Checkpoint Functions of the BRCA1/BARD1 Tumor Suppressor
Abstract
The breast and ovarian-specific tumor suppressor BRCA1 has been implicated in numerous cellular processes, including DNA repair, cell cycle checkpoint control, and mitotic spindle assembly. In vivo, BRCA1 primarily exists in association with BARD1 and the BRCA1/BARD1 heterodimer is thought to mediate the tumor suppression activity of BRCA1. It has been previously shown that the phosphorylation state of the BARD1 polypeptide is cell cycle regulated and that BARD1 is hyperphosphorylated in mitosis at seven distinct residues. To study the function of mitotic BARD1 phosphorylation, I used an siRNA-mediated approach to knockdown endogenous BARD1 expression and then restore expression with siRNA-resistant exogenous forms of BARD1. In this manner, I will evaluate the role of BARD1 mitotic phosphorylation in the G2 accumulation checkpoint, decatenation checkpoint, and homology-directed DNA repair. My initial studies showed that siRNA-mediated knockdown of BARD1 leads to a considerable IR-induced G2 accumulation checkpoint defect, illustrated by an ~8-fold increase in the percent of cells that enter mitosis following IR damage relative to control cells. Reconstitution experiments with mRNAs resistant to knockdown by BARD1-specific siRNAs resulted in ~3-fold decrease in the percentage of cells that escape the IR-induced G2 accumulation checkpoint. We are currently testing the role of individual phosphorylation sites in the checkpoint by reconstituting BARD1-depleted cells with siRNA-resistant BARD1 polypeptides bearing mutations of specific phosphorylation sites.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jul 01, 2007
- Accession Number
- ADA484257
Entities
People
- Ami Modi
Organizations
- Columbia University