摘要
目的研究碱基切除修复途径(base excision repair,BER)关键因子PARP1在Ku70缺失的条件下是否参加DNA双链断裂(double-strand break,DSB)的修复。方法针对人类Ku70构建编码shRNA序列的重组表达载体并转染细胞,建立Ku70蛋白表达沉默的细胞株。以高效的DNA双链断裂诱导剂刺孢霉素calicheamycin诱导DSB形成,在Ku70沉默或联合PARP1抑制剂条件下,通过结晶紫染色法检测DSB对细胞存活率的影响,以及Western blot检测H2AX的磷酸化状态。根据与染色质相互作用的差别,分段提取细胞蛋白组分,Western blot检测PARP1在各组分的分布变化。结果细胞Ku70蛋白水平在Ku70 shRNA干扰后显著下调。Ku70沉默和PARP1抑制剂明显降低细胞在calicheamycin处理后的存活率(P<0.01),两者有明显协同效应。在Ku70沉默的细胞诱导DSB后,PARP1主要分布在与染色质紧密结合的组分。结论在Ku70缺陷时,PARP1可与染色质结合,且抑制其功能影响细胞生存,说明PARP1可能参与了不依赖于Ku的DNA双链断裂修复的替代途径。
Objective To study if the key factor PARP1 for base excision repair（BER） is involved in repair of DNA double strand breaks（DSB） when Ku70 is silenced.Methods A shRNA expression vector against human Ku70 was constructed and then were transfected to produce silent Ku70 cell line.DSB were induced with calicheamycin,a high efficient inductor of DNA DSB.Effect of DSB on cell survival rate was detected when Ku70 was silent or PARP1 was inhibited with crystal violate staining.Phosphorylation of H2AX was detected by Western blotting.Cell fractions were extracted according to the difference in interactions of chromatin.Distribution of PARP1 in cell fractions was assayed by Western blotting.Results The protein level in Ku70 was significantly down-regulated by Ku70 shRNA.Silent Ku70 and PARP inhibitor significantly decreased the survival rate of cells（P0.01）.PARP1 was mainly distributed in fractions of cells closely bound to chromatin.Conclusion PARP1 can bind to chromatin in the absence of Ku70 and decrease the survival time of cells,indicating that PARP1 may participate in repair of DNA DSB independent of Ku70.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2011年第3期286-289,共4页
Journal of Third Military Medical University
