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Structure of the DNA--Eco RI Endonuclease Recognition Complex at 3 Å Resolution

Judith A. McClarin, Christin A. Frederick, Bi-Cheng Wang, Patricia Greene, Herbert W. Boyer, John Grable and John M. Rosenberg
Science
New Series, Vol. 234, No. 4783 (Dec. 19, 1986), pp. 1526-1542
Stable URL: http://www.jstor.org/stable/1698813
Page Count: 17
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Structure of the DNA--Eco RI Endonuclease Recognition Complex at 3 Å Resolution
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Abstract

The crystal structure of the complex between Eco RI endonuclease and the cognate oligonucleotide TCGCGAATTCGCG provides a detailed example of the structural basis of sequence-specific DNA-protein interactions. The structure was determined, to 3 Å resolution, by the ISIR (iterative single isomorphous replacement) method with a platinum isomorphous derivative. The complex has twofold symmetry. Each subunit of the endonuclease is organized into an $\alpha $/ $\beta $ domain consisting a five-stranded $\beta $ sheet, $\alpha $ helices, and an extension, called the ``arm,'' which wraps around the DNA. The large $\beta $ sheet consists of antiparallel and parallel motifs that form the foundations for the loops and $\alpha $ helices responsible for DNA strand scission and sequence-specific recognition, respectively. The DNA cleavage site is located in a cleft that binds the DNA backbone in the vicinity of the scissile bond. Sequence specificity is mediated by 12 hydrogen bonds originating from $\alpha $ helical recognition modules. Arg$^{200}$ forms two hydrogen bonds with guanine while Glu$^{144}$ and Arg$^{145}$ form four hydrogen bonds to adjacent adenine residues. These interactions discriminate the Eco RI hexanucleotide GAATTC from all other hexanucleotides because any base substitution would require rupture of at least one of these hydrogen bonds.

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