Spartan/C1orf124 is important to prevent UV-induced mutagenesis

Abstract

Uninterrupted replication across damaged DNA is critical to prevent replication fork collapse and resulting double-strand DNA breaks. Rad18-mediated PCNA ubiquitination is a crucial event that triggers a number of downstream pathways important for lesion bypass. Here, we report characterization of Spartan, an evolutionarily conserved protein containing a PCNA-interacting peptide motif, called a PIP box, and a UBZ4 ubiquitin-binding domain. Spartan is a nuclear protein and forms DNA damage-induced foci that colocalize with markers for stalled DNA replication. Focus formation of Spartan requires its PIP-box and the UBZ4 domain and is dependent on Rad18 and the PCNA ubiquitination site, indicating that Spartan is recruited to ubiquitinated PCNA. Spartan depletion results in increased mutagenesis during replication of UV-damaged DNA. Taken together, our data suggest that Spartan is recruited to sites of stalled replication via ubiquitinated PCNA and plays an important role to prevent mutations associated with replication of damaged DNA.

Figure 1. Spartan interacts with ubiquitin and PCNA. (A) Domain structure of Spartan and conservation across species. Putative homologs containing both SprT and UBZ4 domains are shown. (B) Multiple alignment of the UBZ4 domains from Spartan homologs in selected species. Zinc-coordinating residues are highlighted with asterisks. The conserved aspartate residue mutated in this study is indicated by an arrow. (C) Multiple alignment of the PIP boxes from Spartan homologs in selected species. Conserved aromatic residues that were mutated to alanines in this study are indicated by arrowheads. (D) Interaction of the Spartan UBZ4 domain with ubiquitin. In vitro GST pull-down assays were performed using indicated recombinant proteins produced in E. coli. MBP-Ub was examined by western blotting against MBP, and GST-fusion proteins in the precipitates were visualized by Coomassie staining. (E) Interaction of full-length Spartan with ubiquitin. In vitro GST pull-down assays were performed as in (D), except that GST fusion proteins were mixed with 293T cell lysates expressing indicated Spartan proteins. Spartan proteins were examined by western blotting against HA tag, and GST-fusion proteins in the precipitates were visualized by Coomassie staining. (F) Interaction of the Spartan C-terminal fragment with PCNA. In vitro GST pull-down assays were performed using the indicated recombinant proteins produced in E. coli. PCNA was examined by western blotting, and GST-fusion proteins in the precipitates were visualized by Coomassie staining.

Figure 2. Spartan localizes to sites of stalled replication. (A) Focus formation of EGFP-Spartan in response to DNA damage or replication stress. U2OS cells stably expressing EGFP-Spartan were treated as indicated. Cells were fixed 3 h after UV (20 J/m²) or 6 h after HU (10 mM) treatment. Where indicated, TX-100 extraction was performed to visualize EGFP-Spartan on chromain. (B) Quantitation of cells containing EGFP-Spartan Foci. Cells were treated as in (A), and percentage of cells containing more than ten EGFP-Spartan foci is presented as mean ± SD (n = 3). (C) Colocalization of EGFP-Spartan with ssDNA. Flag-Spartan was transiently expressed in U2OS cells prelabeled with BrdU for two doubling times. Cells were treated with 10 mM HU for 6 h and coimmunostained with anti-Flag and anti-BrdU in non-denaturing conditions. (D) Colocalization of EGFP-Spartan with RPA. U2OS cells stably expressing EGFP-Spartan were fixed 3 h after UV (20 J/m²) or 6 h after HU (10 mM) treatment and immunostained for RPA. (E) Colocalization of EGFP-Spartan with PCNA. U2OS cells stably expressing EGFP-Spartan were treated with 10 mM HU for 6 h and immunostained for PCNA.

Figure 3. Focus formation of EGFP-Spartan is dependent on Rad18 and PCNA ubiquitination. (A) Focus formation of EGFP-Spartan in response to DNA damage. EGFP-Spartan was stably expressed in HCT116 cells (Rad18 ^+/+ and ^−/−) and visualized 3 h after UV (20 J/m²) or 6 h after HU (10 mM) treatment. (B) Quantitation of cells containing EGFP-Spartan foci. Cells were treated as in (A), and percentage of cells containing more than ten EGFP-Spartan foci is presented as mean ± SD (n = 3). (C) Focus formation of EGFP-Spartan in response to UV. EGFP-Spartan was stably expressed in HCT116 cells expressing wild-type or the K164R mutant PCNA and visualized 3 h after UV (20 J/m²) treatment. See also Figure S2. (D) Quantitation of cells containing EGFP-Spartan Foci. Cells were treated as in (C), and percentage of cells containing more than ten EGFP-Spartan foci is presented as mean ± SD (n = 3).

Figure 4. Focus formation of EGFP-Spartan is dependent on its PIP box and UBZ4 domain. (A) Focus formation of EGFP-Spartan in response to DNA damage. Wild-type or indicated mutants of Spartan were stably expressed as a fusion protein with EGFP in U2OS. Localization of EGFP-Spartan was visualized 3 h after UV (20 J/m²) or 6 h after HU (10 mM) treatment. (B) Quantitation of cells containing EGFP-Spartan Foci. Cells were treated as in (A), and percentage of cells containing more than ten EGFP-Spartan foci is presented as mean ± SD (n = 3).

Figure 5. Spartan depletion increases UV-induced mutagenesis. (A) Knock down of Spartan by RNAi. 293T cells were transfected with the indicated siRNA oligos and Spartan levels were examined by western blotting. β-actin is shown as a loading control. (B) Increased mutagenesis in Spartan-depleted cells. UV-induced mutagenesis was measured using the SupF shuttle vector system in 293T. Cells were transfected with indicated siRNA oligos and then transfected with SupF plasmids after 24 h. Forty-eight hours later, plasmids were recovered and assayed for mutations in the SupF gene. Mutation frequencies are presented as percentage of mutant SupF genes. Experiments were performed in triplicate and results are shown as mean ± SD. Mutations were not detected in undamaged plasmids. (*, p < 0.0005, two-tailed Student’s t-test.)