Fig 2: 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 3: 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).

Fig 4: The ALC1 Linker Region Binds to the AP(A) ALC1fl-histone cross-links. Magenta lines indicate linker-histone cross-links: gray lines indicate other cross-links.(B) Sequence alignment of LANA (1–22) and ALC1 linker(601-635).(C) Fluorescence anisotropy of TAMRA-labeled ALC1RLS in the presence ofvarious amounts ofWT/ WT or APM/APM nucleosomes. Error bars for WT/WT nucleosome curve represent SEM (n = 3 independent experiments). APM/APM nucleosomes: one representative curve out of two independent experiments is indicated. Error bars represent SD (n = 10 technical replicates).(D) Competition between TAMRA-labeled LANA peptide pre-bound to WT/WT nucleosomes and unlabeled ALC1RLS. See also Figure S2A. Error bars represent SEM (n = 3 independent experiments).(E) Cryo-EM map.(F) Left: cartoon representation. Top right inset: cryo-EM density around the ALC1 linker (cyan). Bottom right inset: hydrogen-bonding interactions.(G) Superposition of PDB: 1ZLA onto the linkernucleosome complex.See also Figure S2 and Tables S1 and S2.

Fig 5: 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.