Characterization and Use of Temperature-Sensitive Mutations of BRCA1 for the Study of BRCA1 Function
Abstract
Several physiological roles of the breast and ovarian cancer susceptibility gene (BRCA1) have been described which include control of cell cycle, activation of transcription and nodulation of DNA damage response. The mechanism by which BRCA1 mediates these processes remains unclear. We have identified and characterized a naturally-occurring cancer-predisposing allele of BRCA1 with a missense mutation that displays temperature-sensitive activity. When the carboxy-terminus of BRCA1 carrying the mutation was fused to a GAL4 DNA-binding domain and expressed in human embryonic kidney cells, it was able to activate transcription of a CAL4-responsive reporter gene in a temperature-sensitive manner. At the permissive temperature, transcription activity of this mutant was approximately equal to wild-type, while at the non-permissive temperature, its activity was diminished but not abolished. Based on this observation, we designed a yeast-based screen for additional temperature-sensitive mutations of the C-terminus of BRCA1. One of the mutants identified in yeast, H1686Q, was able to activate transcription of a luciferase reporter gene in human cells in a temperature-dependent manner. Unlike the naturally-occurring mutation, the H1686Q was active only at the permissive temperature. Studies using full-length BRCA1 constructs carrying the naturally-occurring mutation indicate that the cellular distribution of this mutated protein was similar to wild-type at both permissive and non-permissive temperatures. We have generated several stable cell lines that express the full-length naturally-occurring cancer-predisposing mutant protein and are presently using these cells to analyze the role of BRCA1 in transcription activation, cell cycle control and DNA damage response.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jul 01, 2002
- Accession Number
- ADA410408
Entities
People
- Alvaro Monteiro
- Blase C. Billack