Response: Right-Handed Half-Nucleosomes at Centromeres

In conclusion, the composition of cen-tromeric particles—octameric, hexameric, tetrameric (tetrasomes or hemisomes), or a combination of these—as well as the mechanisms by which centromeric chro-matin acquires stable positive supercoil-ing certainly need further clarification. However, the results of Furuyama and Henikoff confirm that nucleosomes are not mere repetitive " canonical " chroma-tin entities: not only do they have a well-acknowledged polymorphism stemming from the existence of histone variants, histone posttranslational modifications, and sequence-dependent properties of the wrapped DNA, but they also come in different histone/DNA stoichiom-etries and opposite chiralities. Overall, the coexistence of negative and positive chromatin supercoiling at specific loci of chromosomes enhances the physiological importance of DNA topology. Supplemental Data Supplemental Data include one figure and one movie and can be found with this article on-line at Our study in Cell demonstrated that centromeric nucleosomes induce positive supercoils. We showed this using both the Drosophila CenH3 his-tone variant assembled in vitro by its native chaperone (RbAp48) and yeast minichromosomes in vivo (Furuyama and Henikoff, 2009). To verify the in vivo results, we used yeast centrom-ere mutants and conditional mutant kinetochore proteins. We showed that positive supercoiling is an inherent property of functional centromeres at mitosis and depends on deposition of CenH3 (Cse4). In their Correspondence, Lavelle et al. accept our conclusions with respect to the topology of cen-tromeric DNA and agree that this is a provocative result. However, they raise a key question that was only indirectly addressed by our study, namely, which protein core structure can impose such an extraordinary reversal of DNA wrap-ping? Our views and theirs on this subject are largely in agreement, in that conventional octameric nucleosomes are inconsistent with the right-handed wrapping that can lead to positive supercoiling. However, we favor hemi-somes as the candidate core structure , based on previous direct in vivo biochemical evidence. The Drosophila CenH3 histone variant is a stoichio-metric component of stable tetrameric nucleosomes purified in their native form, which are proposed to be hemi-somes based on protein content, DNA wrapping, and direct measurements of dimensions and conformation at the single-molecule level (Dalal et al., 2007; Wang et al., 2008). In contrast, Lavelle et al. argue that a particle derived from a " reversome " is an alternative possibility (Bancaud et al., 2007). Reversomes are transient structures that require sustained high torsional stress in order to flip a left-handed octamer into a right-handed configuration. Lavelle et al. propose that …