| The identification of proteins interacting with the 53BP1 tandem Tudor domains | |
Abstract/OtherAbstract
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Tumor protein p53 binding protein 1 (53BP1) is a cell cycle checkpoint protein that is important in the early DNA double strand break (DSB) response signal pathway. Aberrant reduction or lack of 53BP1 is found in significant proportions of carcinomas. 53BP1 is recruited to DSB sites and forms foci through its tandem Tudor domain by recognizing dimethylated lysines in histones. The 53BP1 tandem Tudor (53BP1TT) domain consists of two tightly packed Tudor domains follow by a C-terminal alpha helix, and actively binds to methylated histone lysines H4K20 and H3K79. I hypothesized that 53BP1TT domain can potentially interact with non-histone targets, which contain methylated residues, and may be involved in the maintenance of genomic stability. The primary goal of the work presented in this thesis is to identify the proteins that interact with the 53BP1TT domain. I performed a proteomic screen by employing in vitro transcription/translation coupled reactions on pools of cDNA plasmids and identified two putative 53BP1TT targets, brahma-related gene 1 (BRG1), which is a chromatin remodeling catalytic subunit that has helicase and ATPase activities, and is thought to regulate transcription by altering the chromatin structure, and checkpoint kinase 1 (CHK1), which mediates DNA damage signal to downstream damage responsive proteins and initiates cell cycle checkpoint arrest. I demonstrated that both endogenous BRG1 and CHK1 interacted with the 53BP1TT domain in glutathione-S-transferase pulldown assays. Co-immunoprecipitation between ectopically expressed BRG1 and 53BP1 was observed. Interestingly, the interaction between endogenous BRG1 and 53BP1 was observed only after DNA damage. I mapped BRG1 dimethylated lysine K1375 as the site of interaction of the 53BP1TT domain. Using a BRG1K1375me2-specific antibody, I further detected BRG1 methylation at K1375 in HeLa cells. The detected methylation signal increased in response to DNA damage. By applying cross species sequence alignment on BRG1, the BRG1K1375 was found to be conserved throughout eukaryotes. Based on this finding, the K1375 containing domain was determined to be a putative 53BP1TT domain binding motif. The observation of the interaction between 53BP1 and BRG1 provides a potential link between DNA damage response and chromatin remodeling. |
Authors
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Chang, Kai-wei |
Contributors
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Stephane Richard (Supervisor) |
Publication Detail
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Publisher : McGill University Type : Electronic Thesis or Dissertation Format : application/pdf |
Date Detail
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2009 |
Subject
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Biology - Molecular |
Coverage
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Relation
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Source
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Copyright Information
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© Kai-wei Chang, 2009 |
Other Details
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Languages : en |
Export Citation
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