Kurumizaka H, Wolffe A P
Laboratory of Molecular Embryology, National Institute of Child Health and Human Development, Bethesda, Maryland 20892-5431, USA.
Mol Cell Biol. 1997 Dec;17(12):6953-69. doi: 10.1128/MCB.17.12.6953.
Sin mutations in Saccharomyces cerevisiae alleviate transcriptional defects that result from the inactivation of the yeast SWVI/SNF complex. We have investigated the structural and functional consequences for the nucleosome of Sin mutations in histone H3. We directly test the hypothesis that mutations in histone H3 leading to a SWI/SNF-independent (Sin) phenotype in yeast lead to nucleosomal destabilization. In certain instances this is shown to be true; however, nucleosomal destabilization does not always occur. Topoisomerase I-mediated relaxation of minichromosomes assembled with either mutant histone H3 or wild-type H3 together with histones H2A, H2B, and H4 indicates that DNA is constrained into nucleosomal structures containing either mutant or wild-type proteins. However, nucleosomes containing particular mutant H3 molecules (R116-H and T118-I) are more accessible to digestion by micrococcal nuclease and do not constrain DNA in a precise rotational position, as revealed by digestion with DNase I. This result establishes that Sin mutations in histone H3 located close to the dyad axis can destabilize histone-DNA contacts at the periphery of the nucleosome core. Other nucleosomes containing a distinct mutant H3 molecule (E105-K) associated with a Sin phenotype show very little change in nucleosome structure and stability compared to wild-type nucleosomes. Both mutant and wild-type nucleosomes continue to restrict the binding of either TATA-binding protein/transcription factor IIA (TFIIA) or the RNA polymerase III transcription machinery. Thus, different Sin mutations in histone H3 alter the stability of histone-DNA interactions to various extents in the nucleosome while maintaining the fundamental architecture of the nucleosome and contributing to a common Sin phenotype.
酿酒酵母中的Sin突变可缓解因酵母SWVI/SNF复合物失活而导致的转录缺陷。我们研究了组蛋白H3中Sin突变对核小体的结构和功能影响。我们直接检验了这样一个假设，即在酵母中导致SWI/SNF非依赖型（Sin）表型的组蛋白H3突变会导致核小体不稳定。在某些情况下，这一假设被证明是正确的；然而，核小体不稳定并非总是发生。拓扑异构酶I介导的与突变型组蛋白H3或野生型H3以及组蛋白H2A、H2B和H4组装的微型染色体的松弛表明，DNA被限制在包含突变型或野生型蛋白质的核小体结构中。然而，含有特定突变型H3分子（R116-H和T118-I）的核小体更容易被微球菌核酸酶消化，并且不像用DNase I消化所显示的那样将DNA限制在精确的旋转位置。这一结果表明，靠近二分轴的组蛋白H3中的Sin突变会破坏核小体核心外围的组蛋白-DNA接触。其他含有与Sin表型相关的独特突变型H3分子（E105-K）的核小体与野生型核小体相比，在核小体结构和稳定性方面变化很小。突变型和野生型核小体都继续限制TATA结合蛋白/转录因子IIA（TFIIA）或RNA聚合酶III转录机制的结合。因此，组蛋白H3中的不同Sin突变在核小体中不同程度地改变了组蛋白-DNA相互作用的稳定性，同时维持了核小体的基本结构并导致共同的Sin表型。
