Breast Cancer Research Today is a free monthly online journal that collates and summarizes the latest research about Breast Cancer, including details on symptoms, genetics, screening, treatment, information. | ||||||||
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Mapping and conformational characterization of the DNA-binding region of the breast cancer susceptibility protein BRCA1.Naseem R, Sturdy A, Finch D, Jowitt T, Webb M Faculty of Medicine and Human Health, Centre for Molecular Medicine, Department of Medical Genetics, Stopford Building, University of Manchester, Manchester M13 9PT, UK. 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. Published 6 April 2006 in Biochem J, 395(3): 529-35.
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