RAD18-mediated ubiquitination of PCNA activates the Fanconi anemia DNA repair network

Abstract

The Fanconi anemia (FA) network is important for the repair of interstrand DNA cross-links. A key event in FA pathway activation is the monoubiquitylation of the FA complementation group I (FANCI)-FANCD2 (ID) complex by FA complementation group L (FANCL), an E3 ubiquitin ligase. In this study, we show that RAD18, another DNA damage-activated E3 ubiquitin ligase, also participates in ID complex activation by ubiquitylating proliferating cell nuclear antigen (PCNA) on Lys164, an event required for the recruitment of FANCL to chromatin. We also found that monoubiquitylated PCNA stimulates FANCL-catalyzed FANCD2 and FANCI monoubiquitylation. Collectively, these experiments identify RAD18-mediated PCNA monoubiquitination as a central hub for the mobilization of the FA pathway by promoting FANCL-mediated FANCD2 monoubiquitylation.

Figure 1.

RAD18 is required for cisplatin-induced FANCD2 monoubiquitination and recruitment to chromatin. (A) HeLa cells were transfected with siRNAs that target Luciferase (Luc), RAD18-1 (R18), FANCD2 (FA-D2), or both. 2 d later, trypsinized cells were analyzed by immunoblotting (inset; note that monoubiquitylated FANCD2 is not resolved under these gel conditions) and replated, treated with cisplatin for 24 h, washed, and cultured for 8 d to allow colony formation. A representative experiment that has been repeated three times is shown (n = 3 ± SD; see Fig. S1 A for additional replicates). (B) K562 cells were transfected with siRNAs. 2 d later, the cells were treated with 30 µM cisplatin for 6 h (+), and lysates were immunoblotted. Monoubiquitylated FANCD2 (FANCD2^mUb) was detected by slower migration in SDS-PAGE. (C) RAD18^+/+ HCT-116 and RAD18^−/− HCT-116 cells were treated with 30 µM cisplatin for 6 h and analyzed as in B. (D) K562 cells were cotransfected with siRNAs and plasmids that encode HA-tagged ubiquitin or empty vector (EV). 2 d after transfection, the cells were treated as in B. The lysate was immunoprecipitated (IP) with anti-FANCD2 antibody. The immunoprecipitates and starting lysates were sequentially immunoblotted to detect HA-ubiquitin covalently linked to FANCD2 (anti-HA mAb) and FANCD2. (E) K562 cells were transfected with siRNA, cultured, and treated with cisplatin as in B. Cells were separated into chromatin-bound and soluble fractions, which were analyzed by immunoblotting. Western blot markers are given in kilodaltons.

Figure 2.

RAD18 participates in cisplatin-induced FANCD2 monoubiquitylation through the ubiquitylation of PCNA on Lys164. (A) K562 cells were transfected with siRNAs, cultured for 2 d, and treated with 30 µM cisplatin (6 h). Lysates were immunoblotted as indicated. *, nonspecific band. (B) HeLa cells were transfected with siRNAs. 2 d later, the trypsinized cells were analyzed by immunoblotting (Fig. S2 A), and the remaining cells were replated, treated with cisplatin for 24 h, washed, and cultured for 8 d to allow colony formation. A representative experiment of three independent experiments is shown (n = 3 ± SD). (C) K562 cells were cotransfected twice with siRNAs and plasmids that encode siRNA-resistant wild-type PCNA (PCNA^WT) or PCNA^K164R. 2 d after the second transfection, trypsinized cells were analyzed by immunoblotting and for cell cycle (Fig. S2 B). Short and long exposures of the PCNA immunoblot are shown to demonstrate PCNA loading and PCNA monoubiquitylation (PCNA^mUb), respectively. Western blot markers are given in kilodaltons.

Figure 3.

FANCL binds PCNA via the FANCL DRWD domain. (A) Cell lysates from K562 cells transiently expressing HA-tagged FANCL were immunoprecipitated with nonimmune (IgG) or anti-PCNA rabbit antisera. The precipitates were immunoblotted to detect FANCL (anti-HA) and PCNA. (B) Cell lysates from K562 cells transfected with empty vector (EV) or transiently expressing SFB-tagged FANCL were precipitated with S protein agarose. Precipitates were immunoblotted to detect PCNA and FANCL (anti–S peptide mAb, which recognizes the SFB tag). *, nonspecific band. (C) SFB-tagged versions of the following domains of FANCL were transiently expressed in K562 cells: ELF (amino acids 1–103), DRWD (amino acids 104–293), N terminus (amino acids 1–306), and RING (amino acids 307–375). Cell lysates were immunoprecipitated with nonimmune or rabbit anti-PCNA antiserum, and the precipitates were immunoblotted to detect the SFB tag (anti–S peptide mAb). (D and E) GST and GST-PCNA purified from E. coli were immobilized on GSH agarose, which was incubated with lysates from K562 cells transiently expressing SFB-FANCL (D) or S-tagged FANCL produced by in vitro translation (E). The washed beads were immunoblotted to detect FANCL (anti–S peptide mAb), and the membranes were stained with Coomassie blue (CB) to show GST and GST-PCNA loading. (F) GST and GST-FANCL (amino acids 1–306) purified from E. coli were immobilized on GSH agarose. Washed beads were incubated with E. coli–produced His₆-PCNA. The precipitates were immunoblotted to detect PCNA (anti-PCNA), and the membrane was stained with Coomassie blue to show GST and GST-FANCL (amino acids 1–306) loading. IP, immunoprecipitation. Western blot markers are given in kilodaltons.

Figure 4.

Monoubiquitinated PCNA promotes FANCL chromatin binding and stimulates FANCL-mediated FANCD2 and FANCI monoubiquitylation. (A and B) K562 cells were cotransfected with siRNAs and plasmids, cultured for 2 d, and treated with 30 µM cisplatin for 6 h. Chromatin-bound and soluble fractions were isolated and sequentially immunoblotted to detect FANCL (anti-HA mAb) or the loading controls ORC2 (chromatin-bound fraction) or actin (soluble fraction). (C) E. coli–produced GST-PCNA (wild type) and GST-PNCA^K164R were incubated with ubiquitin-activating enzyme E1, RAD18–RAD6 complex, and HA-ubiquitin (HA-Ub). PCNA preparations (I–III) were repurified by GSH agarose chromatography and analyzed by SDS-PAGE (Coomassie Blue stain). (D and E) The repurified PCNA preparations from C were analyzed for effects on FANCL activity. SFB-FANCL alone, SFB-FANCI alone, and the combination (D) or SFB-FANCL alone, SFB-FANCD2, and the combination (E) were copurified from K562 cells using S protein agarose. After extensive washing, the SFB-tagged proteins were eluted with S peptide and mixed with the indicated E. coli–produced proteins and PCNA preparations (II and III) from C under conditions that support in vitro ubiquitylation of FANCI and FANCD2. The reactions were analyzed by sequentially immunoblotting for ubiquitin (anti-HA mAb), FANCL, FANCD2, and FANCI (anti–S peptide mAb). *, nonspecific band. (F) Model of PCNA acting as a central hub in the activation of the FA pathway. Western blot markers are given in kilodaltons.