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Double-Strand DNA Break Repair By Homologous Recombination Contributes To The Preservation of Genomic Stability In Mouse Embryonic Stem Cells

Tichy, Elisia D.
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1265989840

Abstract Details

2010, PhD, University of Cincinnati, Medicine : Cell and Molecular Biology.
The foundation of proper embryonic development involves the precise control of embryonic stem (ES) cell growth, proliferation, and subsequent differentiation. DNA damage accumulated in the early phases of this process has the potential to affect multiple cell lineages and thus the overall health and survival of the organism. Thus, ES cells must have evolved mechanisms to keep genetic integrity pristine. To test this proposition, the responses of ES cells and differentiated cells to DNA double strand breaks (DSBs) were compared, with a focus on the extent to which different repair pathways are utilized. We show that mouse ES cells rapidly repair DNA DSBs after exposure to etoposide. To establish which of the major DNA DSB repair pathways predominate in these cells, homologous recombinational repair (HRR), non-homologous end joining (NHEJ), and microhomology-mediated end joining (MMEJ) repair were compared, to test the hypothesis that ES cells preferentially repair DNA damage using high fidelity repair pathways. While levels of proteins encoded by genes involved in HRR and MMEJ were highly elevated in ES cells compared to isogenic mouse embryonic fibroblasts (MEFs), those for NHEJ were quite variable, with DNA Ligase IV expression low in ES cells. The half-life of DNA Ligase IV at both the protein and RNA level were also low in ES cells. Reporter plasmids that distinguish between the various DSB repair pathways showed that ES cells predominantly use HRR to repair DSBs, while NHEJ is minimally detectable under basal conditions. MMEJ is also apparent in ES cells at a level similar to that in MEFs. Following induction of DSBs, ES cells tended to differentiate, decreasing HRR with concomitant decreased Rad51 expression. Attempts to elevate NHEJ in ES cells by increasing the abundance of DNA Ligase IV protein expression by overexpression or inhibiting its degradation were unsuccessful. When ES cells were induced to differentiate by administration of all trans retinoic acid (ATRA), however, the level of DNA ligase IV protein increased, as did the capacity to repair by NHEJ. The data suggest that preferential use of HRR rather than error-prone NHEJ may represent an additional layer of genomic protection and that the limited levels of DNA ligase IV may account for the low level of NHEJ activity. The robust HRR activity in ES cells appears to be regulated by a specialized mechanism. Rad51, an E2F target gene, displays robust protein expression that is regulated by a different mechanism than another E2F target gene, the DNA replication gene, PCNA. While PCNA protein is highly expressed in ES cells as a result of increased protein stability, Rad51 protein has a relatively short protein half-life. No significant differences can be identified between the two genes at the RNA transcriptional or stability levels, suggesting that the regulation of Rad51 protein in ES cells occurs at the translational or post-translational level.
Peter Stambrook, PhD (Committee Chair)
Sohaib Khan, PhD (Committee Member)
James Stringer, PhD (Committee Member)
Alvaro Puga, PhD (Committee Member)
Ann Akeson, PhD (Committee Member)
148 p.
Cellular Biology
Embryonic stem cells; DNA repair; Homologous recombination; Non-homologous end joining; microhomology-mediated end joining

Recommended Citations

Citations

  • Tichy, E. D. (2010). Double-Strand DNA Break Repair By Homologous Recombination Contributes To The Preservation of Genomic Stability In Mouse Embryonic Stem Cells [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1265989840

    APA Style (7th edition)

  • Tichy, Elisia. Double-Strand DNA Break Repair By Homologous Recombination Contributes To The Preservation of Genomic Stability In Mouse Embryonic Stem Cells. 2010. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1265989840.

    MLA Style (8th edition)

  • Tichy, Elisia. "Double-Strand DNA Break Repair By Homologous Recombination Contributes To The Preservation of Genomic Stability In Mouse Embryonic Stem Cells." Doctoral dissertation, University of Cincinnati, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1265989840

    Chicago Manual of Style (17th edition)

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This open access ETD is published by University of Cincinnati and OhioLINK.