Characterization of segments from the central region of BRCA1: an intrinsically disordered scaffold for multiple protein-protein and protein-DNA interactions?
The BRCA1 tumor suppressor gene encodes an 1863 amino acid gene product that is implicated in many cellular pathways including transcription, cell-cycle checkpoint control, apoptosis and DNA repair. Much attention has been focused on the structural and biochemical characterization of the N-terminal RING and tandem C-terminal BRCT domains of BRCA1. Here we used NMR spectroscopy in conjunction with CD spectroscopy and limited proteolysis to investigate the biophysical properties of the approximately 1500 residue central region of BRCA1. Our results show that although there are a few small, mildly protease-resistant regions, the majority of the BRCA1 central region lacks any pre-existing independently folded globular domains. Electrophoretic mobility shift assay and intrinsic tryptophan fluorescence experiments also demonstrate that, although intrinsically disordered, polypeptides from the central region are able to mediate interactions with DNA and p53 with affinities in the low micromolar range. This supports a model in which the central region may act as a long flexible scaffold for intermolecular interactions, thereby helping to integrate multiple signals in the DNA damage response pathway.
Mark
Wen-Ying
WY
Division of Molecular and Structural Biology, Department of Medical Biophysics, Ontario Cancer Institute, University of Toronto, Toronto, Ont., Canada M5G 2M9.
Liao
Jack C C
JC
Lu
Ying
Y
Ayed
Ayeda
A
Laister
Rob
R
Szymczyna
Blair
B
Chakrabartty
Avi
A
Arrowsmith
Cheryl H
CH
D017209:Apoptosis; D019313:BRCA1 Protein / Q000737:chemistry* / Q000378:metabolism; D055592:Biophysical Phenomena; D001703:Biophysics; D001943:Breast Neoplasms / Q000378:metabolism; D002453:Cell Cycle; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry*; D004260:DNA Repair; D004305:Dose-Response Relationship, Drug; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008957:Models, Genetic; D020125:Mutation, Missense; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D014158:Transcription, Genetic; D014364:Tryptophan / Q000737:chemistry; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000378:metabolism
D019313:BRCA1 Protein; D016159:Tumor Suppressor Protein p53; D014364:Tryptophan; D004247:DNA