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J. Biol. Chem., Vol. 283, Issue 8, 5058-5068, February 22, 2008

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Structural and Functional Analysis of the Spt16p N-terminal Domain Reveals Overlapping Roles of yFACT Subunits^*

Andrew P. VanDemark¹, Hua Xin, Laura McCullough, Robert Rawlins, Shayla Bentley, Annie Heroux^¶, David J. Stillman, Christopher P. Hill², and Tim Formosa³

From the Departments of Biochemistry and Pathology, University of Utah School of Medicine, Salt Lake City, Utah 84112 and ^¶Biology Department, Brookhaven National Laboratory, Upton, New York 11973

yFACT (heterodimers of Saccharomyces cerevisiae Spt16-Pob3 combined with Nhp6) binds to and alters the properties of nucleosomes. The essential function of yFACT is not disrupted by deletion of the N-terminal domain (NTD) of Spt16 or by mutation of the middle domain of Pob3, but either alteration makes yeast cells sensitive to DNA replication stress. We have determined the structure of the Spt16 NTD and find evidence for a conserved potential peptide-binding site. Pob3-M also contains a putative binding site, and we show that these two sites perform an overlapping essential function. We find that yFACT can bind the N-terminal tails of some histones and that this interaction is important for yFACT-nucleosome binding. However, neither the Spt16 NTD nor a key residue in the putative Pob3-M-binding site was required for interactions with histone N termini or for yFACT-mediated nucleosome reorganization in vitro. Instead, both potential binding sites interact functionally with the C-terminal docking domain of the histone H2A. yFACT therefore appears to make multiple contacts with different sites within nucleosomes, and these interactions are partially redundant with one another. The docking domain of H2A is identified as an important participant in maintaining stability during yFACT-mediated nucleosome reorganization, suggesting new models for the mechanism of this activity.

Received for publication, October 19, 2007 , and in revised form, December 3, 2007.

The atomic coordinates and structure factors (code 3BIP, 3BIQ, 3BIT) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by National Institutes of Health grants (to T. F., D. S., and C. P. H.) and an American Cancer Society grant (to A. P. V.). 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 supplemental Methods, Figs. S1-S5, and Table S1.

¹ Present address: Dept. of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260.

² To whom correspondence may be addressed. Tel.: 801-585-5536; Fax: 801-581-7959; E-mail: chris{at}biochem.utah.edu.

³ To whom correspondence may be addressed. Tel.: 801-581-5435; Fax: 801-581-7959; E-mail: tim{at}biochem.utah.edu.

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