Structural Basis for Stable DNA Complex Formation by the Caspase-activated DNase

Abstract

We describe a structural model for DNA binding by the caspaseactivated DNase (CAD). Results of a mutational analysis and computational modeling suggest that DNA is bound via a positively charged surface with two functionally distinct regions, one being the active site facing the DNA minor groove and the other comprising distal residues close to or directly from helix ␣4, which binds DNA in the major groove. This bipartite protein-DNA interaction is present once in the CAD/inhibitor of CAD heterodimer and repeated twice in the active CAD dimer.The caspase-activated DNase (CAD) 2 (DFF40) is the nuclease subunit of the DNA fragmentation factor DFF, a heterodimeric complex that triggers DNA degradation in apoptotic cells (1-5). Within the complex, CAD is kept inactive by the inhibitory subunit ICAD-L (DFF45) (6 -8). Activation of DFF is achieved by removing ICAD-L via cleavage by caspase-3, allowing two subunits of CAD to form a scissor-like homodimer with the catalytic center near the base of a deep active site crevice (1, 9, 10). This prerequisite for DNA cleavage in an embedded active site may thus explain the preference of the enzyme for cutting accessible linker DNA between nucleosomes, a process referred to as apoptotic DNA laddering.As shown recently by biochemical and crystallographic studies, CAD belongs to the H-N-H or ␤␤␣-Me-finger superfamily of nucleases with a common active site motif (10 -13). This motif, consisting of two antiparallel ␤-strands and an ␣-helix, can be found in a large number of enzymes, ranging from nonspecific to highly sequence-or structurespecific nucleases with diverse structures and biological functions (14 -16). CAD cleaves naked DNA as a nonspecific nuclease, yet in contrast to other nonspecific nucleases both free CAD and, notably, the inhibited enzyme are proficient in forming stable DNA complexes (17, 18). Formation of stable DNA complexes by the DFF (CAD⅐ICAD) complex prior to its activation by caspase-3 leads to stimulation of CAD activity and thus might accelerate DNA fragmentation in apoptotic cells (17).Typically, nonspecific nucleases, e.g. DNase I, exhibit only transient interactions with their substrates sufficient to bring about nucleic acid cleavage in the absence of highly specific complex formation (14,19,20). This concept is also illustrated by the co-crystal structures of three bacterial enzymes related to CAD, the Vibrio vulnificus nuclease (Vvn) and the Escherichia coli nuclease colicins E7 (ColE7) and E9 (ColE9) (14,(21)(22)(23). Vvn nuclease and ColE7/E9 are a periplasmic nuclease and bacterial toxins engaged in host defense and cell killing, respectively. These nonspecific nucleases mainly bind DNA in the minor groove, with their ␤␤␣-Me-finger motifs forming contacts to the phosphodiester backbone of one strand of the substrate and establishing only minor contacts to the DNA major groove (21-23). In contrast, the sequence-specific members of the H-N-H or ␤␤␣-Me-finger nucleases, such as the homing endonucleases I-PpoI or I-HmuI, also use their ␤␤␣-Me-finge...

Roles of the Major Apoptotic Nuclease-DNA Fragmentation Factor-in Biology and Disease

“…The nuclease is inhibited by several poly-anions, like heparin, polyglutamic acid [66], poly(ADP-ribose) [67] and nucleic acids that are not its substrates (single stranded DNA, RNA and DNA-RNA heteroduplexes) [34,66]. Such polyanions, which are thought to bind to the positively charged surface formed by the a4 helices of DFF40 and impair its ability to bind to double-stranded DNA, are competitive inhibitors of the nuclease [66,68]. Knowing that RNA, which inhibits DFF40 in vitro, is the major cellular poly-anion, one could postulate that apoptotic chromatin breakdown might be coordinated with RNA degradation in vivo, and DFF activity would be kept in check until RNA levels fall below some inhibitory threshold.…”

Parole terms for a killer

“…11,12 Following translation the ICAD-L/CAD complex localizes to the nucleus, where it remains highly mobile; however, stable interactions with DNA have been noted suggesting that CAD can be activated not only in the free nuclear space but also in a DNA associated complex. [13][14][15] To activate CAD, caspase 3 targets ICAD for proteolytic cleavage at 2 aspartic acids, D117 and D224. This caspase directed cleavage destabilizes the ICAD/CAD interaction and allows CAD dimerization.…”

Caspase 3/caspase-activated DNase promote cell differentiation by inducing DNA strand breaks

“…4 A and B). Activated CAD as well as the CAD-ICAD complex has been shown to form stable complexes with substrate DNA, suggesting that ICAD may be cleaved by caspase 3 in this DNA-associated complex to activate CAD in a specific manner (12). This stable association allowed for ChIP with an antibody specific for CAD to precipitate associated genomic regions at 12 and 24 h postinduction of differentiation in C2C12 myoblasts.…”

“…CAD is held in check through association with its inhibitor, inhibitor of caspase-activated DNase (ICAD), which is cleaved by caspase 3 to release CAD (10,11). After activation, unobstructed CAD configures into a scissor-like dimer, cleaving DNA with minimal sequence specificity (10,12). CAD is also ubiquitously expressed, suggesting a highly conserved function for this nuclease.…”