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. 1990 Apr;87(8):2882–2886. doi: 10.1073/pnas.87.8.2882

Conversion of a helix-turn-helix motif sequence-specific DNA binding protein into a site-specific DNA cleavage agent.

R H Ebright 1, Y W Ebright 1, P S Pendergrast 1, A Gunasekera 1
PMCID: PMC53797  PMID: 2158096

Abstract

Escherichia coli catabolite gene activator protein (CAP) is a helix-turn-helix motif sequence-specific DNA binding protein [de Crombrugghe, B., Busby, S. & Buc, H. (1984) Science 224, 831-838; and Pabo, C. & 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 Kobs = 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.

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