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 neoca

rzinostatin to induce DNA DSB. By use of fractionation protocol

the recruitment of DDB1/2 to 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-H

2

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-H2AX was significantly affected by down-regulation of DDB1 in the absence of Ku70 (P0.01).        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.