Elucidation of the Mechanisms of Nucleosome Binding and Repositioning by a Chromatin Remodeler: Monomeric ISWI Remodels Nucleosomes Through a Random Walk

The regulation of chromatin structure is controlled by a family of molecular motors called chromatin remodelers. The ability of these enzymes to remodel chromatin structure is dependent on their ability to couple ATP binding and hydrolysis into the mechanical work that drives nucleosome repositioning. The goal of this work was to characterize quantitatively the nucleosome repositioning activity, and associated processes of nucleotide binding, DNA binding, and nucleosome binding, of the chromatin remodeler ISWI. ISWI is capable of repositioning clusters of nucleosomes to create well-ordered arrays or moving single nucleosomes from the center of DNA fragments toward the ends without disrupting their integrity. The necessary first step in determining how these essential enzymes catalyze the repositioning of nucleosomes is to characterize both how they bind nucleosomes and how this interaction is regulated by ATP binding and hydrolysis. Toward this goal we monitored the interaction of the chromatin remodeler ISWI with fluorophore-labeled nucleosomes and DNA through associated changes in fluorescence anisotropy of the fluorophore upon ISWI binding to these substrates. We determined that one ISWI molecule binds to a 20 bp double stranded DNA substrate with an affinity of (18 ± 2) nM. In contrast, two ISWI molecules can bind to the core nucleosome with short linker DNA with stoichiometric macroscopic equilibrium constants 1/&beta1 = (1.3 ± 0.6) nM and 1/&beta2 = (13 ± 7) nM2. Furthermore, in order to better understand the mechanism of DNA translocation by ISWI, and hence nucleosome repositioning, we determined the effect of nucleotide analogs on substrate binding by ISWI. While the affinity of ISWI to binding nucleosome substrate with short lengths of flanking DNA was not affected by presence of nucleotides, the affinity of ISWI for binding DNA substrate is weakened in the presence of non-hydrolysable ATP analogs but not in the presence of ADP. Additionally, using standard electrophoresis assays we have monitored the ISWI-catalyzed repositioning of different nucleosome samples each containing different lengths of DNA symmetrically flanking an initially centrally positioned histone octamer. We find that ISWI moves the histone octamer between distinct and thermodynamically stable positions on the DNA according to a random walk mechanism. Through the application of a novel spectrophotometric assay for nucleosome repositioning we further characterized the repositioning activity of ISWI using short nucleosome substrates and were able to determine the macroscopic rate of nucleosome repositioning by ISWI. Additionally, quantitative analysis of repositioning experiments performed under various ISWI concentrations revealed that monomeric ISWI is sufficient to account for the observed repositioning activity as the presence of a second ISWI bound had no effect on the rate of nucleosome repositioning. We also found that ATP hydrolysis is poorly coupled to nucleosome repositioning suggesting that DNA translocation by ISWI is not energetically rate limiting for the repositioning reaction. This is the first calculation of a microscopic ATPase coupling efficiency for nucleosome repositioning and also further supports our conclusion that a second bound ISWI does not contribute to the repositioning reaction. In conclusion, the characterization of the mechanism of nucleosome binding and repositioning by the chromatin remodeler ISWI presented in this dissertation provides a foundation for future studies aiming to understand how various regulatory elements influence the function of ISWI