Characterization of human Spartan/C1orf124, an ubiquitin-PCNA interacting regulator of DNA damage tolerance

Abstract

Unrepaired DNA damage may arrest ongoing replication forks, potentially resulting in fork collapse, increased mutagenesis and genomic instability. Replication through DNA lesions depends on mono- and polyubiquitylation of proliferating cell nuclear antigen (PCNA), which enable translesion synthesis (TLS) and template switching, respectively. A proper replication fork rescue is ensured by the dynamic ubiquitylation and deubiquitylation of PCNA; however, as yet, little is known about its regulation. Here, we show that human Spartan/C1orf124 protein provides a higher cellular level of ubiquitylated-PCNA by which it regulates the choice of DNA damage tolerance pathways. We find that Spartan is recruited to sites of replication stress, a process that depends on its PCNA- and ubiquitin-interacting domains and the RAD18 PCNA ubiquitin ligase. Preferential association of Spartan with ubiquitin-modified PCNA protects against PCNA deubiquitylation by ubiquitin-specific protease 1 and facilitates the access of a TLS polymerase to the replication fork. In concert, depletion of Spartan leads to increased sensitivity to DNA damaging agents and causes elevated levels of sister chromatid exchanges. We propose that Spartan promotes genomic stability by regulating the choice of rescue of stalled replication fork, whose mechanism includes its interaction with ubiquitin-conjugated PCNA and protection against PCNA deubiquitylation.

Figure 1.

Domain structure and localization of Spartan to stalled replication forks. (A) Schematic representation of the domain architecture of human Spartan. Conserved domains are indicated: SprT, putative metalloprotease domain, PIP, PCNA interacting peptide motif; UBZ, ubiquitin-binding zinc-finger domain. In the multiple alignment of the PIP, UBZ and SprT domains of selected proteins, the identical residues are shaded in black, and similar residues are indicated in gray. Asterisks indicate residues mutated in subsequent experiments; the names of the generated point mutant Spartan proteins are indicated. (B) Recruitment of Spartan to local DNA damage sites. HCT116 cells transiently expressing GFP-Spartan or the GFP-Spartan UBZ mutant were subjected to laser microirradiation, and 20 min later, immunostaining was performed on them using anti-GFP (green) and anti-γ H2AX (red) antibodies, and the nuclei were stained with DAPI. (C) Localization of Spartan to sites of DNA replication. The sites of DNA synthesis of HeLa cells transiently expressing FLAG-Spartan were pulse labeled with BrdU and processed for indirect immunoflourescence with antibodies against FLAG and BrdU (upper panel). Similarly, HeLa cells transfected with GFP-Spartan expressing plasmids were processed for imaging of GFP-Spartan and PCNA (lower panel). (D) Localization of Spartan to DNA damage sites. Cells of an isolated HeLa cell line stably expressing a low concentration of FLAG-Spartan were mock treated or irradiated with 20 J/m² UV light. After the treatment, cells were cultivated for 3 h and immunostained with antibodies against FLAG and PCNA.

Figure 2.

Requirement of the UBZ and PIP domains of Spartan and Rad18 for Spartan localization to replication forks. (A) The UBZ and PIP domains of Spartan are essential for foci formation and colocalization of Spartan with PCNA. HeLa cells transiently transfected with plasmids expressing WT, UBZ, PIP, PIP-UBZ or SprT domain mutant FLAG-Spartan proteins were processed for indirect immunofluorescence with antibodies against FLAG and PCNA. (B) The localization of Spartan depends on Rad18. Knock out HCT116 RAD18-/- and RAD18+/+ cells were transfected with plasmids expressing GFP-Spartan of which localization was compared in the two cell lines (upper panel). Localization of GFP-Spartan in RAD18-/- cells and DsRed-Rad18 expressing RAD18-/- cells (lower panel) was also compared. The percentages of GFP-Spartan expressing cells that display more than five Spartan foci were determined from three independent experiments and standard deviation was also calculated (right panel). (C) Spartan associates with mono- and polyubiquitin PCNA in vivo. HEK 293 cells were transfected with various combinations of control or USP1 shRNAs, HA-PCNA and FLAG-Spartan. Cell extracts were subjected to immunoprecipitation with anti-FLAG antibody, and the coimmunoprecipitated unmodified-, mono- and polyubiquitinated PCNAs were detected by western blotting using anti-HA antibody.

Figure 3.

Purified Spartan preferentially binds to ubiquitin-conjugated PCNA. (A) The UBZ domain of Spartan mediates ubiquitin binding. GST-ubiquitin-bound glutathione-sepharose beads were incubated with purified WT, UBZ, PIP or PIP-UBZ mutant FLAG-Spartan in an 85 mM NaCl containing buffer. After elution, samples were analyzed for direct physical interaction of Spartan and ubiquitin with anti-FLAG and anti-GST antibodies after western blotting. (B) The PIP domain of Spartan mediates PCNA binding. Purified WT, UBZ, PIP or PIP-UBZ mutant GST-FLAG-Spartan samples were immobilized on glutathione-sepharose and incubated with PCNA in an 85 mM NaCl containing buffer. Eluted samples were analyzed for complexes of Spartan and PCNA by western blotting with anti-PCNA and anti-FLAG antibodies. (C) Requirement of the PIP- and UBZ domain of Spartan for monoubiquitin-PCNA binding. As described in (B) but bead-bound Spartan was incubated with purified monoubiquitin-PCNA instead of unmodified PCNA. Both the incubation and the washing steps were carried out at 85 mM (left panel) and at 150 mM NaCl concentrations (right panels) as indicated. (D) Requirement of the PIP- and UBZ domain of Spartan for polyubiquitin-PCNA binding. As described in (C) but bead-bound Spartan was incubated with polyubiquitin-PCNA. (E) Preferential association of Spartan with ubiquitin-conjugated PCNA. As described in (B) but bead-bound WT and mutant Spartan proteins were incubated with a mixture of equal amount of unmodified PCNA, monoubiquitin-PCNA and polyubiquitin-PCNA.

Figure 4.

Role of Spartan in DNA damage tolerance. (A) Spartan depletion sensitizes human cells to UV-irradiation. HeLa cells treated with control or three different siRNAs were assayed for survival after UV treatment by cell competition assay using a reference GFP+ HeLa cell line. The results of three independent experiments for each sample with standard deviation are graphed. Efficiency of siRNA depletion of Spartan was tested with anti-FLAG antibody after 48 h of cotransfecting the FLAG-Spartan plasmid and three different types of Spartan siRNAs. (B) UBZ and PIP domains of Spartan are all required for damage resistance. Complementation of the UV sensitivity of stable Spartan depleted HeLa cells by shRNA resistant form of WT, UBZ, PIP and PIP-UBZ domain mutant FLAG-Spartan (upper panel). The efficiency of stable shRNA Spartan depletion was assayed as in (A), and shRNA-resistant WT or point mutant FLAG-Spartan expressions were confirmed with anti-FLAG antibody (lower panels). (C) The silencing of Spartan causes increased SCE. The control and Spartan depleted stable HeLa cells were compared in spontaneous SCE analysis. (D) Quantification of the SCE. SCE was compared in the following three different stable HeLa cells: 1, expressing control shRNA; 2, Spartan depleted: 3, Spartan depleted but expressing shRNA-resistant FLAG-Spartan. SCE per chromosome values were determined by counting one hundred cells per sample. The results of three independent experiments with standard deviations are graphed. (E) Spartan facilitates Polη foci formation. FLAG-Polη expressing stable HeLa cells were transfected with GFP or GFP-Spartan expressing plasmids, and after 24 h cultivation, mock or UV (20 J/m²) treated. After extracting cells with NP40, a non-ionic detergent, localization of Polη was visualized by immunostaining using anti-FLAG antibody. The expression level of GFP-Spartan was confirmed by anti-GFP antibody after western blotting of cell extracts (left panel). (F) Stimulation of Polη foci formation depends on the UBZ and PIP domains of Spartan. FLAG-Polη expressing stable HeLa cells was transfected with WT, UBZ, PIP and PIP-UBZ GFP-Spartan expressing constructs followed by quantitation for Polη foci forming cell when compared with the green transfected cells. The cells with more than five GFP-Spartan foci were counted as foci positive. (G) Competition of Spartan and USP1 on Polη foci formation. USP1, or Spartan together with USP1, or Spartan was expressed in HeLa cells stably expressing FLAG-Polη, and the percentage of Polη foci forming cells was quantitated. The results of three independent experiments with standard deviations are graphed.

Figure 5.

Inhibition of the USP1-dependent deubiquitylation of ubiquitylated PCNA by Spartan. (A) USP1 knockdown reverses the reduction of PCNA monoubiquitination caused by Spartan knockdown. Spartan, USP1 and they together were knockdown along with transient expression of FLAG-PCNA in HEK 293 cells. After 24 h of transfection, cells were irradiated with 20 J/m² UV, and in 3 h, cell extracts were subjected to immunoprecipitation with anti-FLAG antibody. Monoubiquitylation of endogenous and FLAG-PCNA was detected by western blotting using anti-PCNA antibody. (B) Spartan overexpression reverses the reduction of PCNA monoubiquitylation caused by USP1 overexpression. HEK 293 cells were transfected with FLAG-PCNA and USP1 expression constructs together with mock or Spartan expression constructs. Cell extracts were prepared and analyzed as described in (A). (C) Spartan inhibits USP1-UAF1-dependent in vitro deubiquitylation of monoubiquitin-PCNA. Increasing amounts of purified USP1-UAF1 were incubated with purified monoubiquitin-PCNA in the absence (Lanes 1–4) or presence (5–8) of Spartan at 37°C for 45 min. Deubiquitylation of PCNA was analyzed on 10% denaturing polyacrilamyde gels followed by western blotting and visualization with anti-PCNA antibody. (D) Spartan inhibits USP1-UAF1-dependent in vitro deubiquitylation of polyubiquitin-PCNA. Analysis was carried out as in (A) but using purified polyubiquitin-PCNA substrate instead of monoubiquitin-PCNA. (E) The UBZ and PIP domains of Spartan are essential for inhibition of USP1-UAF1-dependent deubiquitylation of monoubiquitin-PCNA. Monoubiquitin-PCNA was incubated with USP1-UAF1 (50 nM) in the absence or presence of WT, UBZ, PIP or PIP-UBZ mutant Spartan proteins (150 nM). The reaction products were analyzed for deubiquitylation of monoubiquitin-PCNA by western-blotting using anti-PCNA antibody. (F) The UBZ and PIP domains of Spartan are essential for inhibition of USP1-UAF1-dependent deubiquitylation of polyubiquitin-PCNA. Analysis was carried out as in (C) but using purified polyubiquitin-PCNA substrate instead of monoubiquitin-PCNA.

Figure 6.

Model for the role of Spartan in DNA damage tolerance. We suggest that at the stalled replication fork, the Rad6-Rad18-dependent PCNA ubiquitylation and the USP1-dependent PCNA deubiquitylation are dynamic processes, of which balance determines the life-time of ubiquitin-PCNA and the choice of fork rescue mechanism. In the absence of ubiquitin-PCNA, replication fork can be rescued by recombination-dependent mechanisms, which, however, have a potential for DNA rearrangements. In the presence of ubiquitin-conjugated-PCNA, damage bypass or template switching can provide replication through the lesion without the formation of a DSB intermediate. Spartan can provide one regulatory level by binding to ubiquitin-modified PCNA, which protects against PCNA deubiquitylation by USP1. Thus, Spartan can channel the reviving of stalled replication from a recombination-dependent pathway that does not require PCNA ubiquitylation to PCNA ubiquitylation-dependent translesion synthesis or PCNA polyubiquitylation-dependent template switching pathways.