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J. Biol. Chem., Vol. 281, Issue 11, 7040-7048, March 17, 2006

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The DNA-binding Domain of the Yeast Spt10p Activator Includes a Zinc Finger That Is Homologous to Foamy Virus Integrase^*

Geetu Mendiratta, Peter R. Eriksson, Chang-Hui Shen, and David J. Clark¹

From the Laboratory of Molecular Growth Regulation, NICHD, National Institutes of Health, Bethesda, Maryland 20892-2426 and the Department of Biology, College of Staten Island, City University of New York, Staten Island, New York 10314

The yeast SPT10 gene encodes a putative histone acetyltransferase that binds specifically to pairs of upstream activating sequence (UAS) elements found only in the histone gene promoters. Here, we demonstrate that the DNA-binding domain of Spt10p is located between residues 283 and 396 and includes a His₂-Cys₂ zinc finger. The binding of Spt10p to the histone UAS is zinc-dependent and is disabled by a zinc finger mutation (C388S). The isolated DNA-binding domain binds to single histone UAS elements with high affinity. In contrast, full-length Spt10p binds with high affinity only to pairs of UAS elements with very strong positive cooperativity and is unable to bind to a single UAS element. This implies the presence of a "blocking" domain in full-length Spt10p, which forces it to search for a pair of UAS elements. Chromatin immunoprecipitation experiments indicate that, unlike wild-type Spt10p, the C388S protein does not bind to the promoter of the gene encoding histone H2A (HTA1) in vivo. The C388S mutant has a phenotype similar to that of the spt10 mutant: poor growth and global aberrations in gene expression. Thus, the C388S mutation disables the DNA-binding function of Spt10p in vitro and in vivo. The zinc finger of Spt10p is homologous to that of foamy virus integrase, perhaps suggesting that this integrase is also a sequence-specific DNA-binding protein.

Received for publication, October 20, 2005 , and in revised form, January 11, 2006.

^* This work was supported in part by NICHD intramural funds and by National Institutes of Health Grant 1R15GM67730-00-01 (to C-H. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains text describing supplemental Table I.

¹ To whom correspondence should be addressed: Lab. of Molecular Growth Regulation, NICHD, NIH, Bldg. 6A, Rm. 2A14, 6 Center Dr., Bethesda, MD 20892-2426. Tel.: 301-496-6966; Fax: 301-480-1907; E-mail: clarkda{at}mail.nih.gov.

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