Enzymatic active site of caspase-activated DNase (CAD) and its inhibition by inhibitor of CAD

Caspase-activated DNase (CAD) is a deoxyribonuclease that causes DNA fragme ntation during apoptosis. In proliferating cells, CAD is complexed with ICA D (inhibitor of CAD) and its DNase activity is suppressed. Here, we establi shed a quantitative assay for CAD DNase that measures the number of 3' hydr oxyl groups on the CAD-generated DNA fragments. Chemical modification of hi stidine residues and substrate protection experiments demonstrated the pres ence of reactive histidine residues within the active site of the enzyme. A nalysis by site-directed mutagenesis suggested that at least four histidine residues in the C-terminal part of the molecule are essential for the cata lytic activity of CAD DNase. ICAD did not protect CAD from the chemical mod ification of the histidine residues, indicating that it does not mask the a ctive site of CAD. In contrast, ICAD blocked the ability of CAD to bind DNA , suggesting that ICAD causes steric or electrostatic hindrance in CAD for substrate DNA. This molecular mechanism for the inhibition of CAD DNase by ICAD is similar to that proposed for colicin endonuclease and its inhibitor , immunity protein. (C) 2001 Academic Press.