Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope, Duarte, CA 91010-3000.
Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope, Duarte, CA 91010-3000
Proc Natl Acad Sci U S A. 2020 Sep 22;117(38):23588-23596. doi: 10.1073/pnas.2004122117. Epub 2020 Sep 8.
In human cells, the DNA replication factor proliferating cell nuclear antigen (PCNA) can be conjugated to either the small ubiquitinlike modifier SUMO1 or SUMO2, but only SUMO2-conjugated PCNA is induced by transcription to facilitate resolution of transcription-replication conflict (TRC). To date, the SUMO E3 ligase that provides substrate specificity for SUMO2-PCNA conjugation in response to TRC remains unknown. Using a proteomic approach, we identified TRIM28 as the E3 ligase that catalyzes SUMO2-PCNA conjugation. In vitro, TRIM28, together with the RNA polymerase II (RNAPII)-interacting protein RECQ5, promotes SUMO2-PCNA conjugation but inhibits SUMO1-PCNA formation. This activity requires a PCNA-interacting protein (PIP) motif located within the bromodomain of TRIM28. In cells, TRIM28 interaction with PCNA on human chromatin is dependent on both transcription and RECQ5, and SUMO2-PCNA level correlates with TRIM28 expression. As a consequence, TRIM28 depletion led to RNAPII accumulation at TRC sites, and expression of a TRIM28 PIP mutant failed to suppress TRC-induced DNA breaks.
在人类细胞中，DNA 复制因子增殖细胞核抗原（PCNA）可以与小分子泛素样修饰物 SUMO1 或 SUMO2 结合，但只有 SUMO2 结合的 PCNA 会被转录诱导，以促进转录-复制冲突（TRC）的解决。迄今为止，提供针对 TRC 的 SUMO2-PCNA 缀合的底物特异性的 SUMO E3 连接酶仍然未知。我们使用蛋白质组学方法鉴定了 TRIM28 是催化 SUMO2-PCNA 缀合的 E3 连接酶。在体外，TRIM28 与 RNA 聚合酶 II（RNAPII）相互作用蛋白 RECQ5 一起促进 SUMO2-PCNA 缀合，但抑制 SUMO1-PCNA 形成。这种活性需要位于 TRIM28 溴结构域内的 PCNA 相互作用蛋白（PIP）基序。在细胞中，TRIM28 与人类染色质上的 PCNA 的相互作用既依赖于转录又依赖于 RECQ5，并且 SUMO2-PCNA 水平与 TRIM28 表达相关。因此，TRIM28 的耗竭导致 RNAPII 在 TRC 位点的积累，并且表达 TRIM28 PIP 突变体未能抑制 TRC 诱导的 DNA 断裂。
