Date of Award

Summer 8-14-2015

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Programs

Cancer Research

First Advisor

Youri Pavlov, Ph.D.

Abstract

Cellular DNA is under constant attack by endogenous and exogenous DNA damaging agents that threaten genome integrity. Unrepaired DNA lesions often stall replicative DNA polymerases and are bypassed by translesion synthesis (TLS) to prevent replication fork collapse. TLS mechanisms are lesion- and species-specific, with prominent roles of specialized DNA polymerases with relaxed active sites. After incorporation of nucleotide(s) across from the lesion, the distorted primer termini are typically extended by DNA polymerase ζ (Pol ζ). As a result, Pol ζ is responsible for most DNA damage-induced mutations. Mechanisms of sequential polymerase switches and regulation of Pol ζ access to DNA in vivo remain unclear. Pol ζ shares two accessory subunits, called Pol31/Pol32 in yeast, with replicative Pol δ. Inclusion of Pol31/Pol32 in both holoenzymes requires a [4Fe-4S] cluster in the catalytic subunit C-terminal domains (CTDs). Disruption of the Pol ζ cluster or deletion of the POL32 gene attenuates induced mutagenesis.

Here we describe a novel mutation affecting Pol ζ, rev3ΔC. Rev3∆C lacks the entire CTD, the binding platform for Pol31/Pol32. This mutation provides insight into regulation of polymerase switches and further defines regulatory roles of the Pol ζ CTD. rev3ΔC strains are partially proficient in Pol32-dependent UV-induced mutagenesis. This suggests a role for Pol32 in TLS beyond binding Pol ζ, related to Pol δ. We examined several TLS regulatory proteins, including Mgs1 which can compete with Pol32 for binding PCNA. Overproduction of Mgs1 suppressed induced mutagenesis, but had no effect in rev3ΔC suggesting Mgs1 exerts its inhibitory effect by acting specifically on Pol32 of Pol ζ. This evidence for differential regulation of Pol δ/ζ Pol32 emphasizes complexity of polymerase switches. Spectra of mutations induced by UV in rev3∆C were examined to further define the regulatory role of Pol ζ CTD. Rev3∆C produced different mutational spectra than WT, progressively deficient first in transversions/frameshifts, then transitions, and altered upon increasing UV doses. This supports a fine-tuned role for the CTD in regulating Pol ζ function and highlights differential mechanisms activated by different UV doses.

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