Li Jing, Summerlin Matthew, Nitiss Karin C, Nitiss John L, Hanakahi Leslyn A
Department of Medicinal Chemistry and Pharmacognosy, University of Illinois, 1601 Parkview Ave. Rockford, Chicago, IL, 61107, United States.
Biopharmaceutical Sciences, College of Pharmacy, University of Illinois, 1601 Parkview Ave. Rockford, Chicago, IL, 61107, United States.
DNA Repair (Amst). 2017 Dec;60:40-49. doi: 10.1016/j.dnarep.2017.10.003. Epub 2017 Oct 16.
Tyrosyl-DNA phosphodiesterase 1 (TDP1) can remove a wide variety of 3' and 5' terminal DNA adducts. Genetic studies in yeast identified TDP1 as a regulator of non-homologous end joining (NHEJ) fidelity in the repair of double-strand breaks (DSBs) lacking terminal adducts. In this communication, we show that TDP1 plays an important role in joining cohesive DSBs in human cells. To investigate the role of TDP1 in NHEJ in live human cells we used CRISPR/cas9 to produce TDP1-knockout (TDP1-KO) HEK-293 cells. As expected, human TDP1-KO cells were highly sensitive to topoisomerase poisons and ionizing radiation. Using a chromosomally-integrated NHEJ reporter substrate to compare end joining between wild type and TDP1-KO cells, we found that TDP1-KO cells have a 5-fold reduced ability to repair I-SceI-generated DSBs. Extracts prepared from TDP1-KO cells had reduced NHEJ activity in vitro, as compared to extracts from wild type cells. Analysis of end-joining junctions showed that TDP1 deficiency reduced end-joining fidelity, with a significant increase in insertion events, similar to previous observations in yeast. It has been reported that phosphorylation of TDP1 serine 81 (TDP1-S81) by ATM and DNA-PK stabilizes TDP1 and recruits TDP1 to sites of DNA damage. We found that end joining in TDP1-KO cells was partially restored by the non-phosphorylatable mutant TDP1-S81A, but not by the phosphomimetic TDP1-S81E. We previously reported that TDP1 physically interacted with XLF. In this study, we found that XLF binding by TDP1 was reduced 2-fold by the S81A mutation, and 10-fold by the S81E phosphomimetic mutation. Our results demonstrate a novel role for TDP1 in NHEJ in human cells. We hypothesize that TDP1 participation in human NHEJ is mediated by interaction with XLF, and that TDP1-XLF interactions and subsequent NHEJ events are regulated by phosphorylation of TDP1-S81.
酪氨酰-DNA磷酸二酯酶1（TDP1）能够去除多种3'和5'末端DNA加合物。酵母中的遗传学研究将TDP1鉴定为在缺乏末端加合物的双链断裂（DSB）修复中，非同源末端连接（NHEJ）保真度的调节因子。在本通讯中，我们表明TDP1在人细胞中连接粘性DSB方面发挥着重要作用。为了研究TDP1在活的人细胞NHEJ中的作用，我们使用CRISPR/cas9产生了TDP1基因敲除（TDP1-KO）的HEK-293细胞。正如预期的那样，人TDP1-KO细胞对拓扑异构酶毒物和电离辐射高度敏感。使用染色体整合的NHEJ报告底物来比较野生型和TDP1-KO细胞之间的末端连接，我们发现TDP1-KO细胞修复I-SceI产生的DSB的能力降低了5倍。与野生型细胞提取物相比，从TDP1-KO细胞制备的提取物在体外具有降低的NHEJ活性。对末端连接接头的分析表明，TDP1缺陷降低了末端连接保真度，插入事件显著增加，这与之前在酵母中的观察结果相似。据报道，ATM和DNA-PK对TDP1丝氨酸81（TDP1-S81）的磷酸化可稳定TDP1并将TDP1募集到DNA损伤位点。我们发现，不可磷酸化的突变体TDP1-S81A可部分恢复TDP1-KO细胞中的末端连接，但磷酸模拟物TDP1-S81E则不能。我们之前报道过TDP1与XLF存在物理相互作用。在本研究中，我们发现TDP1与XLF的结合因S81A突变而降低了2倍，因S81E磷酸模拟突变而降低了10倍。我们的结果证明了TDP1在人细胞NHEJ中的新作用。我们推测TDP1参与人NHEJ是通过与XLF相互作用介导的，并且TDP1-XLF相互作用以及随后的NHEJ事件受TDP1-S81磷酸化的调节。
