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目的研究碱基切除修复途径(base excision repair,BER)关键因子PARP1、ligase3和XRCC1在Ku70缺失的条件下与DNA双链断裂(double-strand break,DSB)所在染色质的结合及抑制PARP1所产生的影响。方法采用可经药物诱导Ku70 shRNA的细胞株,以高效的DNA双链断裂诱导剂刺孢霉素calicheamicin诱导DSB。分离不同组分蛋白联合稳定同位素标记定量差异蛋白组分析法鉴定Ku70下调后在损伤染色质聚集增多的蛋白,Western blot进行验证并检测PARP1抑制剂DIQ对其染色质聚集的影响。结果在药物Doxy作用下,Ku70明显下调,其余蛋白的表达不受影响。稳定同位素标记定量蛋白组学方法鉴定出PARP1、ligase3和XRCC1在Ku70下调后在损伤染色质聚集增多。Western blot进行了验证并显示PARP1、ligase3和XRCC1在损伤染色质的聚集随DSB程度增加而增加。施加PARP1抑制剂DIQ,随着DIQ浓度的上升,ligase3在损伤染色质的聚集逐渐减少。结论沉默Ku70后,PARP1、ligase3和XRCC1在DSB染色质聚集增加,这种聚集与DSB程度变化一致,并且ligase3的染色质聚集具有一定程度的PARP1依赖性。
OBJECTIVE: To study the binding of PARP1, ligase3 and XRCC1, key factors of base excision repair (BER), to the chromatin in the presence of double-strand break (DSB) in the absence of Ku70 and to inhibit the production of PARP1 Impact. Methods The cell lines which can be induced by drug were constructed and the DSB was induced by calicheamicin, an efficient DNA double strand break inducer. Isolation of different components of proteins combined with stable isotope labeling quantitative differential proteomics analysis identified Ku70 downregulation of chromatin damage in the protein, Western blot and PARP1 inhibitor test detected the impact of its chromatin aggregation. Results Under the action of drug Doxy, Ku70 was significantly down-regulated, and the expression of other proteins was not affected. Stable isotope labeling quantitative proteomics identified that PARP1, ligase3, and XRCC1 were upregulated in Ku70 after chromatin accumulation. The results of Western blot showed that the accumulation of PARP1, ligase3 and XRCC1 in injured chromatin increased with the increase of DSB. Apply PARP1 inhibitor DIQ, with the increase of DIQ concentration, ligase3 gradually decreased in the damaged chromatin. Conclusions After silencing Ku70, PARP1, ligase3 and XRCC1 are increased in DSB chromatin, which is consistent with the change of DSB degree. Chromatin aggregation of ligase3 has a certain degree of PARP1 - dependent.