Fig 2: HERC2 is involved in the degradation pathways. (a) The surface representation of the crystal structure of HERC2ZZ in complex with the H3 peptide (PDB: 6WW4). The H3 peptide is shown as grey sticks, and the negatively charged binding site residues of HERC2ZZ are colored red. (b,c) Superimposed 1H,15N HSQC spectra of the mutated HERC2ZZ collected while the REEE peptide was titrated in the NMR samples. Spectra are color coded according to the protein:peptide molar ratio. (d–f) MCF-7 cells incubated with or without 100 nM everolimus for 24 h (d,e), or either the DMEM or EBSS medium for 4 h (f) were subjected to immunostaining with the indicated antibodies. The nuclei were counter stained with DAPI.

Fig 3: HERC2 promotes USP20 ubiquitination and subsequent degradation. (A) USP20 interacted with HERC2. Total cell lysates were extracted from 293T cells and subjected to immunoprecipitation and immunoblotting with antibodies as indicated. (B) Both USP20 and CLASPIN protein levels increased in response to HU treatment. 293T cells were treated with HU at different time points as indicated, total cell lysates were extracted and subjected to immunoblotting with antibodies as indicated. (C) Inhibition of HERC2 expression increased protein levels of USP20 and CLASPIN. 293T cells were transfected with siCTR or HERC2-specific siRNA (si1-HERC2 and si2-HERC2). Total cell lysates were harvested 2 days later and subjected to immunoblotting with antibodies as indicated. (D) Inhibition of HERC2 expression decreased the ubiquitination levels of FLAG-USP20. HERC2-depleted 293T cells were co-transfected with expression constructs of FLAG-USP20 and HA-UB. Total cell lysates were harvested 2 days later and subjected to immunoprecipitation and immunoblotting with antibodies as indicated. (E) Depletion of HERC2 promotes HU-induced CHK1 activation. HERC2-depleted 293T cells were treated with HU at different time points as indicated. Total cell lysates were harvested and subjected to immunoblotting with antibodies as indicated. (F) Inhibition of HERC2 expression prolonged the half-life of USP20 and CLASPIN. Mocked- or HERC2-depleted 293T cells were treated with CHX at different time points as indicated, total cell lysates were harvested and subjected to immunoblotting with antibodies as indicated.

Fig 4: Effect of suppression of HERC2 E3 ligase activity on RPA2 Ser33 phosphorylation and ubiquitination. (a) Wild type and HERC2ΔE3/ΔE3 HCT116 cells were treated with 0.2 mM HU for the indicated time and subjected to immunoblotting with the indicated antibodies. HERC2 (N): the antibody to an epitope 1781–1974 of HERC2. (b) HCT116-HERC2ΔE3/ΔE3 cells were incubated with or without 10 μM ATR inhibitor VE821, 10 μM ATM inhibitor Ku55933, or 10 μM DNA-PK inhibitor Nu7026 for 2 h as indicated and subjected to immunoblotting with the indicated antibodies. (c) Wild type and HERC2ΔE3/ΔE3 HCT116 cells were treated or not with MG132 and 5 μM ATR inhibitor for 12 h as indicated and subjected to immunoprecipitation in denature condition with control IgG or anti-RPA2 antibody followed by immunoblotting (left panels) or to direct immunoblotting (right panels).

Fig 5: Epistatic relationship of RPA1 and HERC2 or its E3 activity on G4 formation. (a,b) HeLa-shHERC2 cells were transfected with the indicated siRNAs, induced or not with Dox and subjected to immunoblotting with the indicated antibodies (a) or immunostained for G4 (b). (c) Quantification of the mechanically counted G4 foci per cell is shown. Error bars, SEM of duplicate experiments, each based on more than 100 cells. (d,e) Wild-type or HERC2ΔE3/ΔE3 HCT116 cells were transfected with the indicated siRNAs and subjected to immunoblotting with the indicated antibodies (d) or immunostained for G4 (e). (f) Quantification of the G4 foci was performed as in (c).