The cancer-predisposing mutation C61G disrupts homodimer formation in the NH2-terminal BRCA1 RING finger domain.
The breast and ovarian cancer tumor suppressor gene, BRCA1, encodes for a Zn2+-binding RING finger motif located near the protein NH2 terminus. The RING finger motif is characterized by eight conserved Cys and His residues which form two Zn2+-binding sites termed Site I and Site II. We used limited proteolysis in conjunction with matrix-assisted laser desorption ionization time-of-flight mass spectroscopy to investigate the metal binding properties and to probe the solution structures of wild-type and mutant BRCA1 constructs that include the RING finger. Our results show that the RING finger motif is part of a larger proteolysis-resistant structural domain which encompasses the first 110 residues of BRCA1. Analytical gel-filtration chromatography and chemical cross-linking experiments demonstrate that the BRCA1 NH2-terminal domain readily homodimerizes in solution. The cancer-predisposing C61G mutation, which alters a conserved Zn2+-binding residue, abolishes metal binding to Site II of the RING finger motif, while Site I remains intact and functional. The C61G mutation also results in increased proteolytic susceptibility of the COOH-terminal portion of the NH2-terminal domain and perturbs the oligomerization properties of BRCA1.
Brzovic
P S
PS
Department of Biochemistry and Biomolecular Structure Center, University of Washington, Seattle, Washington 98195-7742, USA.
Meza
J
J
King
M C
MC
Klevit
R E
RE
D019313:BRCA1 Protein / Q000737:chemistry* / Q000235:genetics*; D001943:Breast Neoplasms / Q000235:genetics*; D019281:Dimerization; D005260:Female; D016147:Genes, Tumor Suppressor; D006801:Humans; D009154:Mutation; D010051:Ovarian Neoplasms / Q000235:genetics*; D017421:Sequence Analysis
D019313:BRCA1 Protein