The structure of nucleosomes that contain the cenH3 histone variant has been controversial. evolved to favor Cse4 hemisome over octasome formation. The buy Rifampin precise correspondence between Cse4 hemisomes resident on CDEII and reconstituted on CDEII without any other factors implies that CDEII is sufficient for hemisome assembly. INTRODUCTION Centromeres are defining features of eukaryotic chromosomes, and yet exactly what defines a centromere has remained a matter of intense debate (1,2). Rabbit Polyclonal to PLG In most eukaryotes, centromeres are epigenetically defined by the presence of special centromeric nucleosomes, in which the cenH3 histone variant replaces histone H3 in the nucleosome core (3). CenH3 (CENP-A in mammals, Cse4 in yeast and CID in Drosophila) is usually both necessary and sufficient for recruiting the other structural components of the kinetochore. Therefore, a central question in centromere biology is usually: What makes a cenH3 nucleosome different from an H3 nucleosome? Various models for the cenH3 nucleosome have been proposed, including left-handed octameric nucleosome core particles (octasomes) similar to conventional (H3/H4/H2A/H2B)2 octasomes (4), right-handed half-nucleosomes (hemisomes) (5), homotypic (cenH3/H4)2 tetrasomes that lack H2A/H2B dimers (6) and mixed octasomes made up of both cenH3 and H3 (7). Several lines of evidence favor the presence of cenH3 hemisomes at centromeres. Arrays of cenH3 nucleosomes have been isolated and characterized from Drosophila and human cells and shown to have the dimensions, composition and buy Rifampin other features of hemisomes (8C10). Further characterization of these particles has been hampered by the fact that centromeres of most eukaryotes are embedded in highly repetitive satellite DNA sequences that have been refractory to genetic and molecular analysis. In contrast, all 16 budding yeast centromeres are defined by 120-bp Centromere DNA Elements (CDEs), each of which is usually occupied by a single Cse4 nucleosome (11). DNA topology measurements have shown that this Cse4-containing particles induce positive DNA supercoils, which implies a right-handed DNA wrap around the buy Rifampin Cse4 core, opposite to the left-handed wrap of conventional H3 nucleosomes and inconsistent with an octasome model (12). Mapping of all 16 yeast centromeres at base-pair resolution has shown that Cse4 is usually confined to the 80-bp CDEII sequence, only enough DNA for a single wrap around the core, and is flanked by distinct particles occupying CDEI and CDEIII (13), which are occupied respectively by the Cbf1 protein and the CBF3 complex. Furthermore, all 16 yeast centromeres were found to contain uniform amounts of H2A (13), and quantitative fluorescence imaging of kinetochore clusters detected only one Cse4 molecule per centromere over the large majority of the cell cycle (14), consistent with the singly wrapped particle being a Cse4/H4/H2B/H2A hemisome. Yeast Cse4 can support segregation of human chromosomes (15), suggesting that this cenH3 hemisome is the universal unit of centromere identity. The case for left-handed cenH3 octasomes at centromeres is usually primarily based on buy Rifampin several studies in which stable octasomes, but not hemisomes, have been readily produced by reconstitution using human or yeast histones (4,6,16C21). Although Drosophila CID particles induce positive DNA supercoils (12), both human and yeast particles induce unfavorable supercoils (17,19,22), consistent with a conventional left-handed wrap. A high-resolution structure of the left-handed human CENP-A octasome shows that the histone core superimposes well with the H3 octasome (16), and the essentiality of key positions in the cenH3:cenH3 dimerization interface (4,23) support the notion that cenH3 octasomes are biologically important. Evidence for octasome formation has also been reported. When Cse4 is usually mildly overproduced in budding yeast, octasome-sized particles misincorporate into chromosome arms, especially at sites of high nucleosome turnover (13), and it appears that high-level expression of Drosophila CID can also produce octasomes (23). Although hemisomes are found at centromeres during most of the cell cycle, octasomes have been detected as transient intermediates during replication (10). The possibility that octasomes are transient forms is also consistent with the finding buy Rifampin that cenH3 octasomes are partially unwrapped (16,17,19,20) and therefore are inherently less stable than H3 octasomes..