The BLM Helicase Is Involved in the Repair of DNA Lesions Induced by Diverse Genotoxins

 
 
 
 

The BLM Helicase Is Involved in the Repair of DNA Lesions Induced by Diverse Genotoxins

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dc.contributor.advisor Groden, Dr. Joanna L. en_US
dc.contributor.author Behbehani, Gregory Kayvhan en_US
dc.date.accessioned 2008-07-08T16:20:18Z
dc.date.available 2008-07-08T16:20:18Z
dc.date.created 2007 en_US
dc.date.issued 2008-07-08T16:20:18Z
dc.identifier.uri http://rave.ohiolink.edu/etdc/view?acc_num=ucin1169583988 en_US
dc.identifier.uri http://hdl.handle.net/2374.OX/11731
dc.description Bloom’s syndrome (BS) is an autosomal recessive inherited disorder characterized by small size, immunodeficiency, and a striking predisposition to cancer. Cells from persons with BS demonstrate genomic instability, characterized by chromosome breaks, gaps, quadraradials, and a marked elevation in SCE. The gene mutated in BS has been identified and encodes a DNA helicase known as BLM. The mechanism by which BLM deficiency leads to cancer predisposition in persons with BS remains to be elucidated. My work focuses on understanding the role of the BLM helicase in the cellular response to DNA damage induced by diverse genotoxins. Using lymphoblast cell lines isolated from persons with BS, and from age- and sex-matched control individuals, I tested growth capacity in response to several genotoxic agents. I demonstrated BS cell sensitivity to mitomycin C (MMC), hydroxyurea (HU), and a low sensitivity to ionizing radiation (IR). To test sensitivity in vivo, Blm heterozygous mice were treated with large doses of IR and MMC. These experiments demonstrated that heterozygous mice have a slight sensitivity to IR, but surprisingly not to MMC. To confirm that BLM is involved in the response to DNA damage induced by these agents, I demonstrated that a GFP-BLM fusion protein could co-localize with phosphorylated H2AX at sites of DNA damage, and that this co-localization was much greater following DNA damage induced by MMC and HU than damage induced by IR. Lastly, I used flow cytometry to demonstrate that the kinetics of H2AX phosphorylation and de-phosphorylation are largely normal in BS cells, with only a slight delay in cell cycle progression following MMC exposure. I also investigated the formation of foci of H2AX phosphorylation in the breast cancer cell line MCF7 following treatment with the histone deacetylase inhibitors (HDI) trichostatin A (TSA) and sodium butyrate (NaB). I demonstrated that these foci were correlated with double strand DNA breaks as treatment of MCF7 cells with TSA and NaB resulted in formation of comet tails. I then demonstrated that a GFP-BLM fusion protein localized to the sites of H2AX phosphorylation induced by the HDIs, while a similar fusion protein lacking helicase activity did not localize to these sites. Lastly, these experiments demonstrated that BS cells are sensitive to TSA and NaB, and strongly suggested that BLM is required for response to changes in DNA metabolism induced by HDIs. Taken together the results of my experiments demonstrate that BLM plays a central role in the repair of DNA damage induced by a variety of genotoxins. My results support a model of BLM function in which DNA repair initiates normally in the absence of BLM, but that BLM is required for the correct and efficient completion of repair that proceeds through the pathway of homology-directed repair. Despite my work and similar work by others demonstrating the role of BLM in DNA damage response, whether or not incorrect DNA repair leads to cancer in persons with BS is still unknown. I have proposed a series of experiments to address this question. en_US
dc.format application/pdf en_US
dc.format 230p. en_US
dc.rights unrestricted en_US
dc.rights Copyright and permissions information available at the source archive en_US
dc.subject Bloom's syndrome en_US
dc.subject DNA repair en_US
dc.subject Cancer en_US
dc.subject H2AX en_US
dc.subject DNA damage en_US
dc.subject Ionizing Radiation en_US
dc.subject Mitomycin C en_US
dc.subject Hydroxyurea en_US
dc.subject Histone De-acetylase en_US
dc.title The BLM Helicase Is Involved in the Repair of DNA Lesions Induced by Diverse Genotoxins en_US
dc.type Electronic Thesis or Dissertation en_US
dc.degree.name PhD en_US
dc.degree.level doctoral en_US
dc.degree.discipline Medicine : Molecular Genetics, Biochemistry, & Microbiology en_US
dc.degree.grantor University of Cincinnati en_US
dc.contributor.publisher University of Cincinnati / OhioLINK en_US

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