Biallelic deleterious BRCA1 mutations in a woman with early-onset ovarian cancer.

UNLABELLED: BRCA1 and BRCA2 are the most important breast and ovarian cancer susceptibility genes. Biallelic mutations in BRCA2 can lead to Fanconi anemia and predisposition to cancers, whereas biallelic BRCA1 mutations have not been confirmed, presumably because one wild-type BRCA1 allele is required during embryogenesis. This study describes an individual who was diagnosed with ovarian carcinoma at age 28 and found to have one allele with a deleterious mutation in BRCA1, c.2457delC (p.Asp821Ilefs*25), and a second allele with a variant of unknown significance in BRCA1, c.5207T>C (p.Val1736Ala). Medical records revealed short stature, microcephaly, developmental delay, and significant toxicity from chemotherapy. BRCA1 p.Val1736Ala cosegregated with cancer in multiple families, associated tumors showed loss of wild-type BRCA1, and BRCA1 p.Val1736Ala showed reduced DNA damage localization. These findings represent the first validated example of biallelic deleterious human BRCA1 mutations and have implications for the interpretation of genetic test results. SIGNIFICANCE: Accurate assessment of genetic testing data for BRCA1 mutations is essential for clinical monitoring and treatment strategies. Here, we report the fi rst validated example of an individual with biallelic BRCA1 mutations, early-onset ovarian cancer, and clinically significant hypersensitivity to chemotherapy.

The Rap80-BRCC36 de-ubiquitinating enzyme complex antagonizes RNF8-Ubc13-dependent ubiquitination events at DNA double strand breaks.

DNA double strand breaks (DSBs) initiate reversible cellular checkpoint and repair activities. Whereas many of the activating events at DSBs have recently been elucidated, the mechanisms used to terminate responses at these sites are largely undefined. Here we report a pathway required to reverse RNF8-Ubc13 dependent ubiquitination events on chromatin flanking DSBs. Inhibition of the Rap80-BRCC36 de-ubiquitinating enzyme complex partially restored DSB-associated ubiquitin levels following RNF8 knockdown or proteasome inhibition. Similarly, BRCC36 knockdown or expression of a BRCC36 de-ubiquitinating enzyme-inactive mutant rescued both 53BP1 recruitment to DSBs and ionizing radiation-induced gammaH2AX ubiquitination following RNF8 depletion, and mitigated ionizing radiation sensitivity resulting from RNF8 deficiency. Thus, concomitant and opposing RNF8-Ubc13 ubiquitin ligase and Rap80-BRCC36 ubiquitin hydrolysis activities are responsible for determining steady-state ubiquitin levels at DNA DSBs. These findings reveal a Rap80-BRCC36 dependent pathway that is required for appropriate DSB recruitment and repair responses.

Exonuclease-1 deletion impairs DNA damage signaling and prolongs lifespan of telomere-dysfunctional mice.

Exonuclease-1 (EXO1) mediates checkpoint induction in response to telomere dysfunction in yeast, but it is unknown whether EXO1 has similar functions in mammalian cells. Here we show that deletion of the nuclease domain of Exo1 reduces accumulation of DNA damage and DNA damage signal induction in telomere-dysfunctional mice. Exo1 deletion improved organ maintenance and lifespan of telomere-dysfunctional mice but did not increase chromosomal instability or cancer formation. Deletion of Exo1 also ameliorated the induction of DNA damage checkpoints in response to gamma-irradiation and conferred cellular resistance to 6-thioguanine-induced DNA damage. Exo1 deletion impaired upstream induction of DNA damage responses by reducing ssDNA formation and the recruitment of Replication Protein A (RPA) and ATR at DNA breaks. Together, these studies provide evidence that EXO1 contributes to DNA damage signal induction in mammalian cells, and deletion of Exo1 can prolong survival in the context of telomere dysfunction.

RAP80 targets BRCA1 to specific ubiquitin structures at DNA damage sites.

Mutations affecting the BRCT domains of the breast cancer-associated tumor suppressor BRCA1 disrupt the recruitment of this protein to DNA double-strand breaks (DSBs). The molecular structures at DSBs recognized by BRCA1 are presently unknown. We report the interaction of the BRCA1 BRCT domain with RAP80, a ubiquitin-binding protein. RAP80 targets a complex containing the BRCA1-BARD1 (BRCA1-associated ring domain protein 1) E3 ligase and the deubiquitinating enzyme (DUB) BRCC36 to MDC1-gammaH2AX-dependent lysine(6)- and lysine(63)-linked ubiquitin polymers at DSBs. These events are required for cell cycle checkpoint and repair responses to ionizing radiation, implicating ubiquitin chain recognition and turnover in the BRCA1-mediated repair of DSBs.