Development of functional assays for BRCA1 missense mutations

Background

The breast cancer susceptibility gene, BRCA1, is mutated in a high percentage of hereditary breast and ovarian cancers. It is a large gene containing 5,592 nucleotides, and since its discovery over 1,500 distinct mutations have been identified throughout the entire coding region. While genetic screening can be informative it is frustratingly ambiguous, as a complete spectrum of mutation types are presented and, while those that result in the introduction of a premature stop codon or a frame shift can be predicted to adversely affect protein function, there is considerable uncertainty regarding the functional outcome of the majority of the missense mutations. Evaluating the functional significance of such mutations is challenging due to the difficulties in purifying such a large protein. The identification of functional domains in BRCA1 will therefore be critical to the development of functional assays to evaluate their pathogenicity. Prior to our studies only two domains had been identified – the N-terminal RING domain and the C-terminal BRCT domain – and while the structures of both these domains provide a platform from which the structural consequences of missense mutations can be predicted, they only account for 16% of the total protein and hundreds of mutations of unknown pathogenicity remain to be characterised. Our work aims to identify domains in BRCA1 that can be used to determine the functional outcome of missense mutations.

Methods

BRCA1 (230 to 534), identified as a soluble fragment [1], was overexpressed in Escherichia coli BL21 (DE3) codon plus and purified to homogeneity from crude cell extracts by ion exchange and Ni²⁺-nitrilotriacetate affinity chromatography. The purified fragment was digested with trypsin at an enzyme to substrate ratio of 1:500 and a resistant domain identified using a combination of N-terminal sequencing and MALDI mass spectrometry (Bruker REFLEX III). The domain identified as amino acids 340 to 554 was purified and characterised as described previously [2]. The DNA binding affinity and selectivity were determined by surface plasmon resonance and gel retardation assays. Heteronuclear single quantum coherence NMR spectra of free and bound protein were recorded on a Varian INOVA 600 MHz spectrometer. Site-directed mutagenesis was carried out using the Quik change site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA), and the ability of the resultant proteins to bind to DNA was assessed. All mutant proteins were purified by a simple one-step procedure using nickel chelate affinity chromatography.

Results and discussion

A host of reports implicate a role for BRCA1 in the repair of damaged DNA, in particular that of double-strand breaks by homologous recombination. The formation of DNA crossovers (four-way junctions) is a central feature of this repair process, and the ability of BRCA1 to specifically recognise these structures is an important part of its function, as it potentially targets it to sites of DNA repair. Using a combination of limited proteolysis and mass spectrometry we have identified and characterised a domain in BRCA1 that binds specifically to these DNA structures [1, 2]. This region is comprised of amino acids 340 to 554 and contains one polymorphic and 42 unclassified missense variants. Analysis of the residues involved in DNA binding by NMR spectroscopy reveals that three of four arginine residues (R507, R506, R504 and R496) are potentially involved in binding; which three remains to be identified but R507, R506 and R496 are known to be mutated in some cancer patients (R507I, R504H, R496C and H). It is therefore tempting to speculate that the three involved in DNA binding are R507, R506 and R496. To determine whether this is the case, and indeed the value of using the DNA binding activity as a functional screen, each of these have been changed to their respective mutations, and also to glutamic acid and alanine, by Quik change site-directed mutagenesis. R506 has also been changed to E and A. All mutant proteins contain a C-terminal hexahistidine fusion, which has allowed a simple one-step purification procedure using nickel chelate affinity chromatography to be developed. The ability of each mutant domain to bind to four-way junction DNA is currently under investigation. These studies will allow us to determine whether the DNA binding activity of BRCA1 can be used as a potential functional assay for BRCA1 missense mutations.