ABSTRACT

SmaI endonuclease recognizes and cleaves the sequence CCCGGG. The enzyme requires magnesium for catalysis; however, equilibrium binding assays revealed that the enzyme binds specifically to DNA in the absence of magnesium. A specific association constant of 0.9 10M was determined for SmaI binding to a 22-base duplex oligonucleotide. Furthermore, the K was a function of the length of the DNA substrate and the enzyme exhibited an affinity of 1.2 10M for a 195-base pair fragment and which represented a 10-fold increase in affinity over binding to nonspecific sequences. A K of 17.5 nM was estimated from kinetic assays based on cleavage of the 22-base oligonucleotide and is not significantly different from the K estimated from the thermodynamic analyses. Footprinting (dimethyl sulfate and missing nucleoside) analyses revealed that SmaI interacts with each of the base pairs within the recognition sequence. Ethylation interference assays suggested that the protein contacts three adjacent phosphates on each strand of the recognition sequence. Significantly, a predicted protein contact with the phosphate 3` of the scissile bond may have implications in the mechanism of catalysis by SmaI.

FOOTNOTES

*

This work was supported by the National Science Foundation Grant MCB9004611 (to J. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Supported in part by a Rumble Graduate Fellowship from Wayne State University. Present address: Dept. of Cancer Research, Parke-Davis Pharmaceutical, Warner-Lambert Company, 2800 Plymouth Rd., Ann Arbor, MI 48105.

¶

To whom correspondence should be addressed: Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, 3126 Scott Hall, Detroit, MI 48201. Tel.: 313-577-5545; Fax: 313-577-5218; jdunbar{at}cmb.biosci.wayne.edu.

()

The abbreviations used are: bp, base pair(s); DMS, dimethyl sulfate.

()

J. C. Dunbar, unpublished results.

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