DNA Polymerase Epsilon (Pol ϵ) is one of three main eukaryotic Pols responsible for nuclear DNA replication. The Pol ϵ holoenzyme is comprised of four subunits, termed p261, p59, p17, and p12, with the largest subunit containing the DNA polymerase and 3' to 5' exonuclease (exo) proofreading activities. In addition to nuclear DNA replication, Pol ϵ participates in DNA repair, recombination, maintenance of epigenetic states and S-phase regulation, though the contribution of the smaller subunits to these processes is largely unknown. I set out to identify functions of the p12 subunit through determining post-translational modifications and protein-protein interaction partners. This approach found that p12 is likely constitutively phosphorylated and that p12 ubiquitylation dynamics may be important during replication stress and fork stalling. p12 also putatively interacts with proteins involved in maintaining genome stability including TOP1, HSP90, nucleolin and PRKDC.
A larger portion of my project involved studying the role of cancer-associated mutations within the exo domain of POLE1, the gene encoding the p261 subunit. Tumors harboring these POLE1 mutations are hypermutated, with mutation frequencies exceeding 100 mutations/Mb. However, unlike POLE1 wild type hypermutated tumors, the POLE1 mutant tumors are microsatellite stable (MSS). With our collaborators at the Baylor College of Medicine Human Genome Sequencing Center and the Memorial Sloan Kettering Cancer Center, we determined that C→A and C→T base pair substitutions are highly elevated in these tumors relative to others, specifically at TCT and TCG motifs, respectively. I purified recombinant Pol ϵ and showed that several cancer mutant constructs, including S459F, P286H/R, L424V/I, and D275A/E277A, had elevated error rates for all 12 base pair substitutions and frameshifts, with the same propensity to make TCT→TAT mutations in vitro. In order to study the mechanism of how these specific mutations are made upon Pol ϵ exo inactivation in vivo, I constructed a knock-in cell culture model. In this model, I used targeted knock-in approach to introduce the D275A/E277A double amino acid substitution at the genomic POLE1 locus using an engineered recombinant adeno-associated virus. Mutation rates and base pair substitution error rates were both increased in a mismatch repair null background upon Pol ϵ exo inactivation. TCT→TAT basepair substitutions had the highest increase in error rate in the engineered cell culture system, as was seen in the POLE tumors, demonstrating the utility of this system for studying the relationship between Pol ε-dependent mutagenesis and tumor formation.
The high rate of TCT→TAT mutagenesis has interesting consequences in tumors. The nucleotide preference of Pol ϵ variants leads to increases in recurrent nonsense mutations in key tumor suppressors such as TP53, ATM and PIK3R1. Moreover, strand-specific mutation patterns are seen during replication of these genes. Mapping of TCT→TAT hotspot mutations around known origin of replications provided the first direct evidence that Pol ϵ is the leading strand polymerase in human cells. The strand specificity of these mutations and high abundance in human tumors allows for unique identification of eukaryotic origins of replication.
|Advisor:||Pursell, Zachary F.|
|Commitee:||Belancio, Victoria P., Engel, Astrid, Landry, Samuel, Lustig, Arthur J.|
|School Location:||United States -- Louisiana|
|Source:||DAI-B 76/09(E), Dissertation Abstracts International|
|Subjects:||Molecular chemistry, Biochemistry, Oncology|
|Keywords:||Cancer, Dna polymerase, Epsilon functions|
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