Biochem. J. (2006) 395
(529–535) (Printed in Great Britain)
Mapping and conformational characterization of the DNA-binding region of the breast cancer susceptibility protein BRCA1
Riffat NASEEM*, Alice STURDY†, David FINCH*, Thomas JOWITT‡ and Michelle WEBB*1
*Faculty of Medicine and Human Health, Centre for Molecular Medicine, Department of Medical Genetics, Stopford Building, University of Manchester, Manchester M13 9PT, U.K., †Department of Chemistry, Dainton Building, University of Sheffield, Sheffield S3 7HF, U.K., and ‡Biomolecular Analysis Core Facility, Faculty of Life Sciences, Michael Smith Building, University of Manchester, Manchester M13 9PT, U.K.
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.
Key words: breast cancer, breast cancer susceptibility protein 1 (BRCA1), circular dichroism, DNA-binding domain, four-way junction DNA, limited proteolysis.
Abbreviations used: 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.
1To whom correspondence should be addressed (email michelle.webb@manchester.ac.uk).
Received 10 October 2005/19 January 2006; accepted 7 February 2006
Published as BJ Immediate Publication 7 February 2006, doi:10.1042/BJ20051646
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