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In CHRAC, the p14-p16 heterodimer is tightly associated with the N-terminus of ACF1 (see 4.3.1). In contrast, the heterodimer interacts only weakly with DNA, and its DNA binding properties appear to be rather dynamic and modulated by the C-terminal tails of both subunits (see 4.5.2). Whereas the C-terminus of CHRAC14 contributes to DNA binding and

deletion of the tail leads to an almost complete loss of DNA binding ability, the acidic CHRAC16 C-terminus has an opposite effect on the DNA binding properties: Due to its negative charge, it prevents tight DNA binding, and its deletion leads to a strong increase of DNA affinity (see Figure 4.19 and 5.2.1).

The enhancement of ACF nucleosome sliding activity by p14-p16 (Figure 4.24) is both dependent on the association of p14-p16 with ACF1 and with DNA, since a loss of either of these two interactions results in a loss of the observed activating effect (compare Figure 4.25 and 4.26 A, B). Furthermore, the p14-p16ΔC deletion variant inhibits the sliding activity of ACF due to its increased DNA affinity (Figure 4.26 C).

Biochemical studies of human CHRAC17-CHRAC15 by Varga-Weisz and colleagues have led to similar results (Kukimoto et al., 2004). In agreement with the analysis presented here, the human homologues interact with the N-terminus of human ACF1 and facilitate ACF- mediated nucleosome sliding in a manner that is dependent on interaction with both DNA and ACF1. Besides, the authors report a stimulating effect of hCHRAC17-hCHRAC15 on ACF-dependent chromatin assembly reactions. However, this feature does not seem to be specific for the two CHRAC subunits, since other histone fold proteins such as the two small DNA polymerase ε subunits or negative cofactor 2 (NC-2) show the same effect (Kukimoto et al., 2004).

The authors also demonstrate that deletion of any of the hCHRAC17-hCHRAC15 C- terminal tails impairs the stimulation of hACF sliding activity. Like for the Drosophila proteins, this effect seems to be caused by altered DNA affinities of the deletion variants. However, in contrast to the results for Drosophila p14-p16 presented here, the authors did not detect any significant increase in DNA binding and did not observe any direct sliding inhibition with the human deletion variants. This discrepancy could be species-specific, but it might also be caused by the fact that the C-terminal tail truncations were chosen such that additional flanking sequences to the acidic residues were deleted. Therefore, the authors failed to conclude about the potential mechanism of CHRAC-mediated chromatin remodelling (Kukimoto et al., 2004).

It has been mentioned previously that the effects of p14-p16 on nucleosome remodelling were only detectable in the presence of a high excess of p14-p16 (see 4.6.1). Despite several attempts, this issue could not be solved and satisfactorily explained. One possibility could be suboptimal conditions during the nucleosome mobilisation reaction. Although the in vitro sliding assay has been optimised for ACF (Eberharter et al., 2004a), it might be that p14-p16 are not maximally active under the same conditions and the change of one or more parameters such as temperature, ionic strength, buffer conditions, ATP concentration etc. could improve

their activity. However, attempts to increase their effect on nucleosome remodelling by optimising the assay conditions were unsuccessful.

Alternatively, the requirement for the large excess of p14-p16 in the nucleosome sliding reactions might be due to the quality of the two recombinantly expressed subunits. Several protein preparations purified from E. coli were tested, and all of them had essentially the same activity in the sliding reactions. It cannot be ruled out that p14-p16 produced by a prokaryotic system (E. coli) are partially misfolded and therefore less active. However, recombinant p14- p16 produced by an eukaryotic system (Sf9-cells) co-purified a highly active ATP-dependent chromatin remodelling activity (see 4.1.2.2 and Figure 4.4). Heat-inactivation of the contaminating remodelling ATPase (30 to 60 min at 37°C) resulted in p14-p16 that had approximately the same specific activity in nucleosome sliding assays than E. coli-expressed p14-p16 (not shown). Therefore, misfolding of the recombinant protein can only partially explain its weak activity.

An accurate way of measuring the influence of p14-p16 on nucleosome sliding would be the direct comparison of reconstituted ACF and CHRAC, purified in parallel from the same source (Sf9 cells). Yet, the co-expression of all four CHRAC subunits by co-infection of Sf9 cells with three different baculoviruses turned out to be very difficult, and it was impossible to get the four proteins expressed at stoichiometric levels. Furthermore, the experience with the contaminating chromatin remodelling activity in the p14-p16 preparations (see 4.1.2.2, Figure 4.4 and above) shows that co-expression of all four CHRAC subunits in Sf9 cells would be problematic as well. Presumably, the protein preparations would also be contaminated by Sf9 cell-specific chromatin remodelling factors, which would be extremely difficult to monitor (see also 5.4).

It should be mentioned here that the nucleosome mobilisation assays with human ACF and human CHRAC17-CHRAC15 were also carried out with an excess of the CHRAC17- CHRAC15 heterodimer over hACF, although this excess was much less pronounced (approximately 25-fold, (Kukimoto et al., 2004). However, the catalytic activity of human ACF itself appeared to be very weak, so that efficient remodelling was achieved only at an equimolar ratio of hACF and mononucleosomes or even an excess of hACF. Therefore, similar concerns about enzyme activity can be raised in that case as well. Besides, the poor activity of hACF might be very efficiently increased even by a weak stimulus of hCHRAC17- hCHRAC15, whereas the activity of Drosophila ACF appears to be very high even without CHRAC14-CHRAC16, and a stimulatory effect of these two subunits might be more difficult to observe.