Abstract
The breast cancer susceptibility gene, BRCA1, encodes a large nuclear phosphoprotein, the major isoform of which is 1863 amino acids in size. Structure–function studies have been largely restricted to the only two domains identified by homology searches: the RING (really interesting new gene) and BRCT (BRCA1 C-terminus) domains. However, we have recently reported the identification of a large central soluble region of BRCA1 (residues 230–534) that binds specifically to four-way junction DNA, a property that potentially facilitates its role in the repair of DNA lesions by homologous recombination. We have now used a combination of limited proteolysis and extension cloning to identify more accurately the DNA-binding region of BRCA1. Limited trypsinolysis of BRCA1-(230–534) resulted in the production of a soluble domain identified as residues 230–339. However, after cloning, expression and purification of this region, studies revealed that it was unable to bind to four-way junctions, suggesting that the DNA-binding activity, in part, resides within residues 340–534. A series of fragments extending from residue 340 were produced, and each was tested for its ability to bind to four-way junction DNA in gel retardation assays. In these experiments, residues 340–554 of BRCA1 were identified as the minimal DNA-binding region. We then went on to characterize the conformation of this region using CD spectroscopy and analytical centrifugation.
- breast cancer
- breast cancer susceptibility protein 1 (BRCA1)
- circular dichroism
- DNA-binding domain
- four-way junction DNA
- limited proteolysis
Abbreviations: BASC, BRCA1 genome surveillance complex; BRCA1, breast cancer susceptibility protein 1; BRCT, BRCA1 C-terminus; DTT, dithiothreitol; MALDI, matrix-assisted laser-desorption ionization; MSH, human MutS homologue; NBS, Nijmegen breakage syndrome protein; Ni-NTA, Ni2+-nitriloacetic acid; RAD, radiation-dependent; RING, really interesting new gene; TAE, Tris/acetate/EDTA
- The Biochemical Society, London