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.
Worley
Terri
T
Strang Cancer Prevention Center, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.
Vallon-Christersson
Johan
J
Billack
Blase
B
Borg
Ake
A
Monteiro
Alvaro N A
AN
D000483:Alleles; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019313:BRCA1 Protein / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D002460:Cell Line; D017124:Conserved Sequence; D004268:DNA-Binding Proteins; D005786:Gene Expression Regulation; D019398:Genes, BRCA1; D017930:Genes, Reporter; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017354:Point Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation
D019313:BRCA1 Protein; D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors