Previous work has suggested that SWI/SNF complexes generate an unstable reaction intermediate that contains disrupted histone-DNA contacts (4
). It has also been proposed that SWI/SNF complexes generate and inter-convert products with different regions of DNA exposed via the formation of such an intermediate (4
). Our results add to this reaction framework by capturing, for the first time, a transient reaction intermediate generated during the octamer and dimer transfer reactions catalyzed by the major SWI/SNF complex in budding yeast, RSC. As discussed below, we hypothesize that this intermediate entails a close encounter between the two exchange substrates and is formed after the disruption of histone-DNA contacts ().
Figure 5 Models for RSC catalyzed octamer transfer. In these schematic the acceptor DNA is shown in blue. A disrupted intermediate is inferred based on previous work (4, 6, 18, 19) whereas the encounter intermediate is inferred based on this work. It is assumed (more ...)
Comparison of the FRET-based data and gel-based data suggests that dimer and octamer transfer occur via at least two steps: (i) a fast step in which the histones from the donor nucleosome are brought in close vicinity of the acceptor nucleosome or DNA to generate an unstable intermediate species, and (ii) a slow step in which this unstable species is converted to a stable nucleosome containing histone components deriving from the donor nucleosome. There are two extreme models to explain these observations. Below we discuss these models in the context of octamer transfer, but a similar analysis would apply to the dimer exchange reaction.
In the first model, complete dissociation of the donor DNA from the histone octamer and association of acceptor DNA with the histone octamer are coupled. The unstable intermediate contains, in close proximity, a donor nucleosome with disrupted histone-DNA contacts and an acceptor DNA (). In such an encounter intermediate, the acceptor DNA could be partially associated with the exposed regions of the histone octamer in the donor nucleosome. The slow step would then involve complete transfer of the octamer from the donor DNA to the acceptor DNA. The disrupted intermediate could arise due to globally disrupted histone-DNA contacts as recently proposed (19
). Alternatively the disrupted intermediate could be a consequence of the histone octamer translocating beyond the DNA ends and thereby generating exposed octamer surfaces that can interact with another piece of DNA. In the context of dimer exchange, the encounter intermediate would resemble a dinucleosome. Indeed previous work has shown that SWI/SNF family complexes can generate dinucleosome-like species from mononucleosomes (7
). If the encounter intermediate involves movement of the donor octamer beyond a DNA end, then it would be expected to form less readily in the context of nucleosomal arrays. However, previous work has shown that RSC can catalyze exchange of dimers between nucleosomes within arrays, suggesting that the dimer exchange reaction does not rely solely on the histone octamer moving off the DNA end (5
In the second model, dissociation of the donor DNA from the histone octamer and association of acceptor DNA with the histone octamer occur in two separate steps (). The unstable encounter intermediate contains only the histone octamer from the donor nucleosome in close proximity to the acceptor DNA, while the donor DNA is completely removed in a prior step. The slow step would then involve association of the donor DNA with the histone octamer to form a stable nucleosomal complex. In the first model, a slightly positively charged RSC active site could facilitate global histone-DNA disruption as well as accommodate the acceptor DNA. In the second model, the RSC active site has to be compatible with binding a nucleosome as well as the separated histone octamer and DNA, and each imposes very different electrostatic requirements on the RSC active site. In such a case, it is possible, as discussed below, that other RSC subunits participate in binding the different intermediate histone and DNA states.
Interestingly, we find that free DNA from the donor nucleosome appears faster than the octamer transfer product when visualized by native gel. Further the donor free DNA appears with similar kinetics in the absence of the acceptor DNA. These observations are initially more consistent with the second model, in which the disrupted intermediate entails complete dissociation of the nucleosomal DNA. However, it is also possible that the less stably bound DNA in the disrupted intermediate of Model 1 dissociates readily on a native gel.
In both the above models, collapse of the encounter intermediate into a stable nucleosome is not ATP-dependent. The ATP requirement that we observe in arises because, only a small fraction of the disrupted intermediate gets converted to the encounter intermediate in each remodeling cycle. As a result, ATP-dependent RSC activity is necessary to constantly replenish the pool of disrupted intermediate (). By itself, the much slower formation of the stable transfer products, compared to repositioned products, could be interpreted to mean that the transfer products are off-pathway products. However, the observation that the substrates of the transfer reactions come together much faster suggests that the exchange products can be on-pathway RSC products. Further, the time-scale on which the encounter intermediate is formed is comparable to the in vivo
time-scales associated with RNA and DNA polymerase translocation on 150 bp of DNA (26
), raising the possibility that these exchange products can have biologically relevant roles.
Both models () raise the possibility that RSC has binding sites for more than one nucleosomal or DNA substrate. These additional binding sites may be provided by non-catalytic RSC subunits. Precedence for such a mechanism comes from previous work on the SWR and SWI/SNF complexes that identified specific subunits required for dimer transfer (28
). These subunits are believed to facilitate efficient dimer transfer by directly binding to H2A/H2B dimers. Although a homologous subunit in RSC is not readily apparent, it is possible that one of RSC’s many non-catalytic subunits may be involved in binding additional substrates to facilitate transfer. RSC function may also be additionally regulated by other chromatin factors that bias RSC towards forming predominantly dimer exchange or octamer transfer products.