Mapping and conformational characterization of the DNA-binding region of the breast cancer susceptibility protein BRCA1.
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.
Naseem
Riffat
R
Faculty of Medicine and Human Health, Centre for Molecular Medicine, Department of Medical Genetics, Stopford Building, University of Manchester, Manchester M13 9PT, UK.
Sturdy
Alice
A
Finch
David
D
Jowitt
Thomas
T
Webb
Michelle
M
D000595:Amino Acid Sequence; D019313:BRCA1 Protein / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D001943:Breast Neoplasms / Q000378:metabolism*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004198:Disease Susceptibility; D015870:Gene Expression; D008969:Molecular Sequence Data; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D019313:BRCA1 Protein; D004247:DNA