Identification of functionally relevant histidine residues in the apoptotic nuclease CAD

The caspase-activated DNase CAD (DFF40/CPAN) degrades chromosomal DNA durin g apoptosis. Chemical modification with DEPC inactivates the enzyme, sugges ting that histidine residues play a decisive role in the catalytic mechanis m of this nuclease. Sequence alignment of murine CAD with four homologous a poptatic nucleases reveals four completely (His242, His263, His304 and His3 08) and two partially (His127 and His313) conserved histidine residues in t he catalytic domain of the enzyme. We have changed these residues to aspara gine and characterised the variant enzymes with respect to their DNA cleava ge activity, structural integrity and oligomeric state. All variants show a decrease in activity compared to the wild-type nuclease as measured by a p lasmid DNA cleavage assay. H242N, H263N and H313N exhibit DNA cleavage acti vities below 5% and H308N displays a drastically altered DNA cleavage patte rn compared to wild-type CAD. Whereas all variants but one have the same se condary structure composition and oligomeric state, H242N does not, suggest ing that His242 has an important structural role. On the basis of these res ults, possible roles for His127, His263, His304, His308 and His313 in DNA b inding and cleavage are discussed for murine CAD.