Author ORCID Identifier

https://orcid.org/0000-0002-7661-272X

Date of Graduation

12-2020

Document Type

Dissertation (PhD)

Program Affiliation

Epigenetics and Molecular Carcinogenesis

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Richard Wood

Committee Member

Blaine Bartholomew

Committee Member

Francesca Cole

Committee Member

Nayun Kim

Committee Member

Kevin McBride

Abstract

DNA polymerase ζ (pol ζ) is a specialized polymerase that protects cells from the consequences of DNA damage and stress. Without pol ζ function mammalian cells and yeast are exquisitely sensitive to genotoxic stresses including ultraviolet irradiation and platinum compounds such as the chemotherapeutic agent cisplatin. This elevated sensitivity arises from the central role of pol ζ in lesion bypass. Pol ζ is also required to protect cells from endogenous genomic damage or stress. In primary mammalian cells, the function of pol ζ is required for genome protection and normal proliferation. Immortalized Rev3lknockout (KO) MEFs are under considerable constitutive genomic stress, as demonstrated by elevated levels of micronuclei formation. As the cellular consequences of this stress remain unexplored, genome-wide transcriptional changes were measured in Rev3l KO MEFs. The work in this thesis demonstrates that loss of pol ζ promotes activation of an innate immune response.

Rev3l knockout MEFs had over 1000 genes that were either upregulated or downregulated more than 4-fold. Importantly, this revealed that loss of Rev3linduced an increase in expression of known interferon-stimulated genes indicating that the innate immune system may be activated. Increased expression of selected interferon-stimulated genes was confirmed at the mRNA and protein levels in Rev3lKO MEFs.

Recently DNA damage has been shown to activate the innate immune system as another level of the DNA damage response. Micronuclei and other forms of cytosolic DNA, that can arise from genomic stress, can activate the cytosolic DNA sensor cGAS which promotes an interferon response with its signaling partner, STING. Knockdown of cGAS and STING in Rev3l KO MEFs significantly reduced expression of interferon-stimulated genes indicating that loss of pol ζ promotes activation of the cGAS-STING pathway. In sum the work in this thesis demonstrates that cells with defective pol ζ are under a constitutive activation of the cGAS-STING pathway. Pol ζ is already being explored as a therapeutic target to sensitize tumors to chemotherapeutic agents, like cisplatin. This work suggests that targeting pol ζ may also engage the innate immune system which could widen the uses of targeting pol ζ in cancer treatment.

Keywords

DNA damage, genome instability, DNA polymerase ζ, cGAS, STING

Available for download on Monday, December 06, 2021

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