Fig 2: The Linker-AP Interaction Is Important for ALC1 Activity(A) ATPase activity for 20 μM ALC1fl R860W or ALC1fl R611A/S612A/R860W in the presence of 2 μM WT/WT nucleosomes. Error bars represent SD (n = 3 independent experiments).(B) Michaelis-Menten curves of remodeling rates for WT/WT nucleosomes (10 nM) and various ALC1fl R860W or R611A/S612A/R860W concentrations. Inset: enlarged view for ALC1fl R611A/S612A/R860W.(C) Left: remodeling of10nM nucleosomes by 20 μM remodeler. Right: relative remodeling rates. Error bars represent SD(n = 3 independent experiments). Data for ALC1fl R860W and WT/WT nucleosomes as well as for ALC1fl R860W and APM/APM nucleosomes: same as indicated in Figure 1B.(D) Left: remodeling of WT/WT nucleosomes (10 nM) in the presence of 20 μM ALC1fl R860W or ALC1fl R611Q. Right: relative rates for ALC1fl R860W or ALC1fl R611Q. Error bars represent SD (n = 3 independent experiments). Data for ALC1fl R860W and WT/WT nucleosomes: same as indicated in Figure 1B.(E) Representative smFRET traces indicating the remodeling of individual WT/WT nucleosomeswith 3 bp of linker DNA(left) by ALC1fl R860W (middle) orALC1fl R611A/S612A/R860W (right). Only pauses immediately following translocation phases with a FRET decrease were analyzed (shaded yellow).(F) Mean pause durations for ALC1fl R860W or ALC1fl R611A/S612A/R860W as described in (E). Error bars represent SEM (n > 170 events).See also Figure S3.

Fig 3: ALC1RLS Mutations Alter Recruitment Dynamics(A) U2OS cells expressing R860W, R611A/S612A/ R860W, or R611Q/R860W YFP-ALC1, imaged upon laser damage. Scale bar, 10 μm.(B) Kinetics of R860W, R611A/S612A/R860W, and R611Q/R860W YFP-ALC1 association with DNA breaks. Error bars represent SEM (n R 131 traces from 3 independent experiments). See also Figure S4.(C) Fractional recruitment of R860W, R611A/ S612A/R860W, and R611Q/R860W YFP-ALC1 to DNA breaks. Error bars represent SEM (n R 131 traces from 3 independent experiments).(D) Half times from (C). Data are means ± 95% confidence intervals.(E) In the absence of DNA damage, association of the macro domain of ALC1 with its C-terminal ATPase lobe stabilizes an ATPase “OFF” state. PAR binding to the macro domain displaces it from the ATPase. ALC1RLS then tethers the remodeler to the AP, which stabilizes an active conformation (macro domain not shown) and promotes efficient coupling of ATP hydrolysis to nucleosome mobilization.See also Figure S4.

Fig 4: The AP Is Important for Remodeling by ALC1(A) Left: octamer surface colored by electrostatic potential (from –5 kB T/e, indicated in red, to +5 kB T/e, indicated in blue). Right: key residues of the H2A AP. Based on PDB: 1AOI.(B) Left: schematic of FRET-labeled wild-type (WT/WT) nucleosomes and APM/APM nucleosomes with AP mutations on both faces of the octamer. Middle: ensemble remodeling of WT/WT and APM/APM nucleosomes (10 nM) in the presence of 20 μM ALC1fl R860W. Right: relative remodeling rates. Error bars represent SD (n = 3 independent experiments).(C) Ensemble remodeling of WT/WT nucleosomes (10 nM) by 80 nM ALC1fl R860W with or without LANA peptide.(D) smFRET labeling and detection.(E) ALC1fl R860W-catalyzed remodeling of individual WT/WT nucleosomes toward the longer (left) or shorter (right) linker DNA.(F) Percentages oftraceswith the initial remodeling direction toward longerorshorter linker DNAforWT/WT and WT/APM nucleosomes. Error bars indicate SEM (n > 100 traces).See also Figure S1.

Fig 5: CHD1L depletion re-sensitizes cells with acquired resistance to Talazoparib. (a) Clonogenic assay showing growth of PSN1 TalaR-M cells with CHD1L stable knockdown. (b) Immunoblot showing expression level of CHD1L and SNAI2 in CHD1L stable knockdown cells as in (a). (c) IncuCyte curves showing the growth PSN1 TalaR-M with inducible CHD1L knockdown with and without doxycycline induction. (d) qPCR showing mRNA level of CHD1L and SNAI2 in PSN1 TalaR-M with inducible CHD1L knockdown with and without doxycycline induction as in (c). (e) IncuCyte curves showing the growth of PSN1 parental and TalaR-M with CHD1L knockout. (f) IncuCyte curves showing the growth of HCC1806 parental and TalaR-M with CHD1L knockout. (g, h) Immunoblot and qPCR showing CHD1L and SNAI2 level in HCC1806 parental and TalaR-M with CHD1L knockout as in (f).