Characterization and Use of Temperature-Sensitive Mutations of BRCA1 for the Study of BRCA1 Function
Abstract
Recent evidence suggests that the breast and ovarian cancer susceptibility gene product BRCAl is involved in at least two fundamental cellular processes: transcriptional regulation and DNA repair. The precise mechanism of action of BRCAl in either of these processes is still unknown. We have identified a naturally-occurring allele of BRCAl which codes for a single amino acid substitution from arginine to tryptophan at residue 1699 (R1699W). This mutation occurs in a region at tile N- terminal BRCT domain that is highly conserved among BRCAl 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 BRCAl at the permissive temperature (30C) but exhibited significantly less transcription activity at the restrictive temperature (37C or 39C). The transcription activity of the mutant protein appears to undergo complex regulation, as temperature-sensitive transcription activation was found to be cell type specific and not dependent on the tissue of origin. Stable cell lines expressing the mutant protein had similar capacity for DNA-damage repair of double strand breaks induced by ionizing radiation at all temperatures examined, suggesting that the activity of the mutant protein in DNA-damage repair is not temperature sensitive. . Our results demonstrate 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 BRCAl in processes related to transcription.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jul 01, 2003
- Accession Number
- ADA418024
Entities
People
- Alvaro Monteiro
- Blass Billack