Fig 2: Rad18 facilitates polη SUMOylation by promoting polη interaction with PIAS1 SUMO ligase.(a) 293FT cells were depleted of Rad18 using either a single duplex (RAD18d) or a pool of four sequences (RAD18sp) 24 h before co-transfection of plasmids expressing polη and His-SUMO3. Polη SUMOylation was analysed as in Fig. 2. (b) Schematic representation of the point mutations of Rad18 used in the study. RING, really interesting new gene domain conferring ubiquitin ligase activity (mutated in Rad18C28F); UBZ, ubiquitin-binding zinc finger (mutated in Rad18C207F); SAP, SAF-A/B, Acinus and PIAS domain (mutated in Rad18SAP* G269A,K271A); Rad6 BD, E2-conjugating enzyme Rad6-binding domain; polη BD: polη-binding domain; NLS, nuclear localization signal. (c,d) SUMOylation of polη was assessed after overexpression of WT Rad18 or the mutants depicted in b. Red line: SUMOylation on K163, black lines: K163-independent SUMOylation events. (e) 293FT cells were transfected with pcDNA-POLH, His-SUMO3 and various truncation mutants of GFP-RAD18 (upper panel). As Rad181-460 and Rad181-409 lack the C-terminal NLS, the NLS of the T antigen of SV40 (T Ag) was added to the N-terminus of the protein (Rad18nls1-460 and Rad18nls1-409). (f) The impact of these truncation mutants on polη SUMOylation was compared with the one of Rad18WT. (g) 293FT cells were co-transfected with HA-RAD18 and Flag-PIAS1 plasmids. Co-immunoprecipitations were performed using anti-HA or anti-Flag antibodies. (h) Plasmids expressing polη and Flag-PIAS1 were co-transfected in mock- or Rad18-depleted 293FT cells. The interaction between polη and PIAS1 was analysed by immunoprecipitation using an anti-Flag antibody.

Fig 3: Effects of repeat DMBA treatment on survival and malignancy in Rad18+/+ and Rad18−/− mice. (A) Kaplan–Meier plots showing survival rates of Rad18+/+ and Rad18−/− mice after repeat treatment with DMBA. (B) Representative DMBA-induced tumors in Rad18+/+ and Rad18−/− mice: (a) Wright–Geimsa stained cytospin preparation of a normal bone marrow flush from a Rad18+/+ mouse (WT7) (original objective magnification 60x) showing maturation of the expected hematopoietic elements including granulocytes, erythroids and megakaryocytes. (b) Wright–Geimsa stained cytospin preparation of a bone marrow flush from Rad18−/− mouse (KO9) mice. The expected heterogeneity seen in normal bone marrow (evident in panel (a) is absent. The marrow cells are replaced by a monotonous proliferation of discohesive, large, abnormal lymphoid cells characterized by irregular nuclear contours and scant basophilic cytoplasm. (c) Hematoxylin and eosin (H&E) stained sections of liver showing dense infiltrates of large abnormal lymphoid cells (original objective magnification 20x). (d–g) B cell and T cell tumors are morphologically indistinguishable. However, immunohistochemical staining identifies B cell tumors (B220-positive/CD3-negative, shown in panels (d) and (e)) and T cell tumors (CD3-positive/B220-negative, shown in panels (f) and (g)) (brown chromogen is considered positive with blue counterstain for contrast. original objective magnification 40x). (h) Skin lesions from a Rad18−/− mouse demonstrating classic features of basal cell carcinoma with nodular collections of hyperchromatic basaloid cells extending from the epidermal surface as indicated by the arrows (original objective magnification 20x).

Fig 4: Hypothetical Role of Rad18 in PAH-induced mutagenesis. Leading strand DNA synthesis (dashed arrow) stalls at sites of PAH-induced DNA damage such as bulky adducts and AP sites (indicated by the red explosion). In Rad18+/+ cells, the Y-family TLS DNA polymerases (Polη, Polκ, Polι, REV1) are recruited to stalled DNA replication forks and promote error-prone replicative bypass of DNA lesions, resulting in mutagenesis (SNV). In Rad18−/− cells, TLS polymerases are not efficiently recruited to sites of PAH-induced DNA damage leading to persistent replication fork stalling and eventually to fork collapse. The DBSs resulting from collapsed replication forks are re-ligated via error-prone mechanisms such as TMEJ leading to insertions and deletions. See ‘Discussion’ section for details.

Fig 5: RAD18 suppresses transcription and R-loop associated DNA replication stress and DNA damage.(A,B) RPA2-ser33 phosphorylation immunofluorescence in non-targeting control or siRAD18 treated HeLa cells with or without transfection of a GFP-tagged RNaseH1 overexpression plasmid. A, representative images, B, quantification. (C,D) H2AX-ser139 phosphorylation immunofluorescence in non-targeting control or siRAD18 treated HeLa cells with or without transfection of RNaseH1 overexpression plasmid. C, representative image, D, quantification. (E,F) ATM-ser1981 phosphorylation immunofluorescence in P53-/- RPE1 RAD18 knockout clone cells and WT with and without a 2 hour treatment of triptolide and pladienolide B and 24 treatment of APH. E, representative image, F, quantification. Treatment concentrations of triptolide (40 nM), Pladienolide B (5 μM) and APH (400 nM). (B,D,F) Fisher’s Exact Test, p<0.05, n = 3. Error bars represent standard error of the mean and the central line represents the mean. APH, aphidicolin; PladB, pladienolide B; siCon, non-targeting control siRNA; ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.