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Dissertation
Characterization of the role of transcription factor Sp1 and its ATM-mediated phosphorylation in the cellular response to DNA damage
Doctor of Philosophy (Ph.D.), Drexel University
Sep 2008
DOI:
https://doi.org/10.17918/00010130
Abstract
DNA double strand breaks (DSBs), the most lethal form of DNA damage, are caused by endogenous and exogenous sources of reactive oxygen species (ROS). In response to DSBs, the MRN complex, Mre11, Nbs1, and Rad50, localizes to DSBs and activates the ataxia telangiectasia muted kinase (ATM), resulting in phosphorylation of substrates involved in DNA damage recognition and repair, checkpoint activation and apoptosis. We have found that exposure to DNA damaging agents, such as H₂O₂, ionizing radiation (IR), ultraviolet light (UV), and several chemotherapeutic drugs induces phosphorylation of Sp1, a transcription factor regulating expression of proteins involved in DNA synthesis and cell cycle progression. We have identified serine 101 of Sp1 to be a specific target of ATM in response to ail of these agents. Mutation of S101 to alanine results in greatly reduced phosphorylation of Sp1 in response to DNA damaging agents. Cells depleted of Sp1 by siRNA show decreased viability which can be rescued by expression of Wt Sp1 but not by expression of Sp1S101A. We have made a phospho-specific Sp1 antibody to serine 101 ([gamma]Sp1101). The phosphorylation of Sp1 is rapid and transient in response to H₂O₂, IR, and some drugs, which parallels the phosphorylation of histone variant, [gamma]H2AX, a marker of DSBs. Using immunofluorescence, we have demonstrated in cells treated with IR (0.5 Gy) that [gamma]Sp1101 forms discrete foci which co-localize with [gamma]H2AX. Using laser micro-irradiation and by creating DSBs at specific endogenous endonuclease sites, we have demonstrated that ySp1101 is specifically recruited to DSBs. The recruitment of Sp1 to the DSB appears to be through interaction with Nbs1; depletion of Nbs1 using siRNA abolishes the phosphorylation of Sp1 and its recruitment to DSBs. In contrast, the phosphorylation of Sp1 in response to UV is delayed compared to H2AX. Localization of [gamma]Sp l01 in response to UV varies throughout the cell cycle, and largely overlaps with the distribution of phospho-ATM and [gamma]H2AX. The phosphorylation of S101 is inhibited in cell lines deficient in nucleotide excision repair. Together, these data provide evidence for a role of Sp1 in the cellular response to DNA damage and as a potential biomarker.
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Details
- Title
- Characterization of the role of transcription factor Sp1 and its ATM-mediated phosphorylation in the cellular response to DNA damage
- Creators
- Crystal Marie Kelly
- Contributors
- Jane Clifford (Advisor) - Drexel University, Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- viii, 315 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Biochemistry and Molecular Biology; College of Medicine; Drexel University
- Other Identifier
- 991021888745304721