Doctor of Philosophy (PhD)
Polina V. Shcherbakova, Ph.D.
Despite multiple DNA repair pathways, DNA lesions can escape repair and compromise normal chromosomal replication, leading to genome instability. Cells utilize specialized low-fidelity Translesion Synthesis (TLS) DNA polymerases to bypass lesions and rescue arrested replication forks. TLS is a highly conserved two-step process that involves insertion of a nucleotide opposite a lesion and extension of the resulting aberrant primer terminus. The first step can be performed by both replicative and TLS DNA polymerases and, because of non-instructive DNA lesions, often results in a nucleotide misincorporation. The second step is almost exclusively catalyzed by DNA polymerase ζ (Polζ). This unique role of Polζ allows the misincorporated nucleotide to remain in DNA, resulting in a mutation. Because of the low fidelity of Polζ, a processive copying of undamaged DNA beyond the lesion site by this polymerase is expected to be mutagenic. To restore faithful DNA replication, Polζ must be immediately replaced by an accurate replicative DNA polymerase. However, in vivo evidence for this is lacking.
To elucidate the late steps of TLS, we aimed to determine the extent of error-prone synthesis associated with mutagenic lesion bypass in yeast. We demonstrate that TLS tracts can span up to 1,000 nucleotides after lesion bypass is completed, leading to more than a 300,000-fold increase in mutagenesis in this region. We describe a model explaining how the length of the error-prone synthesis may be regulated and speculate that Polζ could contribute to localized hypermutagenesis, a phenomenon that plays an important role in cancer development, immunity and adaptation.
To gain further insight into the mechanisms of Polζ -dependent mutagenesis, we determined how the increase in dNTP levels occurring in response to DNA damage in yeast affects Polζ function. Surprisingly, increasing the dNTP concentrations to “damage-response” levels only minimally affected the activity, fidelity and error specificity of Polζ, suggesting that, unlike the replicative DNA polymerases, Polζ is resistant to fluctuations in the dNTP levels. Importantly, we demonstrated that Polζ -dependent mutagenesis in vivo does not require high dNTP levels either. Altogether, our results suggest a novel function of Polζ in bypassing lesions or other impediments when dNTP supply is limited.
Kochenova, Olga V., "DNA Polymerase Zeta-Dependent Mutagenesis: Molecular Specificity, Extent of Error-Prone Synthesis, and the Role of dNTP Pools" (2016). Theses & Dissertations. 153.