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

• Published in: The Journal of biological chemistry 2005-02, Vol.280 (7), p.5169-5177
• Main Authors: Novoseler, Minna, Hershkovits, Gitit, Katcoff, Don J.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Chromatin - metabolism; Chromosomal Proteins, Non-Histone - chemistry; Chromosomal Proteins, Non-Histone - metabolism; DNA, Fungal - chemistry; DNA, Fungal - genetics; DNA, Fungal - metabolism; DNA, Superhelical - chemistry; DNA, Superhelical - genetics; DNA, Superhelical - metabolism; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - metabolism; Electrophoretic Mobility Shift Assay; Molecular Sequence Data; Nucleic Acid Conformation; Polylysine - metabolism; Protein Structure, Tertiary; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M406249200

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 his4-912δ 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.