The breast and ovarian cancer specific tumor suppressor BRCA1, bound to BARD1, has multiple functions aimed at maintaining genomic stability in the cell. We have shown earlier that the BRCA1/BARD1 E3 ubiquitin ligase activity regulates centrosome-dependent microtubule nucleation. In this study, we tested which domains of BRCA1 and BARD1 were required to control the centrosome function. In the present study, (a) we confirmed that the ubiquitination activity of BRCA1 regulates centrosome number and function in Hs578T breast cancer cells; (b) we observed that both the amino and carboxyl termini of BRCA1 are required for regulation of centrosome function in vitro; (c) an internal domain (770-1,290) is dispensable for centrosome regulation; (d) BARD1 is required for regulation of centrosome function and protein sequences within the terminal 485 amino acids are necessary for activity; and (e) BARD1 is localized at the centrosome throughout the cell cycle. We conclude that the BRCA1-dependent E3 ubiquitin ligase functions to restrain centrosomes in mammary cells, and loss of BRCA1 in the precancerous breast cell leads to centrosomal hypertrophy, a phenotype commonly observed in incipient breast cancer.

It is known that the Fanconi anemia D2 protein is vital for protecting the genome from DNA damage, but what activities this protein has are unknown. In these experiments we purified full-length Fanconi anemia protein D2 (FANCD2), and we found that FANCD2 bound to DNA with specificity for certain structures: double strand DNA ends and Holliday junctions. Proteins containing patient-derived mutations or artificial variants of the FANCD2 protein were similarly expressed and purified, and each variant bound to the Holliday junction DNA with similar affinity as did the wild-type protein. There was no single discrete domain of FANCD2 protein that bound to DNA, but rather the full-length protein was required for structure-specific DNA binding. This finding of DNA binding is the first biochemical activity identified for this key protein in the Fanconi anemia pathway.

The breast-and ovarian-specific tumor suppressor BRCA1 has been implicated in numerous cellular processes, including transcription, ubiquitination, and DNA repair. Its tumor suppression activity is tightly linked to that of BARD1, a protein that heterodimerizes with BRCA1. It has been previously shown that BRCA1 binds to DNA, an interesting functional observation in light of the genetic data linking BRCA1 to DNA repair pathways. In this work, we reexamine the DNA-binding properties of BRCA1, comparing them with the DNA-binding properties of the BRCA1/BARD1 heterodimer. Because nuclear BRCA1 exists as a heterodimer with BARD1, it is likely that in vitro studies of the heterodimer will provide a more accurate model of physiologic conditions. Our results indicate that whereas BARD1 cannot directly bind DNA, it does enhance DNA binding by BRCA1. This is a surprising observation as both DNA-binding domains are distal to the BARD1-interacting RING domain of BRCA1. Further analysis of the dimerization reveals that the BRCA1/ BARD1 interaction is not limited to the amino-terminal RING domains of each protein. The carboxyl terminus of BRCA1 contributes significantly to the stability of the heterodimer. We also show that the presence of BARD1 has a secondary effect, as autoubiquitination of BRCA1/BARD1 heterodimers additionally enhances the affinity of BRCA1 for DNA. Together, these data suggest that BRCA1 and BARD1 heterodimerization is stabilized via domains not previously thought to interact and that BARD1 acts in both ubiquitination-dependent and ubiquitination-independent ways to influence the role of

BackgroundFanconi anemia (FA) is a rare autosomal recessive syndrome characterized by developmental abnormalities, progressive bone marrow failure, and predisposition to cancer. The key FA protein FANCD2 crosstalks with members of DNA damage and repair pathways that also play a role at telomeres. Therefore, we investigated whether FANCD2 has a similar involvement at telomeres.ResultsWe reveal that FANCD2 may perform a novel function separate to the FANCD2/BRCA pathway. This function includes FANCD2 interaction with one of the telomere components, the PARP family member tankyrase-1. Moreover, FANCD2 inhibits tankyrase-1 activity in vitro. In turn, FANCD2 deficiency increases the polyADP-ribosylation of telomere binding factor TRF1.ConclusionsFANCD2 binding and inhibiting tankyrase-1PARsylation at telomeres may provide an additional step within the FA pathway for the regulation of genomic integrity.