms of DFF45 and DFF40 action. We will engineer mutations in recombinant DNA encoding these proteins for their expression in E. coli and human cells to address the following specific aims: 1. For the chaperone andinhibitor subunit of DFF, DFF45: a. Identify DFF40 interaction surface(s) b. Create dominant-negative species c. Isolate DFF45 mutants that can constitutively activate DFF40 2. For the latent endonuclease subunit of DFF, DFF40: a. Identify DFF45 interaction surface(s) b. Identify homo-oligomerization interaction surfaces c. Locate the endonuclease active site d. Create auto-activated forms of DFF40 e. Create dominant-negative species
