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Access Type

WSU Access

Date of Award

January 2020

Degree Type


Degree Name




First Advisor

Ashok S. Bhagwat


AID/APOBEC3 group of cytosine deaminases are central to our innate and adaptive immunity. However, the misregulation of these enzymes may lead to mutations, double-strand breaks, and translocations, that can result in cancer or drug-resistant tumors. Due to the limitations of the current sequencing technologies to detect uracils in the DNA, it is challenging to track the activity of these enzymes. Bhagwat lab has developed a sequencing method called UPD-Seq that can map the genomic uracils. This sequencing method labels and pulls down uracilated fragments of DNA. When the human APOBEC3A or a variant of AID was expressed in E. coli cells and the resulting uracils were mapped, we were able to study the genomic targets of these deaminases and learn about their targeting behavior. Normalized Differential Coverage (NDC) was used in conjunction with other enhancements in the bioinformatics analysis to detect uracil peaks. Uracilation Index as a proxy for mutation proved to be a useful quantitative method to study the deaminase’s genome-wide targeting behavior. A3A and AID both Prefer tRNA coding genes and overwhelmingly target transcription start sites; however, the targeted genes do not have high transcription levels. A3A targets hairpin-forming sequences and this preference can override the enzyme’s nucleotide context preference; AID does not target hairpin-forming sequences. A3A activity is higher at the lagging strand template of replication, while the AID activity does not show any replicative strand bias. We show that A3A Loop1 is important for deaminase activity and substrate specificity. UPD-Seq of an A3A variant with extended loop 1 targets more tRNA coding genes, creates greater replicative strand bias, and prefers different types of hairpin structures compared to the A3A WT. We were also able to use this technology to map the targets and off-targets of the CRISPR/cas9 inspired technology, Cytosine Base Editors (CBE). UPD-Seq is potentially a great tool to test the specificity of the CBEs for when it is used in mammalian systems.

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