The BRCA1 tumor suppressor has been implicated in many cellular pathways, but the mechanisms by which it suppresses tumor formation are not fully understood. In vivo BRCA1 forms a heterodimeric complex with the related BARD1 protein, and its enzymatic activity as a ubiquitin ligase is largely dependent upon its interaction with BARD1. To explore the genetic relationship between BRCA1 and BARD1, we have examined the phenotype of Bard1-null mice. These mice become developmentally retarded and die between embryonic day 7.5 (E7.5) and E8.5. Embryonic lethality results from a severe impairment of cell proliferation that is not accompanied by increased apoptosis. In the absence of p53, the developmental defects associated with Bard1 deficiency are partly ameliorated, and the lethality of Bard1; p53-nullizygous mice is delayed until E9.5. This result, together with the increased chromosomal aneuploidy of Bard1 mutant cells, indicates a role for Bard1 in maintaining genomic stability. The striking similarities between the phenotypes of Bard1-null, Brca1-null, and double Bard1; Brca1-null mice provide strong genetic evidence that the developmental functions of Brca1 and Bard1 are mediated by the Brca1/Bard1 heterodimer.Germ line mutations of the BRCA1 tumor suppressor gene are responsible for many cases of hereditary breast and ovarian carcinomas (34), and its protein product has been implicated in a broad spectrum of cellular processes that includes transcriptional regulation, chromatin remodeling, DNA repair, and cell cycle checkpoint control (for recent reviews, see references 2, 25, 35, 42, and 49). The major isoform of Brca1 has two recognizable amino acid motifs: a RING domain at the N terminus and two tandem copies of the BRCT domain at the C terminus (29, 34). In some patients, the predisposing BRCA1 lesion can be traced to missense mutations in the RING domain (8, 44), indicating that proper folding of this motif is essential for BRCA1-mediated tumor suppression. Many RING proteins are now known to function as ubiquitin E3 ligases, a family of enzymes that catalyze the final step in protein ubiquitination (22,24). Recent studies have shown that the N-terminal RING sequence of BRCA1 can also catalyze the formation of polyubiquitin chains in vitro and that this activity is abolished by tumor-associated missense mutations (7,17,32,39).The in vivo functions of BRCA1 have been explored using genetically modified mice bearing either null Brca1 alleles, which are completely devoid of Brca1 activity and/or expression, or hypomorphic alleles that presumably retain some aspects of normal Brca1 activity (reviewed in references 4 and 19). Mice that are heterozygous for Brca1 mutations, whether null or hypomorphic, develop normally, but unlike human carriers of BRCA1 mutations, they are not predisposed to mammary carcinogenesis. On the other hand, mice that are homozygous for null Brca1 alleles die around the time of gastrulation, typically between days 6.5 and 7.5 of embryogenesis (15,31,33). Brca1-null embryos are not char...

The tumor suppressor p53 is considered as the guardian of the genome which is activated following genotoxic stress. In many cell types, p53 mediates G1 cell cycle arrest as the predominant cellular response. Inactivation of wild-type p53 leads to loss of G1/S checkpoint control and to genomic instability, including increased spontaneous homologous recombination (HR). To determine whether regulation of the G1/S checkpoint is required for suppression of HR, we assessed recombination events using a plasmid substrate that stably integrated into the genome of p53-null mouse ®broblasts. Exogenous expression of a temperature-sensitive p53 protein (Ala135 to Val), which had lost trans-activation function and could not regulate G1/S transition when in mutant conformation, reduced HR rates to the same extent as wild-type p53. Furthermore, a p53 construct with an alternatively-spliced carboxy terminus also retained this ability in the absence of both activities, G1/S control and non-sequence speci®c DNA binding as mediated by the carboxy terminus. Our data dissociate regulation of HR by p53 from its role as a cell cycle checkpoint protein.The results support a model which extends p53's role as a guardian of the genome to include transactivationindependent regulatory functions in DNA repair, replication and recombination. Oncogene (2000) 19, 632 ± 639.

Women with germ-line mutations of the BRCA1 tumor suppressor gene are highly susceptible to breast and ovarian cancer. The protein product of BRCA1 is involved in a broad spectrum of biological processes and interacts with many diverse proteins. One of these, BARD1, associates with BRCA1 to form a heterodimeric complex that is enzymatically active as an ubiquitin E3 ligase. Although the BRCA1/BARD1 heterodimer has been implicated in several aspects of BRCA1 function, its role in tumor suppression has not been evaluated. To address this question, we generated mouse strains carrying conditional alleles of either Bard1 or Brca1 and used Cre recombination to inactivate these genes in mammary epithelial cells. Significantly, the conditional Bard1-and Brca1-mutant mice developed breast carcinomas that are indistinguishable from each other (and from those of double conditional Bard1/Brca1-mutant animals) with respect to their frequency, latency, histopathology, and cytogenetic features. Reminiscent of the basal-like breast carcinomas seen in human BRCA1 mutation carriers, these tumors are ''triple negative'' for estrogen and progesterone receptor expression and HER2/neu amplification. They also express basal cytokeratins CK5 and CK14, have an elevated frequency of p53 lesions, and display high levels of chromosomal instability. The remarkable similarities between the mammary carcinomas of Bard1-, Brca1-, and Bard1/Brca1-mutant mice indicate that the tumor suppressor activities of both genes are mediated through the BRCA1/BARD1 heterodimer.basal-like breast cancer ͉ mammary carcinogenesis G erm-line mutations of the BRCA1 tumor suppressor gene serve as predisposing lesions in women with a familial susceptibility to breast and ovarian cancer (1). The breast tumors of BRCA1 mutation carriers typically display ''basal-like'' features that define a subtype of breast cancer with a distinct histopathology and gene expression profile (2-5). Basal-like breast carcinomas have been described as ''triple negative'' because they often lack expression of estrogen receptor (ER) ␣, the progesterone receptor (PR), and the HER2/neu protooncogene. Patients with basal-like breast cancer face a poor prognosis and reap little benefit from current therapies that target ER-or HER2-expressing tumor cells.Although the mechanism of BRCA1-mediated tumor suppression is unclear, its protein product has been implicated in a remarkably broad range of cellular processes, some of which serve to maintain genome stability (6-8). Consistent with its pleiotropic nature, the BRCA1 polypeptide has been reported to interact with a large and diverse group of proteins. One of these, BARD1, is structurally related to BRCA1 in that it harbors an N-terminal RING motif and two C-terminal BRCT domains (9), and the heterodimer complex formed by these proteins functions as a potent ubiquitin E3 ligase (10). Although it has been proposed that the tumor suppression activity of BRCA1 is mediated by the BRCA1/BARD1 heterodimer (11), experimental evidence to support this h...