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J. Biol. Chem., Vol. 280, Issue 7, 5169-5177, February 18, 2005

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Functional Domains of the Yeast Chromatin Protein Sin1p/Spt2p Can Bind Four-way Junction and Crossing DNA Structures^*

Minna Novoseler, Gitit Hershkovits, and Don J. Katcoff

From the Faculty of Life Sciences, Bar Ilan University, Ramat Gan 52900, Israel

Sin1p/Spt2p is a yeast chromatin protein that, when mutated or deleted, alters the transcription of a family of genes presumably by modulating local chromatin structure. In this study, we investigated the ability of different domains of this protein to bind four-way junction DNA (4WJDNA) since 4WJDNA can serve as a model for bent double helical DNA and for the crossed structure formed at the exit and entry of DNA to the nucleosomes. Sequence alignment of Sin1p/Spt2p homologues from 11 different yeast species showed conservation of several domains. We found that three domains of Sin1p/Spt2p fused to glutathione S-transferase can each bind independently in a structure-specific manner to 4WJDNA as measured in a gel mobility shift assay. A feature common to these domains is a cluster of positively charged amino acids. Modification of this cluster resulted in either abolishment of binding or a change in the binding properties. One of the domains tested clearly bound superhelical DNA, although it failed to induce bending in a circularization assay. Poly-L-lysine, which may be viewed as a cluster of positively charged amino acids, bound 4WJDNA as well. Phenotypic analysis showed that disruption of any of these domains resulted in suppression of a his4-912 allele, indicating that each domain has functional significance. We propose that Sin1p/Spt2p is likely to modulate local chromatin structure by binding two strands of double-stranded DNA at their crossover point.

Received for publication, June 4, 2004 , and in revised form, November 22, 2004.

^* This work was supported by a grant from the Health Sciences Research Center at Bar Ilan University (to D. J. K.). 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.

To whom correspondence should be addressed. Tel.: 972-3-5318248; Fax: 972-3-5351824; E-mail: katcoff{at}mail.biu.ac.il.

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This article has been cited by other articles:

				G. Hershkovits, H. Bangio, R. Cohen, and D. J. Katcoff Recruitment of mRNA cleavage/polyadenylation machinery by the yeast chromatin protein Sin1p/Spt2p PNAS, June 27, 2006; 103(26): 9808 - 9813. [Abstract] [Full Text] [PDF]

				A. Nourani, F. Robert, and F. Winston Evidence that Spt2/Sin1, an HMG-Like Factor, Plays Roles in Transcription Elongation, Chromatin Structure, and Genome Stability in Saccharomyces cerevisiae Mol. Cell. Biol., February 15, 2006; 26(4): 1496 - 1509. [Abstract] [Full Text] [PDF]