Wright J H, Zakian V A
Fred Hutchinson Cancer Research Center, Seattle, WA 98104, USA.
Nucleic Acids Res. 1995 May 11;23(9):1454-60. doi: 10.1093/nar/23.9.1454.
Telomeric DNA in Saccharomyces is organized into a non-nucleosomal chromatin structure called the telosome that can be released from chromosome ends in soluble form by nuclease digestion (Wright, J. H., Gottschling, D. E. and Zakian, V. A. (1992) Genes Dev. 6, 197-210). The protein-DNA interactions of soluble telosomes were investigated by monitoring isolated telomeric DNA fragments for the retention of bound protein using both gel mobility shift and nitrocellulose filter-binding assays. Telosomal proteins remained associated with telomeric DNA at concentrations of ethidium bromide that dissociated nucleosomes. The protein-DNA interactions in the yeast telosome were also disrupted by much lower salt concentrations than those known to disrupt either the interactions of ciliate terminus-binding proteins with telomeric DNA or the interactions of histones with DNA in nucleosomes. Taken together, these data corroborate previously published nuclease mapping data indicating that telosomes are distinct in structure from conventional nucleosomes. These data also indicate that yeast do not possess telomere binding proteins similar to those detected in ciliates that remain tightly bound to telomeric DNA even in high salt. In addition, the characteristic gel mobility shift of soluble telosomes could be mimicked by complexes formed in vitro with yeast telomeric DNA and recombinant Rap1p suggesting that Rap1p, a known component of soluble yeast telosomes (Wright, J. H., Gottschling, D. E. and Zakian, V. A. (1992) Genes Dev. 6, 197-210; Conrad, M. N., Wright, J. H., Wolf, A. J. and Zakian, V. A. (1990) Cell 63, 739-750), is likely to be the major structural protein bound directly to yeast telomeric DNA.
酿酒酵母中的端粒DNA被组织成一种称为端粒小体的非核小体染色质结构，通过核酸酶消化可将其以可溶形式从染色体末端释放出来（Wright, J. H., Gottschling, D. E. 和 Zakian, V. A. (1992) Genes Dev. 6, 197 - 210）。通过凝胶迁移率变动分析和硝酸纤维素滤膜结合分析监测分离的端粒DNA片段上结合蛋白的保留情况，对可溶端粒小体的蛋白质 - DNA相互作用进行了研究。在溴化乙锭浓度能使核小体解离的情况下，端粒小体蛋白仍与端粒DNA结合。与已知能破坏纤毛虫端粒结合蛋白与端粒DNA相互作用或核小体中组蛋白与DNA相互作用的盐浓度相比，更低的盐浓度就能破坏酵母端粒小体中的蛋白质 - DNA相互作用。综合这些数据证实了先前发表的核酸酶图谱数据，表明端粒小体在结构上与传统核小体不同。这些数据还表明，酵母不具有类似于在纤毛虫中检测到的端粒结合蛋白，后者即使在高盐条件下仍与端粒DNA紧密结合。此外，可溶端粒小体的特征性凝胶迁移率变动可被体外由酵母端粒DNA和重组Rap1p形成的复合物模拟，这表明Rap1p是可溶酵母端粒小体的已知成分之一（Wright, J. H., Gottschling, D. E. 和 Zakian, V. A. (1992) Genes Dev. 6, 197 - 210; Conrad, M. N., Wright, J. H., Wolf, A. J. 和 Zakian, V. A. (1990) Cell 63, 739 - 750），它可能是直接与酵母端粒DNA结合的主要结构蛋白。
