Fig 1: Mutations that Destabilize ATPase Motor-Macro Domain Interactions Constitutively Activate ALC1 and Alter the Dynamics of Its Recruitment at DNA Damage Sites(A) ATPase activities of ALC1fl WT (teal), R857Q (green), R857E (magenta), and R860W (brown).(B) Remodeling by ALC1fl WT (same as in Figure 1C) compared to R857Q (left), R857E (middle), and R860W (right) in the presence or absence of PARP1/NAD+. Bar graph: relative rates for ALC1fl WT (teal), R857Q (green), R857E (magenta), and R860W (brown).(C) Representative images of U2OS cells expressing WT, R857Q, R857E, and R860W YFP-ALC1, taken at the indicated early time points following laser damage.(D) Kinetics of YFP-ALC1 (left) and YFP-ALC1 R860W (right) association with DNA breaks as quantified from (C).(E) Half-hour time course with representative images of U2OS cells expressing WT, R857Q, R857E, and R860W YFP-ALC1, taken at indicated time points following laser damage.(F) Kinetics of WT, R857Q, R857E, and R860W YFP-ALC1 association with/dissociation from DNA breaks as quantified from (E).(G) Kinetics of YFP-ALC1 R857Q and R857E association with DNA breaks.(H) Kinetics of WT YFP-ALC1 dissociation from DNA breaks.Error bars ± SEM; t: time constant; scale bars, 10 µm. See also Figure S7.
Fig 2: PtdIns(3,4,5)P3interacts and colocalizes with PARP1 in the nucleolus.A, Western immunoblotting of supernatants (SPN) and resulting nuclear pellets (NUC) obtained after the incubation of isolated HeLa nuclei in the retention buffer in the absence (-) or presence (+) of neomycin. B, lipid overlay assay using PIP strips incubated with recombinant GST or GST-PARP1 and detection of protein–lipid interactions using an anti-GST-HRP–conjugated antibody. C, GST-PARP1 or GST-PLCδ1-PH pulldown with the indicated PPIn-conjugated beads. Eluates were resolved by SDS-PAGE and Western blotted using an anti-GST antibody conjugated to horse radish peroxidase. D, HeLa cells costained with anti-PARP1 and anti-PtdIns(3,4,5)P3, anti-PtdIns(4,5)P2, or anti-PtdIns(3,4)P2 antibodies and imaged by confocal microscopy. The scale bar represents 10 μm. Images are representative of at least three biological replicates. PPIn, polyphosphoinositide; PtdIns(3,4)P2, phosphatidylinositol 3,4-bisphosphate; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PARP1, poly(ADP-ribose) polymerase 1; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate.
Fig 3: The Binding of PARylated PARP1 to the Macro Domain Displaces It from the ATPase Motor(A) Reconstitution of an ALC1cat:ALC1macro complex (ALC1complex). Top: analytical gel filtrations. Bottom: SDS-PAGE of the peak fractions.(B) SDS-PAGE of ALC1complex after Ni-NTA purification. 6xHis-tag bearing ALC1cat was incubated with a version of ALC1macro that lacks the 6xHis-tag (ALC1macroΔ6xHis) and purified using Ni-NTA resin.(C) Co-immunoprecipitation (coIP) assay with plasmids expressing FLAG-tagged macro domain (FLAG-macro), YFP, or an YFP-tagged version of the ATPase (YFP-ALC1Δmacro). Cells were grown in the presence or absence of olaparib, as indicated. Cell lysates (input) were immunoprecipitated with α-FLAG. Immunoblotting was carried out with antibodies as indicated.See also Figure S4.
Fig 4: The Binding of PARylated PARP1 to the Macro Domain of ALC1 Triggers a Major Conformational Change(A) Difference plot of HDX data from ALC1fl in the presence and absence of PARylated PARP1. Experimental error is shown in gray, and areas of increased exposure and protection are shaded in red and blue, respectively. Different line colors show different D2O incubation times. Non-covered sequence (Figure S5C) is omitted.(B) Comparison of intra-ALC1 cross-links in non-activated or activated ALC1fl. Gray, green, and magenta lines indicate cross-links that are detected in both cases, only with activated, or only with non-activated ALC1fl, respectively.(C) SAXS-based ab initio and rigid-body models of ALC1cat.(D) The ATPase of SNF2 (Liu et al., 2017) aligned onto that of ALC1cat (cartoon) and of ALC1fl-MC (lines), using the N-lobe as a guide (not shown for SNF2).(E) SAXS-derived pairwise interatomic distance distribution for ALC1cat in the apo (orange) and DNA-bound (green) state, scaled such that the area under the curve matches the observed particle volume.See also Figure S5.
Fig 5: PARP1 binds to PPIn via three polybasic regions.A, domain structure of PARP1 and deletion constructs. B, schematic representation of lipids spotted (100 pmol) on PIP strips (Echelon Biosciences) including lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), phosphatidylinositol (PtdIns), PtdIns3P, PtdIns4P, PtdIns5P, phosphatidylethanolamine (PE), phosphatidylcholine (PC), sphingosine-1-Phosphate (S1P), PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2, PtdIns(3,4,5)P3, phosphatic acid (PA), phosphatidylserine (PS), and blank. C and D, lipid overlay assay using PIP strips incubated with recombinant GST-PARP1 deletion constructs WT and mutants and detection of protein–lipid interactions using an anti-GST-HRP–conjugated antibody. E, multiple sequence alignment of the polybasic regions located in the zinc finger III and the BRCT-WGR linker found in human PARP-1 compared with other vertebrate species performed using the online program MUSCLE. Accession number for Homo sapiens (P09874), Mus musculus (P11103), Bos taurus (P18493), Gallus gallus (P26446), Xenopus laevis (P31669), and Danio rerio (Q5RHR0). F, ribbon representation of the human PARP-1 zinc-finger III NMR structure (aa 233–357 PDB: 2JVN, (126)). The two polybasic regions found in the N-terminal (aa 233–236) and C-terminal (aa 346–352) parts of the zinc-finger III are colored in red. ART, (ADP-ribosyl) transferase domain (ART); BRCT, BRCA1 C-terminal domain; HD, helical subdomain; HRP, horse radish peroxidase; PARP1, poly(ADP-ribose) polymerase 1; ZnF 1 to 3, zinc-finger I-III; WGR, Trp-Gly-Arg.
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