The Role of Non-Replicative DNA Polymerases in Microsatellite Mutagenesis
Open Access
- Author:
- Jacob, Kimberly Dawn
- Graduate Program:
- Genetics
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- September 09, 2009
- Committee Members:
- Kristin Ann Eckert, Dissertation Advisor/Co-Advisor
Kristin Ann Eckert, Committee Chair/Co-Chair
Sarah Bronson, Committee Member
Ralph Lauren Keil, Committee Member
Lisa M Shantz, Committee Member
Maria J Baker, Committee Member - Keywords:
- mutagenesis
poymerae beta
polymerase IV
DNA replication - Abstract:
- Microsatellites, or short tandem repeats (STR), are sequences of 1-6 base pairs per unit repeat and are found throughout the human genome. Mutations in these sequences are sources of genetic variation and one proposed mechanism to explain these mutations is the polymerase slippage model. Although polymerase utilization of slipped DNA intermediates is a requisite step in this model, the cellular polymerase(s) responsible for microsatellite mutagenesis are unknown. In this study, we have examined the role of 2 non-replicative polymerases in microsatellite mutagenesis, Escherichia coli (E.coli) DNA Polymerase IV (Pol IV) and human DNA Polymerase β (Pol β). We investigated the loss of the E.coli dinB gene product (Pol IV) on mononucleotide and dinucleotide repeat stability. Additionally, we tried to determine the effect of knockdown of Pol β levels in human cells on mononucleotide microsatellite mutagenesis. To do this we used a Herpes Simplex Virus thymidine kinase (HSV-tk) gene episomal reporter system for microsatellite mutations. In E. coli we found that loss of dinB did not have an effect on microsatellite mutagenesis, but did cause a 4-fold reduction in the mutation rate within the coding region of the HSV-tk gene. Using an in vitro assay, we determined the mutational specificity of Pol β on mononucleotide G/C tracts. We found that Pol β makes greater than 80% of mutational events at the mononucleotide allele on both templates, which is in stark contrast to dinucleotide repeats. In human cells, we were unsuccessful at creating a knockdown of Pol β levels, however we did determine that there is an effect of lentiviral infection and selection on mutagenesis. In lentiviral infected and selected cells we observed a 3-fold reduction in overall mutation rate which was statistically significant. We also saw a shift in mutational specificity, from a bias toward coding region mutations in uninfected cells, toward mutations at the microsatellite repeat in those that were infected and selected.