Functional domains of the yeast chromatin protein Sin1p/Spt2p can bind four-way junction and crossing DNA structures.
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 his4-912delta 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.
Novoseler
Minna
M
Faculty of Life Sciences, Bar Ilan University, Ramat Gan 52900, Israel.
Hershkovits
Gitit
G
Katcoff
Don J
DJ
D000595:Amino Acid Sequence; D002843:Chromatin / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry* / Q000378:metabolism*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004278:DNA, Superhelical / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011107:Polylysine / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*
D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004271:DNA, Fungal; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D011107:Polylysine; C069938:SPT2 protein, S cerevisiae