THE CANCER-PREDISPOSING MUTATION C61G DISRUPTS HOMODIMER FORMATION INTHE NH2-TERMINAL BRCA1 RING FINGER DOMAIN

The breast and ovarian cancer tumor suppressor gene, BRCA1, encodes fo r a Zn2+-binding RING finger motif located near the protein NH2 termin us, 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 la ser desorption ionization time-of-flight mass spectroscopy to investig ate 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 prote olysis-resistant structural domain which encompasses the first 110 res idues of BRCA1. Analytical gel-filtration chromatography and chemical cross-linking experiments demonstrate: that the BRCA1 NH2-terminal dom ain readily homodimerizes in solution. The cancer-predisposing C61G: m utation, which alters a conserved Zn2+-binding residue, abolishes meta l binding to Site II of the RING finger motif, while Site I remains in tact and functional. The C61G mutation also results in increased prote olytic susceptibility of the COOH-terminal portion of the NH2-terminal domain and perturbs the oligomerization properties of BRCA1.