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Molecular and Cellular Biology, January 1999, p. 585-593, Vol. 19, No. 1
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

The Yeast a1 and alpha 2 Homeodomain Proteins Do Not Contribute Equally to Heterodimeric DNA Binding

Yisheng Jin,dagger Hualin Zhong,Dagger and Andrew K. Vershon*

Waksman Institute of Microbiology and Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08854-8020

Received 1 June 1998/Returned for modification 3 August 1998/Accepted 29 September 1998

In diploid cells of the yeast Saccharomyces cerevisiae, the alpha 2 and a1 homeodomain proteins bind cooperatively to sites in the promoters of haploid cell-type-specific genes (hsg) to repress their expression. Although both proteins bind to the DNA, in the alpha 2 homeodomain substitutions of residues that are involved in contacting the DNA have little or no effect on repression in vivo or cooperative DNA binding with a1 protein in vitro. This result brings up the question of the contribution of each protein in the heterodimer complex to the DNA-binding affinity and specificity. To determine the requirements for the a1-alpha 2 homeodomain DNA recognition, we systematically introduced single base-pair substitutions in an a1-alpha 2 DNA-binding site and examined their effects on repression in vivo and DNA binding in vitro. Our results show that nearly all substitutions that significantly decrease repression and DNA-binding affinity are at positions which are specifically contacted by either the alpha 2 or a1 protein. Interestingly, an alpha 2 mutant lacking side chains that make base-specific contacts in the major groove is able to discriminate between the wild-type and mutant DNA sites with the same sequence specificity as the wild-type protein. These results suggest that the specificity of alpha 2 DNA binding in complex with a1 does not rely solely on the residues that make base-specific contacts. We have also examined the contribution of the a1 homeodomain to the binding affinity and specificity of the complex. In contrast to the lack of a defective phenotype produced by mutations in the alpha 2 homeodomain, many of the alanine substitutions of residues in the a1 homeodomain have large effects on a1-alpha 2-mediated repression and DNA binding. This result shows that the two proteins do not make equal contributions to the DNA-binding affinity of the complex.

* Corresponding author. Mailing address: Waksman Institute of Microbiology and Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08854-8020. Phone: (732) 445-2905. Fax: (732) 445-5735. E-mail: vershon{at}mbcl.rutgers.edu.

dagger Present address: Department of Molecular Biology, Genentech, Inc., South San Francisco, California.

Dagger Present address: Laboratory of Cell Biology, Rockefeller University, New York, New York.

Molecular and Cellular Biology, January 1999, p. 585-593, Vol. 19, No. 1
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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