Functional interaction of DFF35 and DFF45 with caspase-activated DNA fragmentation nuclease DFF40

DNA fragmentation factor (DFF) functions downstream of caspase-3 and direct ly triggers DNA fragmentation during apoptosis, Here we described the ident ification and characterization of DFF35, an isoform of DFF45 comprised of 2 68 amino acids. Functional assays have shown that only DFF45, not DFF35, ca n assist in the synthesis of highly active DFF40, Using the deletion mutant s, we mapped the function domains of DFF35/45 and demonstrated that the int act structure/conformation of DFF45 is essential for it to function as a ch aperone and assist in the synthesis of active DFF40, Whereas the amino acid residues 101-180 of DFF35/45 mediate its binding to DFF40, the amino acid residues 23-100, which is homologous between DFF35/45 and DFF40, may functi on to inhibit the activity of DFF40. In contrast to DFF45, DFF35 cannot wor k as a chaperone, but it can bind to DFF40 more strongly than DFF45 and can inhibit its nuclease activity. These findings suggest that DFF35 may funct ion in vivo as an important alternative mechanism to inhibit the activity o f DFF40 and further, that the inhibitory effects of both DFF35 and DFF45 on DFF40 can put the death machinery under strict control.