PDF(Pubmed)
Abstract：
The Fanconi anemia (FA) repair pathway governs repair of highly genotoxic DNA interstrand crosslinks (ICLs) and relies on translesion synthesis (TLS). TLS is facilitated by REV1 or site-specific monoubiquitination of proliferating cell nuclear antigen (PCNA) (PCNA-Ub) at lysine 164 (K164). A PcnaK164R/K164R but not Rev1-/- mutation renders mammals hypersensitive to ICLs. Besides the FA pathway, alternative pathways have been associated with ICL repair (1, 2), though the decision making between those remains elusive. To study the dependence and relevance of PCNA-Ub in FA repair, we intercrossed PcnaK164R/+; Fancg-/+ mice. A combined mutation (PcnaK164R/K164R; Fancg-/- ) was found embryonically lethal. RNA-seq of primary double-mutant (DM) mouse embryonic fibroblasts (MEFs) revealed elevated levels of replication stress-induced checkpoints. To exclude stress-induced confounders, we utilized a Trp53 knock-down to obtain a model to study ICL repair in depth. Regarding ICL-induced cell toxicity, cell cycle arrest, and replication fork progression, single-mutant and DM MEFs were found equally sensitive, establishing PCNA-Ub to be critical for FA-ICL repair. Immunoprecipitation and spectrometry-based analysis revealed an unknown role of PCNA-Ub in excluding mismatch recognition complex MSH2/MSH6 from being recruited to ICLs. In conclusion, our results uncovered a dual function of PCNA-Ub in ICL repair, i.e. exclude MSH2/MSH6 recruitment to channel the ICL toward canonical FA repair, in addition to its established role in coordinating TLS opposite the unhooked ICL.
摘要：
范可尼贫血（FA）修复途径控制着高度基因毒性的DNA链间交联（ICL）的修复，并依赖于跨损伤合成（TLS）。通过REV1或在赖氨酸164（K164）处的增殖细胞核抗原（PCNA）（PCNA-Ub）的位点特异性单泛素化来促进TLS。PcnaK164R/K164R而不是Rev1-/-突变使哺乳动物对ICL过敏。除了FA途径，替代途径与ICL修复相关(1，2)，尽管两者之间的决策仍然难以捉摸。为了研究PCNA-Ub在FA修复中的依赖性和相关性，我们交叉了PcnaK164R/+；Fancg-/+小鼠。发现组合突变(PcnaK164R/K164R;Fancg-/-)是胚胎致死的。原代双突变（DM）小鼠胚胎成纤维细胞（MEF）的RNA-seq显示复制应激诱导的检查点水平升高。为了排除压力诱发的混杂因素，我们利用Trp53敲除法获得了一个模型来深入研究ICL修复.关于ICL诱导的细胞毒性，细胞周期停滞,和复制叉进展，发现单突变型和DMMEFs同样敏感，建立PCNA-Ub对FA-ICL修复至关重要。免疫沉淀和基于光谱的分析揭示了PCNA-Ub在排除错配识别复合物MSH2/MSH6被募集到ICL中的未知作用。总之,我们的结果揭示了PCNA-Ub在ICL修复中的双重功能，即排除MSH2/MSH6募集,将ICL引导至规范FA修复,除了在协调与未连接的ICL相对的TLS方面的既定作用。
