Strategies to cleave double-stranded DNA at specific DNA sites longer than those of restriction endonucleases (longer than 8 base pairs) have applications in chromosome mapping, chromosome cloning, and chromosome sequencing-provided that the strategies yield high DNA-cleavage efficiency and high DNA-cleavage specificity. In this report, the DNA-cleaving moiety copper:o-phenanthroline was attached to the sequence-specific DNA binding protein catabolite activator protein (CAP) at an amino acid that, because of a difference in DNA bending, is close to DNA in the specific CAP-DNA complex but is not close to DNA in the nonspecific CAP-DNA complex. The resulting CAP derivative, OP(26)CAP, cleaved kilobase and megabase DNA substrates at a 22- base pair consensus DNA site with high efficiency and exhibited no detectable nonspecific DNA-deavage activity.