Date of Award

Summer 8-15-2016

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Cell Biology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Protecting the genome is a vital aspect of safeguarding organismal health. Inability to efficiently and effectively replicate the genome or repair damage the genome may encounter can lead to mutational accumulation or senescence, both of which are drivers of multiple diseases including cancer. Understanding the mechanisms by which the genome is maintained, as well as the consequences of repeated rounds of replication or exposure to DNA damaging agents, will allow for greater understanding of the diseases they promote as well as development of targeted therapies aimed at mitigating the detrimental effects of genomic insult. The first section of my work focuses on cellular senescence, a consequence of both aging and DNA damage. Aging is a significant risk factor for the development of cancer. The increase in disease in aged individuals is due in part to the time required for epithelial cells to accumulate mutations necessary to become tumorigenic, and by the increase in senescent cells within their tissues. Senescent cells express a coordinately upregulated family of pro-tumorigenic factors termed the senescence-associated secretory phenotype, or SASP. The SASP is rich in growth factors, immune modulators, and matrix remodelers that together create an environment primed for tumor development. Here, I study the mechanisms that regulate expression of the SASP in response to DNA damage. In cells exposed to a senescence-inducing stimulus but that do not yet display classical senescence markers, SASP expression is reliant upon active transcription for upregulation. However, senescent cells maintain SASP factor upregulation through post-transcriptional stabilization. Previous work demonstrated that this transition is dependent on the stress kinas p38MAPK, and p38MAPK inhibition prevents upregulation and stabilization of SASP factor mRNAs by modulating the binding of AUF1, a protein that binds regulatory sequences in target mRNAs and largely targets them for degradation. AUF1 is not a direct target of p38MAPK activity, however. In this work, I demonstrate that AUF1 regulation and therefore mRNA stabilization of SASP factors occurs through the p38MAPK-MK2-HSP27 pathway. Furthermore, inhibition of MK2 activity abrogates the ability of senescent cells to promote preneoplastic cell growth, suggesting MK2 inhibition is an attractive therapeutic target that warrants further investigation. In the second section of this work, the role of the essential helicase/nuclease Dna2 in DNA replication is investigated. Maintaining genomic stability is essential to preventing mutational accumulation and cancer development, and both elevated Dna2 expression levels in human cancers and heterozygous deletion in mice have been linked to cancer incidence and poor disease outcome. Dna2’s role in DNA replication was initially described in yeast, where it was hypothesized to function in lagging strand DNA replication, namely in Okazaki fragment maturation. However, work described here demonstrates that while shRNA-mediated depletion of Dna2 results in activation of the replication stress checkpoint and phenotypes indicative of replication defects, Okazaki fragment maturation is not measurably affected in human cells. Therefore, Dna2 plays an additional role in DNA replication that is required to ensure high fidelity duplication of the genome during cell division. Together, my work highlights the essential nature of genomic preservation, and consequences that arise when genome stability is threatened.

Language

English (en)

Chair and Committee

Sheila A. Stewart

Committee Members

Peter Burgers, Nicholas Davidson, Sergej Djuranovic, Joshua Rubin, Zhongsheng You

Comments

Permanent URL: https://doi.org/doi:10.7936/K7Q23XN8

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