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Molecular basis of TopBP1 BRCT domain interactions in the DNA damage response Open Access


Other title
DNA repair
X-ray crystallography
Type of item
Degree grantor
University of Alberta
Author or creator
Leung, Charles
Supervisor and department
Glover, Mark (Biochemistry)
Examining committee member and department
West, Frederick (Chemistry)
Guarne, Alba (McMaster University, Biochemistry and Biomedical Sciences)
Young, Howard (Biochemistry)
Holmes, Charles (Biochemistry)
Department of Biochemistry

Date accepted
Graduation date
Doctor of Philosophy
Degree level
Topoisomerase II-beta binding protein 1 (TopBP1) is a critical regulatory protein that integrates diverse signals in the DNA damage response. In response to DNA replication stress, TopBP1 participates in a series of protein interactions to collectively activate the key Ser/Thr kinase, Ataxia telangiectasia and Rad3 related (ATR), and control the DNA replication checkpoint. These phosphorylation-dependent interactions are mediated by the numerous conserved BRCT domains within TopBP1. Our studies utilize X-ray crystallography in combination with other biochemical and biophysical techniques to elucidate the molecular basis underlying various TopBP1 BRCT-mediated interactions in DNA damage response signalling. Contrary to previous studies suggesting that the single BRCT6 domain of TopBP1 recognizes a phospho-peptide of the transcription factor, E2F1, and binds to poly(ADP-ribose) chains, the crystal structure of BRCT6 provides evidence that both the phospho-peptide binding pocket and PAR-binding motif are non-functional. Our studies of distinct phospho-peptide interactions involving the tandem BRCT7/8 and BRCT4/5 repeats of TopBP1 shed light on critical interactions required for activation of ATR and the DNA replication checkpoint. In addition, the mode of phospho-peptide recognition presented by these BRCT repeats uncover new and exciting perspectives in BRCT domain function that were previously unknown. Analysis of the structures of TopBP1 BRCT7/8 and in complex with a BACH1 phospho-peptide provides the first demonstration of pThr specificity and an uncharacteristic plasticity at the BRCT domain interface for canonical tandem BRCT domains. Our structural investigations of interactions between TopBP1 BRCT4/5 and a MDC1 phospho-peptide reveal a novel tandem BRCT domain packing arrangement, as well as an unexpected dimerization of two BRCT4/5 domains needed to stabilize interactions with a single phospho-peptide. Taken together, our studies of TopBP1 BRCT domain interactions provide molecular insights into crucial protein interactions involved in DNA replication checkpoint signalling and also highlight the extraordinary functional diversity of BRCT domains.
License granted by Charles Leung ( on 2011-09-22T16:41:05Z (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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