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. & 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.
Ebright
R H
RH
Department of Chemistry, Rutgers University, New Brunswick, NJ 08855.
Ebright
Y W
YW
Pendergrast
P S
PS
Gunasekera
A
A
D001483:Base Sequence; D003090:Coliphages / Q000378:metabolism; D002373:Cyclic AMP Receptor Protein / Q000378:metabolism*; D003545:Cysteine; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010618:Phenanthrolines; D011487:Protein Conformation; D015183:Restriction Mapping
D002373:Cyclic AMP Receptor Protein; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D010618:Phenanthrolines; D003545:Cysteine; C025205:1,10-phenanthroline