Checkpoint Functions of the BRCA1/BARD1 Tumor Suppressor

Abstract

Abstract : The tumor suppressor BRCA1 has been implicated in numerous cellular processes, including cell cycle checkpoint control, DNA repair, 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 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, an siRNA-mediated approach was employed to knockdown endogenous BARD1 expression. In this manner, I evaluated the role of BARD1 in clonogenic survival following genotoxic stress, DNA damage-induced cell cycle checkpoints, mitotic spindle assembly, and homology-directed repair (HDR). Knockdown of BARD1 resulted in a dramatic decrease in survival in response to IR, mitomycin C (MMC) and camptothecin (CPT). Rescue with an siRNA-resistant wild-type BARD1 construct resulted in a substantial increase in cell survival; however, reconstitution with siRNA-constructs bearing point mutations at all seven sites produced a decrease in survival in response to MMC and CPT, suggesting an important role for BARD1 mitotic phosphorylations in response to certain forms of damage. Knockdown of BARD1 resulted in substantial defects in both the IR-induced G2 and transient G2/M checkpoints, indicating a role for BARD1 in these processes. Rescue with phosphomutant forms of BARD1 bearing siRNA -resistance did not appear to result in defect in the G2 accumulation checkpoint, suggesting that mitotic phosphorylations do not function in this role. While BARD1 does not appear to function in the mitotic exit checkpoint or spindle assembly checkpoint, it does have a role in proper mitotic spindle assembly. Reconstitution experiments are currently underway to determine the role of mitotic phosphorylations in the process of mitotic spindle assembly.