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Research article

Mapping and conformational characterization of the DNA-binding region of the breast cancer susceptibility protein BRCA1

Riffat Naseem, Alice Sturdy, David Finch, Thomas Jowitt, Michelle Webb
Biochemical Journal May 01, 2006, 395 (3) 529-535; DOI: 10.1042/BJ20051646
Riffat Naseem
Faculty of Medicine and Human Health, Centre for Molecular Medicine, Department of Medical Genetics, Stopford Building, University of Manchester, Manchester M13 9PT, U.K.
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Alice Sturdy
Department of Chemistry, Dainton Building, University of Sheffield, Sheffield S3 7HF, U.K.
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David Finch
Faculty of Medicine and Human Health, Centre for Molecular Medicine, Department of Medical Genetics, Stopford Building, University of Manchester, Manchester M13 9PT, U.K.
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Thomas Jowitt
Biomolecular Analysis Core Facility, Faculty of Life Sciences, Michael Smith Building, University of Manchester, Manchester M13 9PT, U.K.
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Michelle Webb
Faculty of Medicine and Human Health, Centre for Molecular Medicine, Department of Medical Genetics, Stopford Building, University of Manchester, Manchester M13 9PT, U.K.
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  • For correspondence: michelle.webb@manchester.ac.uk
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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

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May 2006

Volume: 395 Issue: 3

Biochemical Journal: 395 (3)
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Mapping and conformational characterization of the DNA-binding region of the breast cancer susceptibility protein BRCA1
Riffat Naseem, Alice Sturdy, David Finch, Thomas Jowitt, Michelle Webb
Biochemical Journal May 2006, 395 (3) 529-535; DOI: 10.1042/BJ20051646
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Mapping and conformational characterization of the DNA-binding region of the breast cancer susceptibility protein BRCA1
Riffat Naseem, Alice Sturdy, David Finch, Thomas Jowitt, Michelle Webb
Biochemical Journal May 2006, 395 (3) 529-535; DOI: 10.1042/BJ20051646

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Keywords

breast cancer
breast cancer susceptibility protein 1 (BRCA1)
circular dichroism
DNA-binding domain
four-way junction DNA
limited proteolysis

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