Abstract
We describe a structural model for DNA binding by the caspase-activated 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 alpha4, 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.
Highlights
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 [1,2,3,4,5]
These results suggest that helix ␣4 is part of a DNA binding site accessible in free CAD as well as CAD bound to its inhibitor in the DFF complex
Models of CAD1⁄7DNA Complexes—To gain insight into the structural basis for stable DNA complex formation by CAD, we modeled CAD1⁄7DNA complexes by superimposing the active site motifs of colicins E7 (ColE7) and Vibrio vulnificus nuclease (Vvn) nuclease from DNA co-crystal structures with the active site of CAD (Fig. 3)
Summary
The caspase-activated DNase (CAD) (DFF40) is the nuclease subunit of the DNA fragmentation factor DFF, a heterodimeric complex that triggers DNA degradation in apoptotic cells [1,2,3,4,5]. 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 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 [21,22,23]. Our results suggest that CAD forms stable DNA complexes via a DNA binding region that comprises residues close to or directly from helix ␣4 distal to the active site
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