SUMOylation of PCNA by PIAS1 and PIAS4 promotes template switch in the chicken and human B cell lines
SUMOylation of PCNA by PIAS1 and PIAS4 promotes template switch in the chicken and human B cell lines
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DOI：
10.1073/pnas.1716349115
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发表时间：
2018-11
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影响因子：
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通讯作者：
Mohiuddin Mohiuddin-Mohiuddin;T. Evans;Md. Maminur Rahman;Islam Shamima Keka;M. Tsuda;H. Sasanuma;S. Takeda
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文献类型：
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作者：
Mohiuddin Mohiuddin-Mohiuddin;T. Evans;Md. Maminur Rahman;Islam Shamima Keka;M. Tsuda;H. Sasanuma;S. Takeda
意义在每个细胞中都有大量的核苷酸不断被破坏。受损的核苷酸阻碍了模板链上的复制DNA聚合酶。由此产生的复制阻塞通过两种替代途径释放，即无错误模板切换（TS）和易出错翻译DNA合成（TLS）。TLS在将DNA损伤转化为突变中起着重要作用，因此，TLS在TS上的相对使用决定了突变的频率。在酿酒酵母中，增殖细胞核抗原（PCNA）的翻译后修饰控制了其相对使用，PCNA与DNA合成酶物理相互作用。环境DNA损伤诱导PCNA泛素化并促进TLS。我们在这里表明，PCNA的SUMOylation确保通过无错误的TS途径释放复制阻塞，并防止生理细胞周期中的突变。DNA损伤耐受性（DDT）通过两种途径释放由模板链上受损核苷酸引起的复制阻塞，即易出错翻译DNA合成（TLS）和无错误模板切换（TS）。增殖细胞核抗原（PCNA）的Lys164在细胞的生理周期中被sumolated。为了探索PCNA在DDT中summoylation的作用，我们对鸡DT40和人TK6 B细胞中PIAS1和PIAS4小泛素样修饰物（SUMO） E3连接酶的缺失进行了表征。DT40细胞在DDT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）]不断使其免疫球蛋白（Ig）可变基因多样化，都缓解了基本位点的复制阻滞，而不伴有DNA断裂。值得注意的是，PIAS1 - / - /PIAS4 - / -细胞显示PCNA在Lys164的sumo化水平降低了数倍，TS率降低了90%以上。同样，PIAS1 - / - /PIAS4 - / - TK6细胞在插入基因组DNA的化学合成UV损伤处显示DDT从TS向TLS转移。PCNAK164R/K164R突变导致Ig GC率降低约90%，对PIAS1−/−/PIAS4−/−细胞没有额外的影响。PCNAK164R/K164R与PIAS1−/−/PIAS4−/−突变之间的上位性关系表明，PIAS1和PIAS4主要通过在Lys164位点的PCNA summoylation促进TS。在PIAS1−/−/PIAS4−/−细胞中，过表达pna - sumo1嵌合蛋白可恢复TS缺陷，这一数据进一步支持了这一观点。综上所述，PIAS1和PIAS4促进了PCNA Lys164位点的summoylation，确保了后生动物细胞在生理DNA复制过程中无错误地释放复制阻塞。
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.