Defense Date

2012

Document Type

Thesis

Degree Name

Master of Science

Department

Pharmacology & Toxicology

First Advisor

Lawrence F. Povirk

Abstract

Breast cancer is the second leading cause of cancer death in women according to the American Cancer Society. The standard treatment regimen for breast cancer involves ionizing radiation combined with surgery and chemotherapy. Ionizing radiation induces a complex signaling response in cells resulting in either growth arrest, senescence or cell death, and the cell killing after exposure to radiation results largely from DNA double-strand breaks (DSBs). There are two main mechanisms in mammalian cells responsible for repairing the DSBs; the primary mechanism is non-homologous end joining (NHEJ) and the secondary mechanism is homologous recombination (HRR). Previous studies showed that breast tumor cells depend mainly on NHEJ for repairing induced DNA damage. XRCC4 and XLF are two essential proteins in the NHEJ process. The interaction between XRCC4 and XLF (also called Cernunnos) is responsible for stimulating ligase IV for rejoining DNA ends. A single mutation on the XLF-binding interface of XRCC4 at M61, F106, M59 or D58 has been shown to disrupt its interaction with XLF and thus inhibiting NHEJ. Therefore, it is proposed that small natural cyclic peptides that bind to the XLF interface of XRCC4 near M61 and F106 can be identified through an mRNA display in vitro selection, and these peptides will inhibit NHEJ and thereby radiosensitize breast tumor cells. We have synthesized five DNA libraries that produced mRNA-peptide fusions containing a trillion unique peptide sequences that will be used for the selection of peptide inhibitors of the XRCC4/XLF interaction, and we have verified their randomness. Tagged wild-type and mutant versions of the head domain of XRCC4 protein, containing the XLF binding site, were successfully purified, and the wild-type version was applied to initial stages of selection of inhibitory peptides by mRNA display. The percentage of the mRNA-peptide fusions that bound to the XRCC4157 after the first round was 2.1%. The recovery after the second and third rounds was 1.14% and 2%, respectively. Results obtained thus far, although preliminary, suggest that the mRNA display method can be successfully applied to the XLF/XRCC4 interaction.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

August 2012

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