Transient kinetic analysis of SWR1C-catalyzed H2A.Z deposition unravels the impact of nucleosome dynamics and the asymmetry of stepwise histone exchange

Abstract

The SWR1C chromatin remodeling enzyme catalyzes an ATP-dependent replacement of nucleosomal H2A with the H2A.Z variant, regulating key DNA-mediated processes, such as transcription and DNA repair. Here we investigate the transient kinetic mechanism of the histone exchange reaction employing ensemble FRET, fluorescence correlation spectroscopy (FCS), and the steady state kinetics of ATP hydrolysis. Our studies indicate that SWR1C modulates nucleosome dynamics on both the millisecond and microsecond timescales, poising the nucleosome for the dimer exchange reaction. The transient kinetic analysis of the remodeling reaction performed under single turnover conditions unraveled a striking asymmetry in the ATPdependent replacement of nucleosomal dimers, promoted by localized DNA translocation. Taken together, our transient kinetic studies identify new intermediates and provide crucial insights into the SWR1C-catalyzed dimer exchange reaction, as well as shedding light on how the mechanics of H2A.Z deposition might contribute to transcriptional regulation in vivo. 3

Structure and dynamics of the yeast SWR1-nucleosome complex

“…Consequently, our structural and singlemolecule data suggest that the role of the Swr1 ATPase activity is to induce local distortion in the DNA at SHL2 that results from limited translocation against the HD1 domain of Swr1, and that this begins to destabilize the DNA wrap and histone dimer contacts within the nucleosome. Such a distortion of the dimer interface with H4 as a consequence of limited, and transient, DNA translocation has been suggested previously on the basis of biochemical data (17,29), and our structure now provides mechanistic insights into that process. Our structure likely represents an initial stage in the reaction cycle, and the distortions we observe in both the DNA and the histone core could be amplified by additional translocation steps.…”

“…Furthermore, although SWR1 is unable to slide nucleosomes, the ATPase activity of the "motor" domains is nonetheless essential for activity (8). Limited sliding of DNA within the confines of the DNA wrap has recently been demonstrated for SWR1 (29). The tighter interactions between the Nterminal motor domain (HD1) and the other DNA gyre may act as a block to prevent net translocation by SWR1 but appears to allow limited local translocation of the DNA wrap leading up to that block.…”

“…Very early studies showed that not only was the Swr1 subunit a member of the superfamily 2 translocases, like other chromatin remodelers that slide nucleosomes, but also that the ATPase activity of this subunit was essential for activity (8); these findings suggested that DNA translocation at some level might be a component of the histone exchange mechanism. However, no group has been able to demonstrate net nucleosome sliding by SWR1, although limited sliding within the nucleosome has recently been reported (29).…”

“…By contrast, for SWR1, a gap in the tracking strand close to the SHL2 site is sufficient to prevent histone exchange, which suggests a requirement for some form of ATPdriven conformational changes of the DNA at the binding site. Although footprinting shows that there is no net sliding of nucleosome position as a consequence of exchange even at base pair resolution (17), limited transient translocation (i.e., a few base pairs) of part of the DNA wrap has been proposed (29).…”

Emergence of robust nucleosome patterns from an interplay of positioning mechanisms

“…First, in vitro reconstitution experiments result in stereotypic nucleosome array patterns only in the presence of ATP (Zhang et al, 2011). This indicates that remodelers mobilize nucleosomes that are otherwise kinetically frozen, which is also supported by direct observations of strongly increased DNA unwrapping rates upon remodeler binding (Singh et al, 2018). Second, remodelers can displace nucleosomes in successive steps in the same direction (Blosser et al, 2009).…”