Access Type

Open Access Dissertation

Date of Award

January 2020

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry

First Advisor

Ashok S. Bhagwat

Abstract

Majority of B-cell lymphoma and leukemia originates in germinal centers and over express AID resulting high levels of uracils and eventually AP sites. Treating cells with a small alkoxyamine AA3 with a terminal alkyne group specifically killed B cell lymphoma cell lines without killing normal human B cells and non-hematological cancers. To study the cytotoxicity of AA3, I designed and synthesized a series of AA3 analogs with and without the alkyne group. Using cytotoxicity assays I demonstrated that the alkyne functionality plays a major role in AA3 cytotoxicity in B-cell cancers and further showed that the effectiveness is modulated by the location of alkyne group and the distance between the alkyne and the alkoxyamine groups. Overall, we found a new class of alkoxyamines that cause cytotoxicity specifically in B cell cancers. One of the AA3 analogs called AA6 was further used to develop a novel Cu free click chemistry tool to quantify AP sites or uracils in DNA. This technique produces reproducible data and can apply to quantify wide range of genomic uracils.

A great deal of research effort has been devoted to understanding how AID is targeted to the Ig genes or mis-targeted to oncogenes or other genomic loci. These studies are based mutations that results from AID activity. However, uracils created by AID can be repaired through error-free pathways masking the effects of AID. Therefore, to get an accurate picture of AID targeting one must study uracils that results from AID activity.

Therefore, in this study UDP-seq technique was employed to generate uracilomes created by human AID (Crystal AID). From the studies based on bacteria, I found that uracilated peaks are enriched with tRNA genes. This is consistent with recent mutational studies using AID, APOBEC3B as well as pull-down studies in APOBEC3A. Crystal AID was shown to have a bifurcated substrate binding surface which supports structured substrate recognition such as structured tRNAs, which could be the reason for the enrichment of tRNA genes in the pull-down. CH12F3 was used as the mammalian cell line to develop uracilomes created by AID. Along with WT and UNG-/-, MSH2-/- cell line was also developed using CRISPR-Cas9 gene editing technology to study the role played by UNG and MMR pathways in shaping the uracilome. Quantifying uracils in the stimulated cell lines, we found that MMR unlike UNG/BER does not play a major role in eliminating global uracils. However, qPCR analysis of switch regions of the pull-down DNA revealed that MMR plays a major role in excising uracils at switch regions. In future, uracilomes will be created to study the role played by these enzymes in shaping the uracilome and to study the mutational landscape created by AID.

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