Chimeras composed of the cdtB gene of a novel bacterial genotoxin and the human type I DNAse I gene were constructed and their products characterized relative to the biochemical and enzymatic properties of the native proteins. The product of a cdtB/DNAse I chimera formed a heterotrimer with the CdtA and CdtC subunits of the genotoxin, and targeted mutations increased the specific activity of the hybrid protein. Expression of active chimeric gene products established that the CdtB protein is an atypical divalent cation-dependent endonuclease and demonstrated the potential for genetically engineering a new class of therapeutic agent for inhibiting the proliferation of cancer cells.