Structural Requirements for the BARD1 Tumor Suppressor in Chromosomal Stability and Homology-directed DNA Repair

Marsha Laufer,Subhadra V Nandula,Ami P Modi,Shuang Wang,Maria Jasin,Vundavalli V V.S Murty,Thomas Ludwig,Richard Baer

doi:10.1074/jbc.m705198200

Abstract

The BRCA1 tumor suppressor exists as a heterodimeric complex with BARD1, and this complex is thought to mediate many of the functions ascribed to BRCA1, including its role in tumor suppression. The two proteins share a common structural organization that features an N-terminal RING domain and two C-terminal BRCT motifs, whereas BARD1 alone also contains three tandem ankyrin repeats. In normal cells, the BRCA1/BARD1 heterodimer is believed to enhance chromosome stability by promoting homology-directed repair (HDR) of double strand DNA breaks. Here we have investigated the structural requirements for BARD1 in this process by complementation of Bard1-null mouse mammary carcinoma cells. Our results demonstrate that the ankyrin and BRCT motifs of BARD1 are each essential for both chromosome stability and HDR. Tandem BRCT motifs, including those found at the C terminus of BARD1, are known to form a phosphoprotein recognition module. Nonetheless, the HDR function of BARD1 was not perturbed by synthetic mutations predicted to ablate the phospho-recognition activity of its BRCT sequences, suggesting that some functions of the BRCT domains are not dependent on their ability to bind phosphorylated ligands. Also, cancer-associated missense mutations in the BRCT domains of BARD1 (e.g. C557S, Q564H, V695L, and S761N) have been observed in patients with breast, ovarian, and endometrial tumors. However, none of these was found to affect the HDR activity of BARD1, suggesting that any increased cancer risk conferred by these mutations is not because of defects in this repair mechanism.

Highlights

Including DNA double strand break (DSB)2 repair and cell cycle checkpoint control
The data presented here establish that BARD1, the heterodimeric partner of BRCA1, is required for maintenance of chromosomal stability and homology-directed repair (HDR) of DSBs
BARD1 polypeptides lacking the three ankyrin repeats or the two C-terminal BRCT motifs fail to suppress formation of de novo chromatid-type abnormalities, indicating that both domains are required for maintenance of chromosomal stability by BARD1

Summary

EXPERIMENTAL PROCEDURES

Cell Lines—Bard-null mouse mammary carcinoma cell lines 10-05 and 18-09 were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 ␮g/ml penicillin/streptomycin, and 2 mM L-glutamine. To generate stably transformed subclones of these lines, 10-05 and 18-09 cells were transfected using FuGENE 6 (Roche Applied Science) with either the empty pIRESpuro vector (Clontech) or. To generate HDR reporter lines, Bard1-null 18-09 cells were transfected with the DR-GFPhygro reporter construct [23], and transformants harboring randomly integrated copies of the reporter were selected with 0.25 mg/ml hygromycin B. HDR Assays—To measure repair of an I-SceI-induced DSB in the stably reconstituted 18-09/DR-GFP-2 reporter subclones (e.g. Fig. 3), ϳ4 ϫ 105 cells/well were seeded in 2 ml of nonselective medium on 6-well plates. B, subclones of Bard1-null 10-05 cells stably transformed with an expression vector encoding human BARD1 (lanes 1– 4) or the empty vector (lane 5) were analyzed by immunoblotting with antibodies specific for mouse Brca, human BARD1, or ␣-tubulin.

ϪMMC Empty vectorb

RESULTS

Findings

DISCUSSION