Functional assay for BRCA1: Mutagenesis of the COOH-terminal region reveals critical residues for transcription activation

The breast and ovarian cancer susceptibility gene product BRCA1 is a tumor suppressor, but its precise biochemical function remains unknown. The BRCA1 COOH terminus acts as a transcription activation domain, and germ-line can cer- predisposing mutations in this region abolish transcription activation , whereas benign polymorphisms do not, These results raise the possibility that loss of transcription activation by BRCA1 is crucial for oncogenesis. Therefore, identification of residues involved in transcription activation by BRCA1 will help understand why particular germ-line missense mutations a re deleterious and may provide more reliable presymptomatic risk assessment , The BRCA1 COOH terminus (amino acids 1560-1863) consists of two BRCTs prece ded by a region likely to be nonglobular. We combined site-directed and ran dom mutagenesis, followed by a functional transcription assay in yeast: (a) error-prone PCR-induced random mutagenesis generated eight unique missense mutations causing loss of function, six of which targeted hydrophobic resi dues conserved in canine, mouse, rat, and human BRCA1; (b) random insertion of a variable pentapeptide cassette generated 21 insertion mutants, All pe ntapeptide insertions NH2-terminal to the BRCTs retained wild-type activity , whereas insertions in the BRCTs were, with few exceptions, deleterious; a nd (c) site-directed mutagenesis was used to characterize five known germ-l ine mutations and to perform deletion analysis of the COOH terminus, Deleti on analysis revealed that the integrity of the most COOH-terminal hydrophob ic cluster (11855, L1854, and Y1853) is necessary for activity, We conclude that the integrity of the BRCT domains is crucial for transcription activa tion and that hydrophobic residues may be important for BRCT function, Ther efore, the yeast-based assay for transcription activation can be used succe ssfully to provide tools for structure-function analysis of BRCA1 and may f orm the basis of a BRCA1 functional assay.