Graduation Date

Winter 12-17-2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Cancer Research

First Advisor

Polina Shcherbakova

Abstract

Replicative DNA polymerases ε (Polε) and δ (Polδ) achieve high fidelity DNA synthesis through a precise balance of polymerization and exonucleolytic proofreading. Errors that escape proofreading are corrected by DNA mismatch repair (MMR). Ultramutated human cancers with proficient MMR carry alterations in the exonuclease domain of Polε, which were initially predicted to abolish proofreading. However, functional studies in yeast of the most recurrent Polε-P286R variant suggested defects beyond a loss of exonuclease activity. Indeed, biochemical analysis of the yeast Polε-P286R analog revealed increased polymerization capacity in addition to decreased proofreading, which enables efficient mismatch extension and bypass of replication-blocking non-B DNA structures. The functional consequences of many additional Polε variants seen in cancers remained poorly understood.

In this work, we modeled 20 cancer-associated Polε mutations in yeast. We show that the mutator effects are highly variable and only variants directly altering the DNA binding cleft of the exonuclease domain are mutators. Nearly all mutator effects exceed those of an exonuclease-null allele, suggesting that additional mechanisms contribute to mutagenesis. Furthermore, all mutator alleles are semidominant, suggesting that heterozygosity for the polymerase mutations is sufficient to drive tumorigenesis. Mutations affecting residues located further from the DNA binding cleft did not significantly elevate mutagenesis. We further show that Polε mutations occurring in MMR-deficient tumors are not strong mutators alone but can act synergistically with MMR loss to increase mutagenesis.

We used purified Polε variants to examine the consequences of mutations to enzyme function. We demonstrate that cancer-associated mutations produce polymerases with a wide range of biochemical alterations. Some variants have no proofreading activity while others have only minor exonuclease defects. Most cancer-associated variants, however, have greatly increased polymerase activity, similar to Polε-P286R. Unlike wild-type Polε, hyperactive Polε variants are highly efficient at synthesizing DNA when challenged with reduced dNTP levels. We propose that this characteristic could promote the survival of metabolically stressed cancer cells. It could also make mutator polymerases favored over wild-type during stress conditions, further increasing the genomic error rate and oncogenic phenotype. Together these results suggest that the fitness of the polymerase, rather than low fidelity alone, determines Polε variant pathogenicity.

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