NYMC Student Theses and Dissertations

Date of Award

3-29-2019

Document Type

Master's Thesis - Open Access

Degree Name

Master of Science

Department

Biochemistry and Molecular Biology

First Advisor

Zhongtao Zhang

Second Advisor

Marietta Lee

Third Advisor

Ernest Lee

Abstract

DNA damages that cause double-strand breaks (DSBs) to the chromosome are most harmful. Subsequent choices have critical consequences for cell fate. Without repair, cells will face certain death. Low-fidelity repair will introduce mutations that could transform the cells, leading to carcinogenesis.

How cells make the decision is not well-understood. A single DSB can lead to apoptosis for some cells, whereas others can repair up to 25 DSBs and survive. It has been postulated that decision to repair DSBs is a stochastic process.

In the nucleus, DSBs elicit a cascade of signaling events that require the recognition, protection, processing, and subsequent repair of these breaks. Repair through homologous recombination (HR) process preserves genomic stability. The proteins involved in HR, such as MRN, ATM, RAD51, BRCA1/BRCA2 and others have been extensively characterized. Loss of function mutations of these proteins renders cells unable to repair DSBs through HR and increases propensity for genomic mutagenesis.

We hypothesize that nuclear signaling events are insufficient for cellular decision to repair DSBs through HR. In addition, the decision also relies upon the presence of pro-survival factors and subsequent activation of signaling pathways. Previous studies have indicated that phosphorylation of proteins such as BRCA1, BRCA2, or RAD51 might play a role in the regulation of HR activity. An example involves the phosphorylation of RAD51 by kinases downstream of phosphatidylinositol 3-kinase-related kinase (PI3K) pathway. However, the functions of many signaling molecules in DNA damage response are still uncertain. Using specific inhibitors of protein kinases, we aim to delineate the signaling molecules involved during HR repair.

Using a cellular HR-reporter assay, we demonstrate that the activation of mitogenic MAP kinase pathway has no impact on HR activity, whereas PI3K pathway is critical for cellular decision of HR repair. Among the downstream PI3K pathways, inhibition of Akt and PDK significantly suppresses HR repair.

Our results indicate that extracellular stimuli can regulate DNA repair process, especially HR. These findings could have broad implications in cancer therapy and gene therapy. Kinase inhibitors could enhance the cancer killing ability of DSB inducers such as platinum derivatives and topoisomerase inhibitors. On the other hand, growth factors could enhance the gene-editing efficacy through enhanced HR in techniques relying on HR such as CRISPR.

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