Characterization of the In Vivo Functions of Y-Family DNA Polymerases Kappa and Rev1

DNA translesion synthesis (TLS) is a mode of damage tolerance utilized by cells experiencing replicative stress as a result of DNA damage. TLS is characterized by the synthesis of DNA opposite template lesions, a process that requires the function of specialized DNA polymerases. My studies focus on particular aspects of Rev1 and Polymerase kappa (Polκ) function in vivo. One of the main goals of my work was to characterize the conservation of the interaction between the Rev1 C-terminus and other Y-family polymerases (demonstrated in vertebrates) in lower eukaryotic species. I showed that these interactions are not conserved in the yeasts S. cerevisiae or S. pombe, nor in the nematode C. elegans, yet they are conserved in the fruit-fly Drosophila melanogaster. Furthermore, I experimentally determined the requirements of the Y-family polymerase interactions in Drosophila and mouse for comparative analysis. The results of this study concluded that special consideration should be exercised when making mechanistic extrapolations regarding translesion DNA synthesis from one eukaryotic system to another. Another central goal of this work was to identify new Rev1 protein interaction partners in S. cerevisiae. I created a yeast two-hybrid library for screening with Rev1 bait. After identifying and verifying interaction partners for Rev1, I further investigated the epistatic relationship of these genes to Rev1 with respect to UV-radiation. The candidate genes investigated do not appear to function in a synonymous pathway to Rev1 in the response to UV-induced stress. A final goal of this work was to determine the spontaneous mutation frequencies of Polκ-/- mice using a well-validated in vivo mutation detection system. I found that somatic spontaneous mutation frequencies are elevated in 9 and 12-month old Polκ-/- kidney and liver, but not wild-type or Polη-/- tissues. Furthermore, I characterized the mutation spectra of these mice and observed a specific elevation in transversion mutations G:C>C:G and G:C>T:A. These data are consistent with previous observations of Polκ-/- mice, and hint at what types of spontaneous damage may be naturally occurring substrates for Polκ in vivo.