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Molecular Basis for Ubiquitin and SUMO Recognition by RAP80, a DNA repair protein
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- Author / Creator
- Singh, Anamika
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The information contained in our genome is essential for the proper functioning of different physiological processes in cells that are required for our growth and survival, making the maintenance of genome integrity a task of utmost priority. Our cells have evolved a highly elaborate, complex and well regulated process of DNA repair that accompanies cell cycle regulation, chromatin remodeling and transcription of repair proteins, collectively known as, DNA damage response. Amidst different kinds of DNA damage, double strand breaks pose a serious threat to the cell survival, however most of the time these breaks are repaired by Non Homologous End Joining (NHEJ) or Homologous Recombination (HR). HR involves the end resection of broken DNA strands followed by a homology search, DNA synthesis and the ligation of the broken strand. The end resection of the broken DNA strand is a special feature of the HR process, which is precisely controlled by many factors and performed by proteins such as CtIP and the MRN complex. RAP80, a DNA repair protein was initially believed to promote HR over NHEJ by recruiting proteins to DNA damage sites that favor end resection, such as BRCA1 that forms a multi protein complex with Abraxas, BRCC36, BARD1 and RAP80, known as BRCA1 A complex. BRCA1, a major player of the DNA repair pathway is a crucial tumor suppressor protein whose mutations show strong links to breast and ovarian cancer. Recent advances suggest that although RAP80 recruits BRCA1 to the DNA damage sites, eventually it represses HR by sequestering BRCA1 within the complex and restricting its association with other complexes that endorse end resection. This decisive role played by RAP80 in the repair pathway choice is due to its ability to recognize Lys-63 linked ubiquitin chains and SUMO moieties through its ubiquitin and SUMO interacting motifs present at the N-terminal region. A single deletion mutation in the ubiquitin interacting motif of RAP80 has been linked with cases of familial breast cancer. Additionally, a mutation in the SUMO interacting motif of RAP80 has been linked to somatic mutations in cancer cells, highlighting the importance of ubiquitin and SUMO recognition in genome protection. We employed NMR spectroscopy, binding studies, stability studies and MD simulations to understand the molecular basis of ubiquitin and SUMO recognition of RAP80. We demonstrate that the ΔE81 mutation in the first ubiquitin interacting motif of RAP80 leads to a structural frameshift of the helix with causing modest changes in the stability. The prominent reason for the impaired binding of this mutant with ubiquitin is its loss of favourable electrostatic interaction, resulting in a loss of multivalent binding advantage. We also determine the binding affinity of the SUMO interacting motif of RAP80 with SUMO-2 using NMR methods and show that the phosphorylation of RAP80 by casein kinase 2 enhances its affinity for SUMO-2 by ~25 fold due to increased electrostatic interactions between the phosphorylated serine residues and the basic loop of SUMO-2 present at the binding site. Understanding these binding interactions from a structural and thermodynamic point of view is fundamental in order to develop a holistic understanding of the ubiquitin and SUMO recognition process. Our research has provided a closer look at the critical interactions in ubiquitin and SUMO signaling in DNA repair and has opened up new avenues for future research in DNA repair field.
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- Graduation date
- Fall 2017
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- Type of Item
- Thesis
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- Degree
- Doctor of Philosophy
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- 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.