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Originally published In Press as doi:10.1074/jbc.M002053200 on June 21, 2000

J. Biol. Chem., Vol. 275, Issue 36, 28152-28156, September 8, 2000
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GATA-1 Bends DNA in a Site-independent Fashion*

Rodolfo Ghirlando and Cecelia D. TrainorDagger

From the Laboratory of Molecular Biology, NIDDKD, National Institutes of Health, Bethesda, Maryland 20892

The DNA binding domain of GATA-1 consists of two adjacent homologous zinc fingers, of which only the C-terminal finger binds DNA independently. Solution structure studies have shown that the DNA is bent by about 15° in the complex formed with the single C-terminal finger of GATA-1. The N-terminal finger stabilizes DNA binding at some sites. To determine whether it contributes to DNA bending, we have performed circular permutation DNA bending experiments with a variety of DNA-binding sites recognized by GATA-1. By using a series of full-length GATA-1, double zinc finger, and single C-terminal finger constructs, we show that GATA-1 bends DNA by about 24°, irrespective of the DNA-binding site. We propose that the N- and C-terminal fingers of GATA-1 adopt different orientations when bound to different cognate DNA sites. Furthermore, we characterize circular permutation bending artifacts arising from the reduced gel mobility of the protein-DNA complexes.

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 301-496-5889; Fax: 301-496-0201; E-mail: ceceliat@intra.niddk.nih.gov.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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