The Chd1 chromatin remodeler forms long-lived complexes with nucleosomes in the presence of ADP?BeF3? and transition state analogs

Chromatin remodelers use helicase-like ATPase domains to reorganize histone?DNA contacts within the nucleosome. Like other remodelers, the chromodomain helicase DNA-binding protein 1 (Chd1) remodeler repositions nucleosomes by altering DNA topology at its internal binding site on the nucleosome, coupling different degrees of DNA twist and DNA movement to distinct nucleotide-bound states of the ATPase motor. In this work, we used a competition assay to study how variations in the bound nucleotide, Chd1, and the nucleosome substrate affect stability of Chd1?nucleosome complexes. We found that Chd1?nucleosome complexes formed in nucleotide-free or ADP conditions were relatively unstable and dissociated within 30 s, whereas those with the nonhydrolyzable ATP analog AMP-PNP had a mean lifetime of 4.8 ? 0.7 min. Chd1?nucleosome complexes were remarkably stable with ADP?BeF3? and the transition state analogs ADP?AlF and ADP?MgF, being resistant to competitor nucleosome over a 24-h period. For the tight ADP?BeFstabilized complex, Mg was a critical component that did not freely exchange, and formation of these long-lived complexes had a slow, concentration-dependent step. The ADP?BeF stabilized complex did not require the Chd1 DNA-binding domain nor the histone H4 tail and appeared relatively insensitive to sequence differences on either side of the Widom 601 sequence. Interestingly, the complex remained stable in ADP?BeF even when nucleosomes contained single-stranded gaps that disrupted most DNA contacts with the guide strand. This finding suggests that binding via the tracking strand alone is sufficient for stabilizing the complex in a hydrolysis-competent state.