摘要
目的探讨DNA损伤结合蛋白DDB1/2复合物在Ku70缺失的条件下与双链断裂DNA(double-strand breaks,DSB)亲和力的变化及下调DDB1对DSB修复的影响。方法针对人类Ku70构建编码shRNA序列的重组腺病毒并感染细胞,建立Ku70蛋白表达沉默的细胞株,以博来霉素(bleomycin,Bleo)或新制癌菌素(neocarzinostatin,Ncs)诱导DNA双链断裂,利用DNA损伤修复蛋白与损伤染色质的高亲和力,分段提取细胞蛋白组分,Western blot检测DDB1/2在各组分的分布变化。免疫荧光检测诱导DNA双链断裂前后DDB1与损伤染色质亲和力的变化。沉默DDB1,Western blot检测在不同条件下γ-H2AX的去磷酸化效率。结果人乳腺癌MDA-MB-231细胞Ku70蛋白水平在Ku70shRNA重组腺病毒感染后4d显著下调,6d达到最低。与Ku70正常细胞相比,在Ku70沉默后诱导DSB,Western blot检测结果显示DDB1/2主要分布在与染色质紧密结合的组分,免疫荧光同样表现出其与染色质亲和力增高。并且在Ku缺失的条件下,Western blot显示下调DDB1能显著降低γ-H2AX去磷酸化效率(P<0.01)。结论下调Ku70可促进DDB1/2在双链断裂DNA聚集,且下调DDB1影响DSB修复,说明DDB1/2可能参与了不依赖于Ku的双链断裂的修复途径。
Objective To study the binding affinity of DNA damaged binding protein 1 /2（ DDB1 /2）complex to double strand break（ DSB） DNA and explore the effect of DDB1 down-regulation on DSB repair in the absence of Ku70. Methods Recombinant adenovirus containing shRNA targeting human Ku70 was constructed to silent Ku70 in human breast cancer cell line MDA-MB-231. The cells were treated with bleomycin or neocarzinostatin to induce DNA DSB. By use of fractionation protocol,the recruitment of DDB1 /2to chromatin was investigated. And the affinity of DDB1 to the cell nucleus in the absence of Ku70 was analyzed by in situ extraction and immunofluorescence assay. The dephosphorylation of γ-H2 AX was also assayed in the absence of DDB1. Results The protein level of Ku70 in MDA-MB-231 cells was significantly down-regulated on the 4th day after adenovirus infection,and reached the lowest on the 6th day. Compared with Ku70 normal condition,Western blotting showed that DDB1 /2 was mainly distributed in the cell fractions closely bound to the chromatin in response to DSB,and immunofluorescence assay showed that the affinity of DDB1 to the chromatin was increased as well in the absence of Ku70. And Western blotting showed that the dephosphorylation level of γ-H2 AX was significantly affected by down-regulation of DDB1 in the absence of Ku70（ P〈0. 01）. Conclusion Down-regulation of Ku70 facilitates the recruitment of DDB1 /2 at DSB DNA,and DDB1 down-regulation affects DSB repair efficiency,indicating that DDB1 /2 may be involved in Ku-independent DSB repair pathway.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2016年第2期148-154,共7页
Journal of Third Military Medical University
基金
国家自然科学基金青年科学基金(81101653)
重庆市自然科学基金(CSTC2011jj A10037)~~
