CONVERSION OF A HELIX-TURN-HELIX MOTIF SEQUENCE-SPECIFIC DNA-BINDING PROTEIN INTO A SITE-SPECIFIC DNA CLEAVAGE AGENT

Escherichia coli catabolite gene activator protein (CAP) is a helix-turn-helix motif sequence-specific DNA binding protein [de Crombrugghe, B., Busby, S. and Bluc, H. (1984) Science 224, 831-838; and Pabo, C. and Sauer, R. (1984) Annu. Rev. Biochem. 53, 293-321]. In this work, CAP has been converted into a site-specific DNA cleavage agent by incorporation of the chelator 1,10-phenanthroline at amino acid 10 of the helix-turn-helix motif. ([N-Acetyl-5-amino-1,10-phenanthroline)-Cys178]CAP binds to a 22-base-pair DNA recognition site with K(obs) = 1 x 10-8 M-1. In the presence of Cu(II) and reducing agent, [(N-acetyl-5-amino-1,10-phenanthroline)-Cys178]CAP cleaves DNA at four adjacent nucleotides on each DNA strand within the DNA recognition site. The DNA cleavage reaction has been demonstrated using 40-base-pair and 7164-base-pair DNA substrates. The DNA cleavage reaction is not inhibited by dam methylation of the DNA substrate. Such semisynthetic site-specific DNA cleavage agents have potential applications in chromosome mapping, cloning, and sequencing.