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

  • Author / Creator
    Leung, Charles
  • 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.

  • Subjects / Keywords
  • Graduation date
    Fall 2011
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
  • Institution
    University of Alberta
  • Degree level
  • Department
  • Supervisor / co-supervisor and their department(s)
  • Examining committee members and their departments
    • West, Frederick (Chemistry)
    • Guarne, Alba (McMaster University, Biochemistry and Biomedical Sciences)
    • Holmes, Charles (Biochemistry)
    • Young, Howard (Biochemistry)