Michael I. Leffak, Ph.D. (Advisor); Michael P. Markey, Ph.D. (Committee Member); Kwang-Jin Cho, Ph.D. (Committee Member)
Master of Science (MS)
To study microsatellites instability and their repair pathways a dual fluorescent (DF2) and selectable (ganciclovir sensitive/ thymidine kinase (TK) expressing) cell system was assayed using replication fork stalling agents hydroxyurea and telomestatin. These cell lines carried ectopically integrated microsatellites derived from the Dystrophia Myotonica Protein Kinase (DMPK) gene ((CTG)102 microsatellite), or an 88 bp polypurine/ polypyrimidine (Pu/Py) repeat from the PKD-1 locus, inserted into a FLP recombinase target site. These microsatellites form non-B DNA structures in -vivo and in-vitro causing replication fork stalling and double strand breaks. DF2 myc (CTG)102 -TK cells treated with hydroxyurea were assayed for mutagenesis of the thymidine kinase gene (ganciclovir resistance) in the presence or absence of Polymerase Deltas 3rd subunit (POLD3). Knockdown in POLD3 lead to a decrease in TK mutagenesis numerous enough in cells possessing wild type POLD3 activity that mutation lead to increased survivability in the presence of ganciclovir but cell senescence without as demonstrated via Resazurin assay. Because break induced replication (BIR) and its mutagenic potential rely on the POLD 3rd subunit Polymerase Delta these results indicate that hydroxyurea damage of (CTG)102 microsatellite is repaired by BIR. Cell lines containing an ectopically integrated Pu/Py repeat were also constructed and analyzed for BIR mutagenesis.
Department or Program
Department of Biochemistry and Molecular Biology
Year Degree Awarded
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