SIN MUTATIONS OF HISTONE H3 - INFLUENCE ON NUCLEOSOME CORE STRUCTURE AND FUNCTION

Sin mutations in Saccharomyces cerevisiae alleviate transcriptional de fects that result from the inactivation of the yeast SWI/SNF complex. We have investigated the structural and functional consequences for th e nucleosome of Sin mutations in histone H3. We directly test the hypo thesis that mutations in histone H3 leading to a SWI/SNF-independent ( Sin) phenotype in yeast lead to nucleosomal destabilization. In certai n instances this is shown to be true; however, nucleosomal destabiliza tion does not always occur. Topoisomerase I-mediated relaxation of min ichromosomes assembled with either mutant histone H3 or wild-type H3 t ogether with histones H2A, H2B, and H4 indicates that DNA is constrain ed into nucleosomal structures containing either mutant or wild-type p roteins. However, nucleosomes containing particular mutant H3 molecule s (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 n ucleosomes containing a distinct mutant H3 molecule (E105-K) associate d 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-bind ing 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 n ucleosome and contributing to a common Sin phenotype.