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Permanent link (DOI): https://doi.org/10.7939/R33D32

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Investigation of the BRCT repeats in human hereditary breast cancer and DNA damage response Open Access

Descriptions

Other title
Subject/Keyword
BRCT
breast cancer
DNA damage
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Lee, Megan Sae Bom
Supervisor and department
Glover, J.N. Mark (Biochemistry)
Examining committee member and department
Hazes, Bart (Medical Microbiology and Immunology)
Spyracopoulos, Leo (Biochemistry)
Young, Howard (Biochemistry)
Holmes, Charles (Biochemistry)
Monteiro, Alvaro N. (Risk assessment, Detection and Intervention Program)
Department
Department of Biochemistry
Specialization

Date accepted
2009-07-27T20:35:33Z
Graduation date
2009-11
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
The C-terminal region of breast cancer susceptibility gene 1 (BRCA1) contains a pair of tandem BRCT repeats that are critical for the tumour suppressor function of BRCA1. BRCT repeats are present in a large of number of proteins that are implicated in the cellular response to DNA damage. A subset of tandem BRCT domains, including those of BRCA1, functions as phosphorecognition modules. Aside from BRCA1, the precise molecular mechanisms of the BRCT repeats of other proteins remain largely unknown. We determined the crystal structure of the tandem BRCT domain of human mediator of DNA checkpoint 1 (MDC1) at 1.45 Å resolution. Our structural and biochemical studies suggest that the tandem BRCT domain of MDC1 functions as the predominant histone variant, γH2AX phosphorecognition module and that the interaction is critically dependent on the free carboxylate group of the γH2AX C-terminal tail. We also determined the crystal structure of the tandem BRCT domain of human BARD1, the in vivo binding partner of BRCA1. Our structure uncovers a degenerate phosphopeptide binding pocket that lacks the key arginine critical for phosphopeptide interactions in other BRCT proteins. Our biochemical studies reveal that a flexible tether links ankyrin and BRCT domains in BARD1. Furthermore, the linker is required for the interactions between the CstF-50 WD-40 domain and BARD1, allowing the BARD1 C-terminus to convey DNA damage signals directly to RNA polymerase. Finally, using protease-based and phosphopeptide pull-down assays, we directly assessed the structural and functional effects of 117 single amino acid substitutions in the BRCA1 BRCT domain derived from breast cancer screening programs. None of the variants showing enhanced sensitivity to proteolytic digestion were found to be active in peptide binding, indicating that these missense mutations contribute to BRCA1 loss of function through protein destabilizing effects. A subset of structurally stable variants was defective in peptide binding activity, suggesting that these variants may disrupt the phosphopeptide binding pocket. Taken together, the results reveal that 32% of the variants show structural stability and peptide binding activity that were indistinguishable from those of wild type.
Language
English
DOI
doi:10.7939/R33D32
Rights
License granted by Megan Lee (megan.lee@ualberta.ca) on 2009-07-22T17:25:13Z (GMT): Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of the above terms. The author reserves all other publication and other rights in association with the copyright in the thesis, and except as herein provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
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