摘要
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 investigate the recruitment of base excision repair（BER） key factors,poly（ADP-ribose） polymerase 1（PARP1）,ligase 3 and X-ray repair cross-complementing protein 1（XRCC1） at chromatin with DNA double-strand break（DSB） after silencing Ku70,and the effect of PARP1 inhibition on the protein recruitment.Methods A cell line capable of inductively expressing Ku70 shRNA was treated with calicheamicin to induce DSB.Proteins recruited at the damaged chromatin after down-regulation of Ku70 expression were identified through stable isotope labeling by amino acids in cell culture（SILAC） and Western blotting,and the influence of PARP1 inhibitor,1,5-dihydroxyisoquinolin（DIQ）,on the protein recruitment was analyzed.Results Ku70 was down-regulated significantly by virtue of doxycycline,while the expression of other proteins was not influenced.The SILAC and Western blotting results showed that the recruitment of PARP1,ligase 3 and XRCC1 at the damaged chromatin increased after Ku70 down-regulation,and the recruitment of PARP1,ligase 3 and XRCC1 at the damaged chromatin increased with the increase of DSB degree.The recruitment of ligase 3 at the damaged chromatin decreased when the DIQ concentration increased.Conclusion Silencing Ku70 promotes the recruitment of PARP1,ligase 3 and XRCC1 at chromatin with DSB,and the protein recruitment is correlated positively to DSB degree.The recruitment of ligase 3 depends on PARP1 to some extent.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2011年第14期1497-1501,共5页
Journal of Third Military Medical University
