SUMOylation of PCNA by PIAS1 and PIAS4 promotes template switch in the chicken and human B cell lines

Abstract

DNA damage tolerance (DDT) releases replication blockage caused by damaged nucleotides on template strands employing two alternative pathways, error-prone translesion DNA synthesis (TLS) and error-free template switch (TS). Lys164 of proliferating cell nuclear antigen (PCNA) is SUMOylated during the physiological cell cycle. To explore the role for SUMOylation of PCNA in DDT, we characterized chicken DT40 and human TK6 B cells deficient in the PIAS1 and PIAS4 small ubiquitin-like modifier (SUMO) E3 ligases. DT40 cells have a unique advantage in the phenotypic analysis of DDT as they continuously diversify their immunoglobulin (Ig) variable genes by TLS and TS [Ig gene conversion (GC)], both relieving replication blocks at abasic sites without accompanying by DNA breakage. Remarkably, PIAS1^-/-/PIAS4^-/- cells displayed a multifold decrease in SUMOylation of PCNA at Lys164 and over a 90% decrease in the rate of TS. Likewise, PIAS1^-/-/PIAS4^-/- TK6 cells showed a shift of DDT from TS to TLS at a chemosynthetic UV lesion inserted into the genomic DNA. The PCNA^K164R/K164R mutation caused a ∼90% decrease in the rate of Ig GC and no additional impact on PIAS1^-/-/PIAS4^-/- cells. This epistatic relationship between the PCNA^K164R/K164R and the PIAS1^-/-/PIAS4^-/- mutations suggests that PIAS1 and PIAS4 promote TS mainly through SUMOylation of PCNA at Lys164. This idea is further supported by the data that overexpression of a PCNA-SUMO1 chimeric protein restores defects in TS in PIAS1^-/-/PIAS4^-/- cells. In conclusion, SUMOylation of PCNA at Lys164 promoted by PIAS1 and PIAS4 ensures the error-free release of replication blockage during physiological DNA replication in metazoan cells.

Keywords: PCNA; PIAS1; PIAS4; SUMOylation; template switching (TS).

Fig. 1.

SUMO E3 ligase mutants displayed a reduced number of SCE events. (A) Sensitivity of DT40 mutants to camptothecin. The viability of cells was measured at 48 h. (B and C) Sensitivity of TK6 mutants to camptothecin (B) and olaparib (C). The viability of cells was measured by colony formation in the methylcellulose. (D–G) SCE induced by 0.25 J/m² UV (D) and 10 μM cisplatin (E) in the chicken DT40 cells and 0.25 J/m² UV (F) and 2 μM cisplatin (G) in the human TK6 cells. At least 50 mitotic cells were analyzed per condition in each experiment. The histogram shows subtracted numbers of mean values for SCEs before the exposure from SCEs after the exposure. The error bars indicate SEM. Statistical analyses were performed by Student’s t test (*P < 0.01).

Fig. 2.

PIAS1^−/−/PIAS4^−/− and POLH^−/− mutations cause a synergistic increase in the sensitivity of TK6 cells to cisplatin and UV. (A and B) Clonogenic cell survival assay following exposure of indicated cell lines to UV (A) and cisplatin (B). Lethal dose 50% (LD50) is the dose of DNA-damaging agents that reduces cellular survival to 50% relative to cells nontreated with DNA-damaging agents. The error bars show the SD of the mean of, at least, three independent experiments. Statistical analyses were performed by Student’s t test (*P < 0.01).

Fig. 3.

Analysis of the Ig GC rate in DT40 B cell clones. (A) Diagram of the Ig GC assay. The frameshift mutation caused by the insertion of G in the rearranged V_λ segment in the parent cells is indicated by the red circle. GC events using an upstream ΨV donor segment can eliminate the frameshift mutation, resulting in a gain in sIgM expression. The ΨV segment is indicated by the blue box. (B) The proportion of sIgM-positive cells was determined in 36 parallel cultures derived from single sIgM-negative parental cells after clonal expansion (3 wk). Median percentages are noted below each data set. (C) Rates of Ig GC and nontemplated mutation in AID-expressing clones carrying the indicated genotypes. The y axis represents the rate of nontemplated mutations (10⁻⁵/base/div), ambiguous mutation (10⁻⁵/base/div), and gene conversion (10⁻³/sequence/div). The error bars indicate SEM. Statistical analyses were performed by Student’s t test (*P < 0.01).

Fig. 4.

SUMOylation of PCNA by PIAS1 and PIAS4 E3 ligases. DT40 cells stably transfected with His-tagged hSUMO1 were analyzed by pull-down assay using Ni-NTA magnetic beads. Whole cell lysates and lysates after pulled down using Ni-NTA magnetic beads are shown on the Left. Quantification of SUMOylated PCNA and unmodified PCNA by pull-down experiments were shown on the Right.

Fig. 5.

Ectopic expression of hPCNA-hSUMO1 reverses the mutant phenotype of PIAS1^−/−/PIAS4^−/− cells. (A) The proportion of sIgM-positive DT40 cells was determined as in Fig. 3B. The error bars indicate SEM. (B) The number of SCEs induced by 0.25 J/m² UV in the DT40 cells was determined as in Fig. 1A. The error bars indicate SEM. (C) Clonogenic cell survival assay following exposure of hPCNA-hSUMO1 expressing XPA^−/−/POLH^−/− and PIAS1^−/−PIAS4^−/−/XPA^−/−/POLH^−/− TK6 cells to UV. The x axis represents the dose of the indicated DNA-damaging agent on a linear scale; the y axis represents the survival fraction on a logarithmic scale. The error bars indicate SD. FP: Flag-hPCNA. FPS: Flag-hPCNA-hSUMO1. Statistical analyses were performed by Student’s t test (*P < 0.01).