Journal: Nucleic Acids Research
Article Title: HPF1 regulates the formation of FUS-dependent compartments by PARP1 and PARP2 activation on damaged DNA
doi: 10.1093/nar/gkag183
Figure Lengend Snippet: PARG was able to efficiently hydrolyze PAR synthesized by PARP1(2) in the presence of HPF1 under conditions of FUS compartment formation. ( A ) Representative AFM images of DNA-rich compartments formed by FUS after PARP1 activation in the absence or presence of HPF1 (30–480 nM) and subsequent hydrolysis of PAR by 40 nM PARG for 30 min. Scale bar: 400 nm; Z scale: 2 nm. AFM images of PARylated PARP1 (30 nM) after incubation with damaged pBR plasmid (12.5 nM) and HPF1 (30–480 nM) for 5 min in the presence of NAD + (0.3 mM) and followed by the addition of FUS (400 nM) and incubation for 1 min before addition of PARG (40 nM). ( B ) The diameter of the compartments was measured in images obtained in conditions similar to Fig. (red circle, before PARG adding) and in images shown in panel (A) (green circle, 30 minutes after adding PARG). ∗ P <.05; *** P <.001; **** P <.0001; ns, nonsignificant, paired t -test; n = 13 per sample (at 0, 30, 60, 120, and 240 nM HPF1 in the absence or presence of PARG) and n = 6 per sample (480 nM HPF1 in the absence of PARG). Horizontal bars indicate mean; scanned area: 30 μm 2 per sample, three samples per condition. ( С, D ) The graphs present quantification of [ 32 P]-labeled PAR after PARG treatment , in the absence of HPF1 (red curve) or in the presence of 60 nM HPF1 (orange curve), 240 nM HPF1 (yellow curve), or 960 nM HPF1 (green curve). The relative level of PAR hydrolysis in the presence of FUS was normalized to the amount of PAR synthesized by PARP1 ( C ) or PARP2 ( D ) in the absence of HPF1 (red curve) for 15 and 30 min, respectively.
Article Snippet: AFM images were recorded in air by using a Nanoscope V Multimode 8 (Bruker, Santa Barbara, CA) in PeakForce Tapping (PFT) mode using Scanasyst-Air probes (Bruker).
Techniques: Synthesized, Activation Assay, Incubation, Plasmid Preparation, Labeling
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![FUS forms DNA-rich compartments upon PARP1 activation in the presence of HPF1. ( A ) Upper panels: <t>AFM</t> <t>images</t> of PARylated PARP1 (30 nM) after incubation with damaged pBR plasmid (12.5 nM) and HPF1 (30–480 nM) for 5 min in the presence of NAD + (300 μM). Lower panels: same conditions as on the upper panel, but followed by the addition of FUS (400 nM) and incubation for 1 min before deposition on a mica surface. Scale bar: 400 nm; Z scale: 2 nm. ( B ) Diameter of PARylated molecules were measured in images shown in panel (A) (upper panels). ∗∗ P <.01; *** P <.001; **** P <.0001, paired t -test; n = 140 per sample. Horizontal bars indicate mean; scanned area: 20 μm 2 per sample, three samples per condition. ( C ) Diameter of compartments were measured in images shown in panel (A) (lower panels). ∗∗ P <.05, paired t -test; n = 40 per sample (except 480 nM HPF1, n = 9 per sample). Horizontal bars indicate mean; scanned area: 30 μm 2 per sample, three samples per condition. ( D ) Diagrams showing calculation of the relative level of PARP1 activity in the presence of HPF1 after separation of the products by 10% SDS–PAGЕ . PARP1 (30 nM) was incubated with damaged DNA (12.5 nM), 0.3 mM NAD + , and 0.4 μCi [ 32 P]-NAD + in the absence or presence of HPF1 (30–960 nM). The relative level of PAR synthesis was normalized to the level of PAR synthesis catalyzed by PARP1 alone for 15 min without HPF1.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6358/pmc12956358/pmc12956358__gkag183fig1.jpg)
![FUS forms DNA-rich compartments upon PARP2 activation in the presence of HPF1. ( A ) Upper panels: AFM images of PARylated PARP2 (30 nM) after incubation with damaged pBR plasmid (12.5 nM) and HPF1 (30–480 nM) for 5 min in the presence of NAD+ (300 μM). Lower panels: Same conditions as on the upper panel, but followed by the addition of FUS (400 nM) and incubation for 1 min before deposition on mica surface. Scale bar: 400 nm; Z scale: 2 nm. ( B ) Diameter of PARylated molecules was measured in images shown in panel (A) (upper panels). ∗∗∗∗ P <.0001, ns, nonsignificant, paired t -test; n = 140 per sample. Horizontal bars indicate mean; scanned area: 20 μm² per sample, three samples per condition. ( C ) Diameter of compartments were measured in images shown in panel (A) (lower panels). ns P >.05, paired t -test; n = 40 per sample (except 480 nM HPF1). Horizontal bars indicate mean; scanned area: 30 μm² per sample, three samples per condition. ( D ) Diagrams showing calculation of the relative level of PARP2 activity in the presence of HPF1 after separation of the products by 10% SDS–PAGЕ . PARP2 (30 nM) was incubated with damaged DNA (12.5 nM), 0.3 mM NAD + , and 0.4 μCi [ 32 P]-NAD + in the absence or presence of HPF1 (30–960 nM). The relative level of PAR synthesis was normalized to the level of PAR synthesis catalyzed by PARP2 alone for 30 min without HPF1.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6358/pmc12956358/pmc12956358__gkag183fig2.jpg)
![PARG was able to efficiently hydrolyze PAR synthesized by PARP1(2) in the presence of HPF1 under conditions of FUS compartment formation. ( A ) Representative AFM images of DNA-rich compartments formed by FUS after PARP1 activation in the absence or presence of HPF1 (30–480 nM) and subsequent hydrolysis of PAR by 40 nM PARG for 30 min. Scale bar: 400 nm; Z scale: 2 nm. AFM images of PARylated PARP1 (30 nM) after incubation with damaged pBR plasmid (12.5 nM) and HPF1 (30–480 nM) for 5 min in the presence of NAD + (0.3 mM) and followed by the addition of FUS (400 nM) and incubation for 1 min before addition of PARG (40 nM). ( B ) The diameter of the compartments was measured in images obtained in conditions similar to Fig. (red circle, before PARG adding) and in images shown in panel (A) (green circle, 30 minutes after adding PARG). ∗ P <.05; *** P <.001; **** P <.0001; ns, nonsignificant, paired t -test; n = 13 per sample (at 0, 30, 60, 120, and 240 nM HPF1 in the absence or presence of PARG) and n = 6 per sample (480 nM HPF1 in the absence of PARG). Horizontal bars indicate mean; scanned area: 30 μm 2 per sample, three samples per condition. ( С, D ) The graphs present quantification of [ 32 P]-labeled PAR after PARG treatment , in the absence of HPF1 (red curve) or in the presence of 60 nM HPF1 (orange curve), 240 nM HPF1 (yellow curve), or 960 nM HPF1 (green curve). The relative level of PAR hydrolysis in the presence of FUS was normalized to the amount of PAR synthesized by PARP1 ( C ) or PARP2 ( D ) in the absence of HPF1 (red curve) for 15 and 30 min, respectively.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6358/pmc12956358/pmc12956358__gkag183fig3.jpg)
