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

Significance A large number of nucleotides is continuously damaged in every cell. Damaged nucleotides stall replicative DNA polymerases on template strands. Resulting replication blockage is released by two alternative pathways, error-free template switching (TS) and error-prone translesion DNA synthesis (TLS). TLS plays a major role in converting DNA damage to mutations, and thus, the relative usage of TLS over TS determines the frequency of mutagenesis. The relative usage is controlled by post-translational modification of proliferating cell nuclear antigen (PCNA), which physically interacts with DNA synthesis enzymes, in Saccharomyces cerevisiae. Environmental DNA damage induces ubiquitylation of PCNA and promotes TLS. We here show that SUMOylation of PCNA ensures the release of replication blockage by error-free TS pathways and prevents mutagenesis during the physiological cell cycle. 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 PCNAK164R/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 PCNAK164R/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.

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