Graduation Date

Fall 12-18-2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Genetics, Cell Biology & Anatomy

First Advisor

Dr. Polina V. Shcherbakova

Abstract

Genome integrity is necessary to prevent mutations and disease. During eukaryotic DNA replication, DNA polymerases ε (Polε) and δ (Polδ) synthesize the leading and lagging strand, respectively. Polε and Polδ also have exonuclease activity that acts in series with post-replicative mismatch repair (MMR) to remove replication errors. Defects in proofreading and MMR lead to an increase in mutations and cause cancer in humans. This dissertation focuses on several unresolved issues involving the relationship between Polε and Polδ in replication error avoidance. First, despite an abundance of data supporting the one-strand-one-polymerase replication fork model, defects in the fidelity of Polε have a much weaker impact on mutagenesis than analogous Polδ defects. It has been proposed, but not directly tested, that Polδ contributes more to mutation avoidance because it proofreads mismatches created by Polε in addition to its own errors. In this work, we sought to explicitly test this idea. Second, the most common cancer-associated Polε variant, P286R, has recently been discovered to possess unusual and puzzling properties. Despite the location in the exonuclease domain, it produces a mutator effect far exceeding the effect of Polε exonuclease deficiency. The purified yeast analog, Polε-P301R, has increased DNA polymerase activity, which is thought to underlie its high mutagenicity, but the exact mechanism remains unclear. We aimed to investigate the impact of the P301R substitution on the function of Polε as the leading strand polymerase, and the removal of Polε errors by error correction mechanisms in vivo.

To test the hypothesis that Polδ proofreads errors made by Polε, we measured mutation rates in yeast strains harboring a nucleotide selectivity defect in one polymerase and a proofreading defect in the other. We show that Polδ can proofread errors made by Polε, but Polε cannot proofread errors made by Polδ. To investigate the role of Polε-P301R at the replication fork, we measured the accumulation of strand-specific replication errors across a well-defined replicon in yeast. We found that, despite exceptional polymerase activity, Polε-P301R is a dedicated leading strand polymerase. We further show that both Polδ proofreading and MMR remove errors incorporated by Polε-P301R and are required for viability of Polε-P301R cells. In summary, by demonstrating Polδ-dependent extrinsic proofreading, we resolved the discrepancy between the one-strand-one-polymerase model and the stronger impact of Polδ defects on genome stability. Using the hyperactive Polε-P301R, we further demonstrate the unexpected ease of polymerase exchange in vivo and its critical role in preventing catastrophic accumulation of errors on the leading strand. Our results also explain the apparent incompatibility of Polε variants and MMR defects in cancers.

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