Study On The Role Of BACP In Cell Genomic Stability

BACP (BRCA1 Associated Centrosome Protein) is a centrosome protein interacting with the C-terminal of BRCA1. Analysis based on the bioinformatics indicates that BACP gene localizes on 20pl 1.22-p11.1 and has 23 extrons. BACP gene encodes a 155,910Da protein which contains several domains, including three coiled-coils domains, two SMC domains and three EF hand domains.The GST pull-down assay and immunoprecipitation assay were developed to prove the physical interaction between BACP protein and BRCA1. Cell immunofluorescence assay was applied to detect the cellular localization of BACP protein. BACP protein localizes at centrosome and colocatlizes with BRCA1. The localization of BACP protein remains unchange at various cell cycle phases.RNAi technique was used to silence BACP expression with an oligo siRNA targeting BACP. Polynuclei were observed in HeLa cells transfected with BACP siRNA. Cell division process was analyzed by computerized video time-lapse approach in HEK293t cells with BACP protein expression knockdown. In such experiment, the chromosome segregation process was disturbed, cytokinesis could not complete and polynuclei were formed at the end of mitosis. Micronuclei were found in cells transfected with BACP siRNA in contrast to the controls. In addition, mitotic spindle did not have the classical two-polar structure following knockdown of BACP protein expression. The computerized video time-lapse approach was also performed to detect the spindle dynamics in mitotic HeLa cells with EGFP-alpha-tubulin stable expression following RNAi treatment. After BACP expression was silenced, the aberrations of spindle structure were apparantly observed. The disorganization included loose spindle polar and multipolar spindle indicating decreased centrosome potential of nucleating microtubules. The mitotic phase period was delayed and cell did not divide. More than one nucleus was formed in a single cell at the end of mitosis.