We have shown that CenH3 nucleosomes induce positive supercoils, both when D. melanogaster CID is reconstituted into nucleosomes in vitro, and when S. cerevisiae Cse4p is assembled at functional minichromosome centromeres in vivo. This behavior is in stark contrast to canonical nucleosomes, in which the left-handed wrapping leads to induction of negative supercoils in topological assays. Our observations of positive supercoiling induced by CenH3 from eukaryotic taxa as different as animals and fungi can be explained by either of two general models: overtwisting with left-handed wrapping or right-handed wrapping.
In a covalently closed circle, overtwisting of DNA (positive ΔTw) causes compensatory negative writhe that is removed by topoisomerase, resulting in a net positive ΔLk after deproteination (Malcolm and Snounou, 1983
). If CenH3 nucleosomes are left-handed octamers (Wr = −1), ΔTw would need to be +2 in order to result in a ΔLk of +1 (ΔLk = ΔTw + ΔW). Although the reported value of ΔWr for left-handed octamers varies (Bancaud et al., 2006
; Prunell, 1998
), we use the most conservative cited value of −1 to calculate the degree of overtwisting consistent with left-handed wrapping. The change required in the helical periodicity of DNA (Δh) to gain ΔTw = +2 and cancel one negative writhe induced by a left-handed nucleosome can be calculated as Δh = −h2
xΔTw/N, where h = N/Tw, where N is the number of base pairs wrapped around the nucleosome. If we assume an octameric CenH3 nucleosome (N=150 bp), Δh equals −1.47 for ΔTw = +2. This corresponds to a helical periodicity of 9.03 bp/turn (whereas h = 10.5 bp/turn for B-DNA free in solution). The situation is even more extreme for CenH3 hemisomes, which wrap 80–120 bp of DNA, because the same amount of twist must be taken up by the shorter span of DNA (helical periodicity of 7.74–8.66 bp/turn). These estimated values for helical twist are conservative in that they assume that the extra twist is distributed over the whole nucleosome, including the DNA that wraps H2A/H2B dimers, whereas in the crystal structure of the H3 nucleosome core particle the twist of DNA wrapping H2A/H2B is similar to that in free solution (Luger et al., 1997
). In addition, DNaseI digestion of CID chromatin assembled in vitro resulted in a normal helical periodicity estimate of ~10 bp/turn (Furuyama et al., 2006
), and electron microscopy of CID chromatin revealed a beads-on-a-string appearance (Dalal et al., 2007b
; Furuyama et al., 2006
), suggesting entry/exit crossing. Thus, existing data are inconsistent with positive ΔTw being the reason for the observed positive supercoiling.
The implausibility of such strongly overtwisted DNA wrapping around a left-handed nucleosome leads us to conclude that positive supercoiling instead indicates a right-handed wrap. A right-handed nucleosome would satisfy the observed positive supercoiling of approximately one supercoil per CenH3 nucleosome without a significant change in B-DNA periodicity. Tetrameric archaeal nucleosomes also wrap DNA in a right-handed configuration, with a helical periodicity of 10–11bp/turn (Musgrave et al., 1991
). Also, in the absence of H2A/H2B dimers, (H3/H4)2
tetramers are capable of spontaneously shifting between both left- and right-handed configurations (Hamiche et al., 1996
), presumably without significant changes in helical twist.
Histone octamers capable of wrapping DNA into a right-handed configuration have never been observed. Because H3/H4 tetramers can wrap DNA in either direction, it is the creation of a left-handed ramp by addition of two H2A/H2B dimers that is incompatible with the right-handed structure (See ). The crystal structure of the H3 nucleosome (H2A’-H2B’-H4’-H3’-H3-H4-H2B-H2A plus DNA) reveals that the N-terminal helix of H3, as well as the C-terminus of H4, contact the C-terminal docking domain of H2A’, which are essential interactions that hold the octamer together (Luger et al., 1997
). In addition, the interaction between H2A and H2A’ within the octamer through their Loop 1 regions hold together the two gyres of the DNA superhelix (Luger et al., 1997
). These interactions that hold the octamer together are expected to be disrupted in a right-handed nucleosome because they would face away from each other in the right-handed structure (); therefore, there is a strong structural basis for the absence of right-handed octameric nucleosomes in eukaryotes. Without altering the twist of DNA significantly, the only structures that yield ΔLk = +1 other than a right-handed octamer are right-handed hemisomes with right entry/exit crossing, and left-handed hemisomes with right entry/exit crossing. A single superhelical turn of DNA around a hemisome results in a closer physical distance between the entry/exit DNA compared to that in an octameric structure, which has an additional turn between the two entry/exit sites (compare ). Therefore, it is structurally very difficult to make a left-handed hemisome with a right-handed crossing.
Structures and model of a right-handed hemisome
In budding yeast, various model of Cse4p nucleosomes have been suggested, including octamers (H2A/H2B/H4/Cse4p/Cse4p/H4/H2B/H2A) (Meluh et al., 1998
), hemisomes (Cse4p/H4/H2B/H2A) (Dalal et al., 2007a
) and nucleosomes containing the non-histone Scm3 protein substituting for H2A/H2B dimers (H4/Cse4p/Cse4p/H4)(Scm3)1–2
(Mizuguchi et al., 2007
). Given our finding that Cse4p nucleosomes induce positive supercoils, it is unlikely that they can exist as octamers. Furthermore, the observation that Scm3 binds to the region of Cse4p required for the 4-helix bundle homodimerization interface of the octameric particle (Stoler et al., 2007
) would a priori argue against a stable octameric particle. That leaves either Cse4p/H4/H2A/H2B hemisomes or Cse4p/H4/Scm3 particles as candidate yeast CenH3 nucleosomes. Both of these models are consistent with the localization of Cse4p to a small ~80bp CDEII region of CDE. It is attractive to suggest that right-handed hemisomes are conserved in all eukaryotes, because Cse4p can functionally replace human CENP-A (Wieland et al., 2004
There are several structural implications of right-handed hemisomes. The strong H3/H3 4-helix bundle at the dyad axis and the weak H4/H2B 4-helix bundles linking the central tetramer to flanking dimers precludes formation of H3/H4/H2B/H2A hemisomes, and indeed no stable H3 hemisomes have been observed. Therefore, the existence of CenH3 hemisomes suggests that CenH3 induces structural alterations that stabilize the tetrameric particle. The crossing of entry/exit DNA in the CenH3 hemisome may be an important feature, because it can potentially stabilize the hemisome. In contrast, the entry/exit DNA of H3 octameric nucleosomes does not cross most of the time, but rather is occupied by a linker histone (Bancaud et al., 2006
; Prunell, 1998
). Consistent with this difference, the H1 linker histone is depleted from centromeric chromatin (Maresca et al., 2005
), and the H5 linker histone is incapable of associating with human CENP-A nucleosomes in vitro (Conde e Silva et al., 2007
). In addition, surfaces involved in contacts within left-handed octameric nucleosomes will be exposed in right-handed hemisomes, such as the C-terminal docking domain of H2A and the N-terminal helix of H3 (). A right-handed configuration also changes the relative position of these domains (). The combination of additional exposed surfaces and altered presentation of the same surfaces might provide essential interaction domains for kinetochore proteins to assemble functional centromeres.
Our finding that CenH3 nucleosomes are right-handed also might help explain why key residues involved in H3/H3 4-helix bundle formation are invariant in CenH3s, despite considerable divergence elsewhere in the core. This observation suggests that the CenH3 dimerization interface is occupied under at least some circumstances. We suggest that this interface has been retained to permit CenH3/H3 hybrid formation (Foltz et al., 2006
), which would result in left/right core particles that should be unable to stably wrap DNA. Misincorporation of CenH3 outside of centromeres occurs under many circumstances (Blower and Karpen, 2001
; Henikoff et al., 2000
; Tomonaga et al., 2003
; Van Hooser et al., 2001
), yet is potentially catastrophic, causing dicentric formation, chromosome loss and dominant lethality (Heun et al., 2006
; Tomonaga et al., 2003
). By retaining the ability to dimerize with H3, misincorporated CenH3s would predominantly form structurally defective nucleosomes, thus helping to maintain the extraordinary fidelity of centromere maintenance.
At the boundary between CenH3 and H3 nucleosomal arrays, the change in the direction of DNA around histones from left-handed to right-handed might also have profound implications for maintaining functional centromeres. The uniform packaging of H3 nucleosomes in pericentric heterochromatin, induced in part by the uniform size of centromeric satellite repeats, is expected to be disturbed by the sudden change in the direction of DNA wrapping around CenH3. This would result in a higher-order structural transition from near-crystalline rigid heterochromatin to less densely packaged centromeric chromatin as implied by the unusually long linker DNA found in Drosophila
centromeric chromatin (Dalal et al., 2007b
). The octameric form of canonical H3 nucleosomes is believed to represent a critical evolutionary leap in being able to more densely package the genome, whereas tetrameric archael nucleosomes fail to condense into a comparable higher order packaging (Pereira et al., 1997
; Sandman et al., 1998
). Therefore, the presence of a CenH3 hemisome array that packages DNA in a right-handed orientation and resists octameric packaging would provide a singular location that remains decondensed during mitosis and accessible to binding by kinetochore proteins. The mutual incompatibility of nucleosome cores that wrap DNA in opposite directions suggests a novel mechanism for perpetual maintenance of the centromere within a chromosomal landscape that is dominated by conventional chromatin.