suGF1 binds in the major groove of its oligo(dG).oligo(dC) recognition sequence and is excluded by a positioned nucleosome core.
We have elsewhere reported the purification of a poly(dG).poly(dC)-binding nuclear protein (suGF1) from sea urchin embryos (J. Hapgood and D. Patterton, Mol. Cell. Biol. 14:this issue, 1994). We proposed that suGF1 may be a member of a family of G-string factors involved in developmental gene regulation, possibly via alterations in chromatin structure. In this article, we characterize the binding of purified suGF1 to 11 contiguous Gs in the H1-H4 intergenic region of a sea urchin early histone gene battery in vitro. It is shown that suGF1-DNA binding is dependent on ionic strength and requires divalent cations. Purified suGF1 forms discrete protein-DNA multimers, consistent with suGF1-suGF1 interactions. In a model for the suGF1-DNA complex derived from our footprinting and methylation interference data, suGF1 contacts the Gs in the major groove as well as one of the bordering phosphate backbones. The data are consistent with the direction of curvature of the DNA in the suGF1-DNA complex being the same as that preferred by the free DNA and exhibited by the DNA when bent around a positioned nucleosome core in vitro. However, on the basis of steric considerations, the binding of suGF1 and that of the histone octamer are predicted to be mutually exclusive. We show that suGF1 is indeed unable to bind to the G string when occupied by a histone octamer located in the major in vitro positioning frame in the H1-H4 intergenic region.
Patterton
D
D
Department of Biochemistry, University of Cape Town, Rondebosch, South Africa.
Hapgood
J
J
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003850:Deoxyribonuclease I; D006657:Histones / Q000096:biosynthesis / Q000235:genetics; D008745:Methylation; D008961:Models, Structural; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D009707:Nucleosomes / Q000378:metabolism*; D011089:Polydeoxyribonucleotides / Q000378:metabolism*; D012617:Sea Urchins / Q000378:metabolism
D002843:Chromatin; D004268:DNA-Binding Proteins; D006657:Histones; D009687:Nuclear Proteins; D009707:Nucleosomes; D011089:Polydeoxyribonucleotides; C085454:suGF1 protein, Parechinus angulosus; C019530:poly(dC-dG); D004247:DNA; D003850:Deoxyribonuclease I