Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, 606-8501 Kyoto, Japan.
Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, 606-8501 Kyoto, Japan
Proc Natl Acad Sci U S A. 2018 Dec 11;115(50):12793-12798. doi: 10.1073/pnas.1716349115. Epub 2018 Nov 28.
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, cells displayed a multifold decrease in SUMOylation of PCNA at Lys164 and over a 90% decrease in the rate of TS. Likewise, TK6 cells showed a shift of DDT from TS to TLS at a chemosynthetic UV lesion inserted into the genomic DNA. The mutation caused a ∼90% decrease in the rate of Ig GC and no additional impact on cells. This epistatic relationship between the and the 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 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.
DNA 损伤容忍 (DDT) 利用两种替代途径，易错跨损伤 DNA 合成 (TLS) 和无差错模板转换 (TS)，释放模板链上受损核苷酸引起的复制阻滞。增殖细胞核抗原 (PCNA) 的赖氨酸 164 在生理细胞周期中被 SUMO 化。为了探索 PCNA SUMO 化在 DDT 中的作用，我们对缺乏 PIAS1 和 PIAS4 小泛素样修饰物 (SUMO) E3 连接酶的鸡 DT40 和人 TK6 B 细胞进行了特征分析。DT40 细胞在 DDT 的表型分析中具有独特的优势，因为它们通过 TLS 和 TS [Ig 基因转换 (GC)] 不断多样化其免疫球蛋白 (Ig) 可变基因，这两种方法都能在不伴随 DNA 断裂的情况下缓解碱基缺失处的复制阻滞。值得注意的是，细胞中 PCNA 赖氨酸 164 的 SUMO 化减少了近 90%，而 TS 的速率则降低了 90%以上。同样，TK6 细胞在基因组 DNA 中插入化学合成的 UV 损伤时，将 DDT 从 TS 转移到 TLS。突变导致 Ig GC 的速率降低了约 90%，但对 细胞没有额外影响。和 突变之间的这种上位性关系表明，PIAS1 和 PIAS4 主要通过 PCNA 赖氨酸 164 的 SUMO 化促进 TS。这一观点进一步得到了以下数据的支持：过表达 PCNA-SUMO1 嵌合蛋白可恢复 细胞中 TS 的缺陷。总之，PIAS1 和 PIAS4 促进的 PCNA 赖氨酸 164 的 SUMO 化确保了真核细胞生理 DNA 复制过程中复制阻滞的无错误释放。
