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SUMOylation of FEN1 facilitates its interaction with <t>RAD1</t> and HUS1. ( A ) In vitro binding assays were conducted for FEN1 or SUMO-1-FEN1 with PCNA or HUS1. Purified recombinant 6×His-tagged FEN1 was incubated with Ubc9, SUMO-1, and other components of the SUMOylation kit, with or without ATP, at 37°C for 60 min. Unmodified FEN1 or SUMO-1-FEN1 was incubated with Ni-NTA beads. After extensive washing, the beads were incubated with a mixture of MBP-tagged PCNA and GST-tagged HUS1 proteins. Incubation of PCNA and HUS1 with beads only (no FEN1) and incubation of FEN1-Ni-NTA beads with non-conjugated MBP and GST proteins without ATP served as negative controls for non-specific binding of the proteins or tags to the beads. FEN1-bound MBP-PCNA and GST-HUS1 were analyzed by western blot analysis using anti-PCNA, anti-HUS1, anti-MBP, and anti-GST antibodies. The intensities of PCNA and HUS1 in the pull-down were quantified and normalized to their input levels. The relative levels of PCNA and HUS1 binding to FEN1, with versus without SUMO-1 modification (ATP), are shown. ( B ) WT HeLa cells were treated with 120 J/m 2 UV irradiation and allowed to recover for 3 h. FEN1 complexes were immunoprecipitated using an anti-FEN1 antibody. FEN1 and SUMO-1-FEN1, as well as PCNA, HUS1, RAD1 that were co-IPed with FEN1, were detected by western blot analysis. A bead-only (no anti-FEN1) control was used as a negative control for IP. The relative levels of PCNA, HUS1, and RAD1 binding to FEN1, normalized to the loading control IgG and relative to that of untreated WT cells, are shown. ( C ) HeLa cells stably expressing 3×FLAG-tagged WT or 4KR mutant FEN1 were treated with 120 J/m 2 UV irradiation and allowed to recover for 3 h. 3×FLAG-FEN1 complexes were immunoprecipitated using anti-FLAG M2 beads. 3×FLAG-FEN1 and SUMO-1-FEN1, as well as PCNA, HUS1, and RAD1 that were co-immunoprecipitated with FEN1, were detected by western blot analysis. ( D ) The interactions of WT or 4KR mutant FEN1 with PCNA, HUS1, and RAD1 with and without UV treatment were analyzed using the Duolink ® in situ PLA with antibody mixtures containing anti-FLAG/anti-PCNA, anti-FLAG/anti-HUS1, and anti-FLAG/anti-RAD-1. Nuclei were stained with DAPI. Upper panels show representative PLA assay images (scale bars: 10 μm). and the bottom panels show PLA intensities, relative to that of control WT cells. Values shown are mean ± SD of three independent assays. P- values were calculated using Student’s t -test. ns, not significant, * P < 0.05, ** P < 0.01.
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SUMOylation of FEN1 facilitates its interaction with RAD1 and HUS1. ( A ) In vitro binding assays were conducted for FEN1 or SUMO-1-FEN1 with PCNA or HUS1. Purified recombinant 6×His-tagged FEN1 was incubated with Ubc9, SUMO-1, and other components of the SUMOylation kit, with or without ATP, at 37°C for 60 min. Unmodified FEN1 or SUMO-1-FEN1 was incubated with Ni-NTA beads. After extensive washing, the beads were incubated with a mixture of MBP-tagged PCNA and GST-tagged HUS1 proteins. Incubation of PCNA and HUS1 with beads only (no FEN1) and incubation of FEN1-Ni-NTA beads with non-conjugated MBP and GST proteins without ATP served as negative controls for non-specific binding of the proteins or tags to the beads. FEN1-bound MBP-PCNA and GST-HUS1 were analyzed by western blot analysis using anti-PCNA, anti-HUS1, anti-MBP, and anti-GST antibodies. The intensities of PCNA and HUS1 in the pull-down were quantified and normalized to their input levels. The relative levels of PCNA and HUS1 binding to FEN1, with versus without SUMO-1 modification (ATP), are shown. ( B ) WT HeLa cells were treated with 120 J/m 2 UV irradiation and allowed to recover for 3 h. FEN1 complexes were immunoprecipitated using an anti-FEN1 antibody. FEN1 and SUMO-1-FEN1, as well as PCNA, HUS1, RAD1 that were co-IPed with FEN1, were detected by western blot analysis. A bead-only (no anti-FEN1) control was used as a negative control for IP. The relative levels of PCNA, HUS1, and RAD1 binding to FEN1, normalized to the loading control IgG and relative to that of untreated WT cells, are shown. ( C ) HeLa cells stably expressing 3×FLAG-tagged WT or 4KR mutant FEN1 were treated with 120 J/m 2 UV irradiation and allowed to recover for 3 h. 3×FLAG-FEN1 complexes were immunoprecipitated using anti-FLAG M2 beads. 3×FLAG-FEN1 and SUMO-1-FEN1, as well as PCNA, HUS1, and RAD1 that were co-immunoprecipitated with FEN1, were detected by western blot analysis. ( D ) The interactions of WT or 4KR mutant FEN1 with PCNA, HUS1, and RAD1 with and without UV treatment were analyzed using the Duolink ® in situ PLA with antibody mixtures containing anti-FLAG/anti-PCNA, anti-FLAG/anti-HUS1, and anti-FLAG/anti-RAD-1. Nuclei were stained with DAPI. Upper panels show representative PLA assay images (scale bars: 10 μm). and the bottom panels show PLA intensities, relative to that of control WT cells. Values shown are mean ± SD of three independent assays. P- values were calculated using Student’s t -test. ns, not significant, * P < 0.05, ** P < 0.01.

Journal: Journal of Molecular Cell Biology

Article Title: SUMO-1 modification of FEN1 facilitates its interaction with Rad9–Rad1–Hus1 to counteract DNA replication stress

doi: 10.1093/jmcb/mjy047

Figure Lengend Snippet: SUMOylation of FEN1 facilitates its interaction with RAD1 and HUS1. ( A ) In vitro binding assays were conducted for FEN1 or SUMO-1-FEN1 with PCNA or HUS1. Purified recombinant 6×His-tagged FEN1 was incubated with Ubc9, SUMO-1, and other components of the SUMOylation kit, with or without ATP, at 37°C for 60 min. Unmodified FEN1 or SUMO-1-FEN1 was incubated with Ni-NTA beads. After extensive washing, the beads were incubated with a mixture of MBP-tagged PCNA and GST-tagged HUS1 proteins. Incubation of PCNA and HUS1 with beads only (no FEN1) and incubation of FEN1-Ni-NTA beads with non-conjugated MBP and GST proteins without ATP served as negative controls for non-specific binding of the proteins or tags to the beads. FEN1-bound MBP-PCNA and GST-HUS1 were analyzed by western blot analysis using anti-PCNA, anti-HUS1, anti-MBP, and anti-GST antibodies. The intensities of PCNA and HUS1 in the pull-down were quantified and normalized to their input levels. The relative levels of PCNA and HUS1 binding to FEN1, with versus without SUMO-1 modification (ATP), are shown. ( B ) WT HeLa cells were treated with 120 J/m 2 UV irradiation and allowed to recover for 3 h. FEN1 complexes were immunoprecipitated using an anti-FEN1 antibody. FEN1 and SUMO-1-FEN1, as well as PCNA, HUS1, RAD1 that were co-IPed with FEN1, were detected by western blot analysis. A bead-only (no anti-FEN1) control was used as a negative control for IP. The relative levels of PCNA, HUS1, and RAD1 binding to FEN1, normalized to the loading control IgG and relative to that of untreated WT cells, are shown. ( C ) HeLa cells stably expressing 3×FLAG-tagged WT or 4KR mutant FEN1 were treated with 120 J/m 2 UV irradiation and allowed to recover for 3 h. 3×FLAG-FEN1 complexes were immunoprecipitated using anti-FLAG M2 beads. 3×FLAG-FEN1 and SUMO-1-FEN1, as well as PCNA, HUS1, and RAD1 that were co-immunoprecipitated with FEN1, were detected by western blot analysis. ( D ) The interactions of WT or 4KR mutant FEN1 with PCNA, HUS1, and RAD1 with and without UV treatment were analyzed using the Duolink ® in situ PLA with antibody mixtures containing anti-FLAG/anti-PCNA, anti-FLAG/anti-HUS1, and anti-FLAG/anti-RAD-1. Nuclei were stained with DAPI. Upper panels show representative PLA assay images (scale bars: 10 μm). and the bottom panels show PLA intensities, relative to that of control WT cells. Values shown are mean ± SD of three independent assays. P- values were calculated using Student’s t -test. ns, not significant, * P < 0.05, ** P < 0.01.

Article Snippet: Anti-IdU was from BD Biosciences; anti-FLAG was from Santa Cruz Biotechnology; antibodies against PCNA, FEN1, SUMO-1, HUS1, RAD9, RAD1, and β-actin were from Proteintech; anti-γH2AX and secondary antibodies were from Cell Signaling Technology.

Techniques: In Vitro, Binding Assay, Purification, Recombinant, Incubation, Western Blot, Modification, Irradiation, Immunoprecipitation, Control, Negative Control, Stable Transfection, Expressing, Mutagenesis, In Situ, Staining