A naturally occurring allele of BRCA1 coding for a temperature-sensitive mutant protein

Recent evidence suggests that the breast and ovarian cancer susceptibility gene product BRCA1 is involved in at least two fundamental cellular processes: transcriptional regulation and DNA repair. However, the mechanism of action of BRCA1 in either of these processes is still unknown. Here, we report the characterization of a disease-predisposing allele of BRCA1, identified in a family with several cases of ovarian cancer, coding for a protein that displays temperature-sensitive activity in transcriptional activation. The mutant protein differs from the wild type protein at a single amino acid, R1699W that occurs in a region at the N-terminal BRCT domain that is highly conserved among BRCA1 homologs. When the C-terminus of the mutant protein (aa 1560-1863) was fused to a heterologous GAL4 DNA-binding domain and expressed in yeast or mammalian cells, it was able to activate transcription of a reporter gene to levels observed for wild type BRCA1 at the permissive temperature (30 degrees C) but exhibited significantly less transcription activity at the restrictive temperature (37 degrees C or 39 degrees C). Our results indicate that the transcriptional activity of the R1699W mutant can be modulated as a function of temperature and provide a novel experimental approach which can be utilized to dissect the molecular mechanism(s) of BRCA1 in processes related to transcription.