Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health San Antonio, San Antonio, TX 78245, USA.
Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health San Antonio, San Antonio, TX 78245, USA; Sam and Ann Barshop Institute for Longevity and Aging Studies, University of Texas Health San Antonio, San Antonio, TX 78245, USA.
Cell Rep. 2020 Dec 22;33(12):108543. doi: 10.1016/j.celrep.2020.108543.
DNA damage tolerance (DDT) and homologous recombination (HR) stabilize replication forks (RFs). RAD18/UBC13/three prime repair exonuclease 2 (TREX2)-mediated proliferating cell nuclear antigen (PCNA) ubiquitination is central to DDT, an error-prone lesion bypass pathway. RAD51 is the recombinase for HR. The RAD51 K133A mutation increased spontaneous mutations and stress-induced RF stalls and nascent strand degradation. Here, we report in RAD51 cells that this phenotype is reduced by expressing a TREX2 H188A mutation that deletes its exonuclease activity. In RAD51 cells, knocking out RAD18 or overexpressing PCNA reduces spontaneous mutations, while expressing ubiquitination-incompetent PCNA increases mutations, indicating DDT as causal. Deleting TREX2 in cells deficient for the RF maintenance proteins poly(ADP-ribose) polymerase 1 (PARP1) or FANCB increased nascent strand degradation that was rescued by TREX2, implying that TREX2 prohibits degradation independent of catalytic activity. A possible explanation for this occurrence is that TREX2 associates with UBC13 and ubiquitinates PCNA, suggesting a dual role for TREX2 in RF maintenance.
DNA 损伤容忍（DDT）和同源重组（HR）稳定复制叉（RF）。RAD18/UBC13/末端修复外切酶 2（TREX2）介导的增殖细胞核抗原（PCNA）泛素化是 DDT 的核心，是一种易错的损伤旁路途径。RAD51 是 HR 的重组酶。RAD51 K133A 突变增加了自发突变和应激诱导的 RF 停滞和新生链降解。在这里，我们在 RAD51 细胞中报告，通过表达缺失其外切酶活性的 TREX2 H188A 突变，这种表型减少。在 RAD51 细胞中，敲除 RAD18 或过表达 PCNA 可减少自发突变，而表达无泛素化能力的 PCNA 会增加突变，表明 DDT 是致病原因。在 RF 维持蛋白多聚（ADP-核糖）聚合酶 1（PARP1）或 FANCB 缺乏的细胞中删除 TREX2 会增加新生链降解，TREX2 可挽救这种降解，这表明 TREX2 独立于催化活性来阻止降解。对此情况的一种可能解释是，TREX2 与 UBC13 结合并泛素化 PCNA，表明 TREX2 在 RF 维持中具有双重作用。
