Graduation Date

Summer 8-12-2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Genetics, Cell Biology & Anatomy

First Advisor

Youri I. Pavlov

Abstract

Translesion DNA synthesis (TLS) effectively bypasses DNA lesions induced by environmental and endogenous mutagens, protecting cells from replication fork arrest, double-strand breaks, and ultimately cell death. Polymerase ζ (Pol ζ), regulated by ubiquitylation of accessory protein PCNA, plays a key role in TLS. Pol ζ acts as the extender from distorted DNA template/primer termini after damage bypass accomplished by other TLS polymerases exploiting a scaffold of cytidyl transferase Rev1. Pol ζ also makes errors during the synthesis of the downstream region. Both transactions lead to mutations. More than 90% of induced and half of spontaneous mutations in yeast are dependent on Pol ζ. Yeast Pol ζ is a four-subunit enzyme, comprised of catalytic subunit Rev3 and accessory subunits Rev7, Pol31 and Pol32. The C-terminus domain (CTD) of Rev3 accommodates a 4Fe-4S cluster required for the interaction with Pol31. Yeast strains with point mutations abolishing binding of Rev3 to Pol31, are immutable after exposure to UV and other DNA damaging agents. Surprisingly, in our laboratory, it was found that yeast rev3-ΔC strains encoding a variant of Rev3 without CTD are UV mutable. Mutation spectra were altered at high UV doses, but showed a hallmark signature of Pol ζ, indicating that the main properties and functionality of the polymerase in TLS were not abolished when the CTD was absent.

Because TLS is a promising target for cancer therapy, it is imperative to better understand the roles of Rev3 CTD in Pol ζ functions in TLS. We constructed strains with the regulatable expression of genes encoding for Pol ζ and studied the effects of levels of production of Pol ζ variants on UV and spontaneous mutagenesis; we examined the roles of the ubiquitylation of PCNA in these processes. We purified different variants of Pol ζ and explored subunit interactions between Rev3 CTD-less Pol ζ and the Rev1 scaffold protein. We also tested parameters of DNA synthesis by Pol ζ variants on correctly paired, mispaired and abasic-site-containing DNA primer-template substrates. Rev1 co-purifies with Rev3-ΔC-Rev7 (Pol ζ2-ΔC) and facilitates the interaction between Pol ζ2-ΔC and Pol32. Pol ζ2-ΔC is catalytically active and can bypass the abasic site, but its catalytical activity is much less active than full-length four-subunit Pol ζ. The addition of Rev1 to Pol ζ2-ΔC inhibits its activity in vitro.

In conclusion, Pol ζ2 without the C-terminal part is a relatively weak but functional polymerase with specificity similar to four-subunit Pol ζ. Rev1 promotes interaction of Pol ζ2-ΔC with Pol32, stimulates UV-induced and spontaneous mutagenesis in vivo, but inhibits the activity of Pol ζ2-ΔC in vitro. These findings reveal the complexity of protein-protein interactions that regulate the functions of Rev3 CTD in Pol ζ during TLS.

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